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.

Pfs2400 can mediate antibody-dependent malaria transmission inhibition and may be the Plasmodium falciparum 11.1 gene product.

Monoclonal antibodies (mAb) have been raised against Plasmodium falciparum gametocyte stage protein extracts, in an effort to identify novel parasite antigens that might mediate malaria transmission-blocking immunity. mAb 1A1 identified Pfs2400, a sexual stage-specific antigen of greater than 2 megadaltons, that is associated with the outer leaflet of the parasitophorous vacuole membrane in mature circulating gametocyte-infected red blood cells. Upon induction of gametogenesis, Pfs2400 partitions between the gamete plasmalemma and the degenerating erythrocyte membrane. The antigen is no longer detectable in the fully emerged gamete. mAb 1A1 dramatically reduces the number of oocysts formed in P. falciparum gametocyte-fed mosquitoes. The cognate antigen is probably the product of the Pf11.1 gene (Scherf et al. 1988. EMBO [Eur. Mol. Biol. Organ.]J. 7:1129) on the basis that a peptide composed of two copies of the degenerate nine amino acid repeat sequence in the Pf11.1 protein, can inhibit binding of mAb1A1 to the native antigen. The mechanism of transmission inhibition mediated by the Pfs2400 is discussed.

Chromosome-sized DNA molecules of Plasmodium falciparum.

At least seven chromosome-sized DNA molecules (750 to 2000 kilobases in length and one fraction of undetermined molecular weight) from cultured clones and isolates of Plasmodium falciparum have been separated by pulsed-field gradient gel electrophoresis. Whereas asexual blood stages and sexual stages of the same line have identical molecular karyotypes, the length of chromosome-sized DNA molecules among different geographical isolates and several clones derived from a single patient is different. These length alterations of chromosomes are the result of DNA rearrangements that must occur unrelated to sexual differentiation.

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum.

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.

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.

Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin gene.

The nucleotide sequence of a Plasmodium falciparum actin gene has been established. The gene codes for a protein of 376 amino acids and is not interrupted by introns. The nucleotide sequence reveals an extreme bias in codon usage. Not less than 85% of the codons possess an A or T at the third position. As has been found for the actins in other unicellular eukaryotes, P. falciparum actin is related both to vertebrate cytoplasmic and vertebrate muscle specific actins. However, the malarial actin is one of the most alpha-like actins hitherto found in lower eukaryotes.

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures.

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens (Mr 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2-3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [3H]palmitic acid covalently. The iso-electric point (pI) of these proteins was assessed as being 6.0 +/- 0.1 (for both 48 and 45 kDa) and 5.6 +/- 0.1 (for 25 kDa).

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.

Identification of Plasmodium falciparum-infected mosquitoes using a probe containing repetitive DNA.

A cloned repetitive DNA sequence (rep20) was evaluated as a diagnostic probe specific for Plasmodium falciparum sporozoites using experimentally infected mosquitoes squashed directly on nylon filters. Head/thorax portions of mosquitoes, killed 14-16 days after ingesting P. falciparum-infected blood, gave positive signals when examined for the presence of P. falciparum sporozoite DNA by hybridisation. This correlated with the number of oocysts found in a sample of the same batch of mosquitoes examined by dissection. No positive signals were obtained with 50 Plasmodium berghei-infected mosquitoes probed with the rep20 sequence. The results indicate that a probe containing rep20 may be useful in the rapid and specific incrimination of vectors carrying P. falciparum sporozoites. The value of repetitive DNA in the diagnosis of malaria is discussed.

Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum.

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

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.

Cultivation of fertile Plasmodium falciparum gametocytes in semi-automated systems. 1. Static cultures.

A semi-automated cultivation apparatus for the in vitro culture of Plasmodium falciparum gametocytes is described. This apparatus has been designed to produce large numbers of fertile sexual stages for use in the development of a gamete vaccine or for the infection of suitable mosquitoes. These mosquitoes in turn may be used for the development of a possible sporozoite vaccine. Loss of red cells during medium change has been eliminated and the addition of warmed fresh medium simplified compared to similar systems described previously. Material harvested from this apparatus has been used for infecting mosquitoes. Up to 98% of Anopheles stephensi were infected with a mean oocyst count of 24 per positive gut (range one to 109). The importance of satisfactory presentation of gametocytes for mosquito infection is stressed. The possible presence of substances in normal human sera which inhibits exflagellation to a variable degree and reduces mosquito infectivity is also discussed.

Feeding behaviour and sporozoite ejection by infected Anopheles stephensi.

Anopheles stephensi mosquitoes infected with Plasmodium falciparum sporozoites were allowed to feed individually through fresh whole thickness mouse skin. More sporozoites were ejected into the skin in clusters than into the blood. Deposition of sporozoites in the blood was an infrequent occurrence and always coincided with ejection of these stages into the skin--perhaps a spill-over effect. The number of probes before feeding (median 4.5) was not correlated with the sporozoite inoculum (median 8), nor was the number of sporozoites in the glands (median 14,500). However, the number of sporozoite clusters in the skin (median 1) was positively correlated with the inoculum size. The median value of the sporozoite inoculum was 22, when only those mosquitoes that ejected sporozoites were included. When feeding was interrupted and recommended on a new membrane, sporozoite ejection occurred with equal frequency on both occasions. Sporozoites disappeared from the site of bites in living mice within 2 h of feeding. The epidemiological significance of these observations is discussed.

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.

Transmission blockade of Plasmodium falciparum: its variability with gametocyte numbers and concentration of antibody.

The transmission of Plasmodium falciparum through Anopheles stephensi was measured in the presence of anti-gamete (anti-45/48 kDa) and anti-zygote/ookinete (anti-25 kDa) antibodies. With lowering numbers of infectious gametocytes in the presence of the same concentration of antibodies, different results were obtained with the two types of antibodies. Transmission blocking appeared to weaken when using anti-45/48 kDa antibody, whilst, with the anti-25 kDa antibody, transmission inhibition was markedly strengthened with lower parasite numbers. Keeping the parasite numbers constant and progressively lowering the concentration of both types of antibodies resulted in a gradual loss of the latters' blocking ability. No enhancement of transmission occurred at low antibody concentrations.

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.

Sporozoite load of mosquitoes infected with Plasmodium falciparum.

In the laboratory, mosquitoes given a second blood meal 5-11 d after an infective one have more sporozoites in their salivary glands than do those given a single infective blood meal only. The presence of specific anti-sporozoite antibody in the second blood meal does not reduce the number of sporozoites in salivary glands. On the contrary, the presence of the raised immunoglobulin levels--even non-specific ones--may result in higher gland infections. Oocyst maturation is extremely asynchronous in mosquitoes given a single blood meal, the maturation time being 10-22 d or more. The explanation for the increased density of sporozoites in salivary glands in mosquitoes having a second blood meal may be acceleration of oocyst maturation. Multiple blood meals are a normal event for infectious mosquitoes in nature, and therefore have no special epidemiological significance. However, in the laboratory a second blood meal could be a simple procedure for increasing the efficiency of sporozoite production.

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.