Manthri S. Ramasamy

Interactions of human malaria parasites, Plasmodium vivax and P.falciparum, with the midgut of Anopheles mosquitoes.

Abstract Present understanding of the development of sexual stages of the human malaria parasites Plasmodium vivax and P.falciparum in the Anopheles vector is reviewed, with particular reference to the role of the mosquito midgut in establishing an infection. The sexual stages of the parasite, the gametocytes, are formed in human erythrocytes. The changes in temperature and pH encountered by the gametocyte induce gametogenesis in the lumen of the midgut. Macromolecules derived from mosquito tissue and second messenger pathways regulate events leading to fertilization. In An.tessellatus the movement of the ookinete from the lumen to the midgut epithelium is linked to the release of trypsin in the midgut and the peritrophic matrix is not a firm barrier to this movement. The passage of the P. vivax ookinete through the peritrophic matrix may take place before the latter is fully formed. The late ookinete development in P.falciparum requires chitinase to facilitate penetration of the peritrophic matrix. Recognition sites for the ookinetes are present on the midgut epithelial cells. N‐acetyl glucosamine residues in the oligosaccharide side chains of An.tessellatus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition sites for P.vivax and P.falciparum ookinetes. It is possible that ookinetes penetrating epithelial cells produce stress in the vector. Mosquito molecules may be involved in oocyst development in the basal lamina, and encapsulation of the parasite occurs in vectors that are refractory to the parasite. Detailed knowledge of vector‐parasite interactions, particularly in the midgut and the identification of critical mosquito molecules offers prospects for manipulating the vector for the control of malaria.

High seasonal malaria transmission rates in the intermediate rainfall zone of Sri Lanka.

Malaria transmission was studied at Nikawehera, a long-established farming village, located in the intermediate rainfall zone of Sri Lanka. Observations were made over a 12-month period (October-September) that included the main rainy season which occurred during the north-east monsoon in November-January. Anolpheles culicifacies, the recognized vector of malaria in Sri Lanka, was the predominant anopheline mosquito collected by human night baiting at Nikawehera. High entomological inoculation rates with An. culcifacies (0.12/hour for Plasmodium vivax) were observed during the height of the transmission season which occurs during, and immediately after, the north-east monsoon. Anolpheles subpictus was identified as a possible additional vector at Nikawehera. Anopheles annularis, a major vector at Weheragala, a site in a new irrigation development (the Mahaweli Scheme) located 70 km away in the dry zone, was not collected by human baiting at Nikawehera. Clinical, entomological and parasitological data suggest that malaria is hyperendemic at Nikawehera, with high seasonal transmission rates.

MOSQUITO MIDGUT GLYCOPROTEINS AND RECOGNITION SITES FOR MALARIA PARASITES

Midgut glycoproteins of the malaria vector Anopheles tessellatus were partially characterised by gel electrophoresis and lectin binding. Specific binding to wheat germ agglutinin (WGA) and Concanavalin A (Con A) indicated the presence of N-linked core oligosaccharides in many proteins. Rabbit antibodies were produced against wheat germ agglutinin binding proteins (WGABP). These antibodies also recognised distinct proteins in the peritrophic membrane which is secreted into the midgut to enclose a bloodmeal. Rabbit anti-WGABP antibodies ingested in a bloodmeal containing infective gametocytes of the human malaria parasites Plasmodium falciparum and P. vivax tended to reduce infectivity of the parasites to vector mosquitoes. Chitotriose added to a bloodmeal also inhibited parasite development in the mosquito. The results are consistent with a hypothesis that N-acetyl glucosamine residues in mosquito midgut glycoproteins and/or midgut chitin and proteoglycan function as recognition sites for malaria parasites.

Antibodies to Anopheles midgut reduce vector competence for Plasmodium vivax malaria

Anopheles tessellatus mosquitoes ingested Plasmodium vivax gametocytes in human erythrocytes suspended in rabbit sera with and without anti‐mosquito midgut antibodies. When the mosquito bloodmeal contained anti‐midgut antibodies, fewer oocysts of P. vivax developed on the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. Complement inactivated serum also reduced the infection rate and load. A second bloodmeal containing anti‐midgut antibodies, given 48 or 72 h later, did not enhance the transmission‐blocking effect. IgG purified from antimidgut sera was shown to mediate the transmission‐blocking effect.

Antibodies and Plasmodium falciparum merozoites

There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium falciparum merozoites affect red blood cell invasion.

Antibody and clinical responses in volunteers to immunization with malaria peptide–diphtheria toxoid conjugates

Twenty residue peptides from the 185–200‐kD and 45‐kD merozoite surface antigens of the malaria parasite Plasmodium falciparum were covalently linked to diphtheria toxoid as a carrier and used to immunize human volunteers with aluminium hydroxide as an adjuvant. Significant antibody levels were elicited by two boosting injections. The antibodies reacted with acetone‐methanol fixed merozoite membranes in an immunofluorescence assay, but no inhibition of merozoite reinvasion could be detected in in vitro cultures containing the antibodies. Antibody levels against the immunizing peptides declined markedly within 77 days after the third injection. No hypersensitivity was observed against the peptides. However, the volunteers developed hypersensitivity against diphtheria toxoid, and in particular a pronounced type III (Arthus) hypersensitivity after three injections with the toxoid. This effect might appear to limit the use of peptide–diphtheria toxoid conjugates for human immunization. Several biochemical, haematological and immunological tests done on the volunteers showed no other adverse effects from the immunizations.

Fecundity of Anopheles tessellatus reduced by the ingestion of murine anti-mosquito antibodies

Abstract. High titres of antibodies to antigens derived from head/thorax, midgut or abdomen of Anopheles tessellatus were produced in inbred mice. These antibodies, when ingested in a bloodmeal, reduced the fecundity of An.tessellatus by up to 29% in different experiments. It is postulated that antibodies directed against antigens shared between the head/thorax, abdomen and midgut tissues are involved in the reduction of fecundity.

Population dynamics of anthropophilic mosquitoes during the northeast monsoon season in the malaria epidemic zone of Sri Lanka

Abstract. Mosquito‐borne diseases are a major health problem in Sri Lanka. Human biting mosquitoes were collected during the night (18.00–06.00 hours) at Nikawehera village, in the malaria endemic intermediate rainfall zone of the country. Collections were made at monthly intervals in the period October 1991 to April 1992, which included the main rainy season due to the northeast monsoon (October‐January). Thirteen Anopheles, eleven Culex, three Aedes, three Mansonia and one Armigeres species were identified, including known vectors of malaria, Bancroftian filariasis, Japanese encephalitis and dengue fever. Mosquito human‐biting rates were highest in December. The main malaria vector Anopheles culicifacies showed peak biting between 18.00 and 23.00 hours whereas the predominant culicines Culex fuscocephala and Cx quinquefasciatus preferred to bite after midnight. In 1991‐92 the prevalence of some species of anophelines at Nikawehera differed markedly from that observed in 1990‐91 and the possible reasons are discussed.

Model multiple antigenic and homopolymeric peptides from non-repetitive sequences of malaria merozoite proteins elicit biologically irrelevant antibodies

Three model peptides containing B-epitopes from conserved, non-repetitive regions of the merozoite surface antigens, MSA2 and MSA1, and the erythrocyte binding protein EBP of Plasmodium falciparum were synthesised. The peptides incorporated GPG spacers and C residues at the N and C termini, and were polymerised by oxidation to form cystine bridges. Multiple copies of essentially the same peptide sequences were also synthesised on a branching lysyl matrix to form a tetrameric multiple antigen peptide. Rabbits were immunised with the polymerised and multiple antigen peptides, in alum followed by Freunds adjuvant, and the antibody responses examined by IFA and ELISA. Reproducible antibody responses were obtained against the MSA1 and EBP but not MSA2 peptides. IgG antibody levels detected by ELISA after three injections of antigen in alum, increased significantly after further immunisation in Freunds adjuvant. IgG levels were largely maintained for at least 23 weeks after the final immunisation. IgM antibodies, generally detectable only after immunisation in Freunds adjuvant, were absent 23 weeks later. Antibody titres against the native protein on fixed parasites, assayed by IFA, were three to five orders of magnitude lower than the corresponding ELISA titres against the peptides. Antibody-dependent inhibition of P. falciparum growth in vitro could not be demonstrated with the immune rabbit sera. The MSA1 and EBP peptides elicited cross-reactive antibodies. The results suggest that the selected non-repetitive sequences are conformationally constrained in the native proteins and only a small proportion of the anti-peptide antibodies bind to the native proteins. The significance of the findings for the development of peptide vaccines and the use of peptides in immunoassays is discussed.

Antigenic relationships between adult and larval Anopheles tessellatus midgut glycoproteins and the midguts of other vector mosquitoes

Glycoproteins expressed on the surface of midgut (MG) epithelium and the peritrophic matrix (PM) of vector mosquitoes (Diptera: Culicidae) are candidate molecules for interacting with pathogens. Antisera produced against Anopheles tessellatus Theobald female MG lectin‐binding proteins (concanavalin A and wheat germ agglutinin) were used in Western blots to investigate MG/PM antigenic relationships between adult and larval An. tessellatus and with the MG glycoproteins of other vector mosquitoes: Anopheles culicifacies Giles, An. subpictus Grassi, An. varuna Iyengar, Aedes aegypti (L.) and Culex quinquefasciatus Say. Within An. tessellatus, strong antigenic cross‐reactions were observed between adult and larval MG proteins, and between adult MG and PM proteins. Anopheles tessellatus adult MG antisera reacted with MG antigens from adult females of the other five mosquito species, with interspecific contrasts of relative molecular mass (Mr) of nearly all reacting antigens, except the strong 36 kDa band shared by An. tessellatus and Cx. quinquefasciatus. Cross‐reactivity within female An. tessellatus may be due to the MG containing precursors to the PM glycoproteins and/or some common fully processed proteins, or perhaps carbohydrate epitopes that are shared between related or unrelated MG and PM glycoproteins. Cross‐reactions between adult MG proteins from different mosquito species, mostly with differential Mr, reflect the presence of homologous proteins that may be relevant to specific vector competence.