Stefania Lombardi

Prevalence and levels of antibodies to the circumsporozoite protein of plasmodium falciparum in an endemic area and their relationship to resistance against malaria infection

A study on malaria transmission, prevalence of infection and anti-sporozoite antibodies was carried out in Burkina Faso (West Africa). The prevalence and the levels of antibodies to (NANP)3 were found to be related to the entomological sporozoite inoculation rates measured at the same time in a defined area. The major inducer of anti-(NANP)3 antibody production under field conditions is sporozoite inoculation by infected mosquitoes. Levels of antibodies to (NANP)3 vary considerably with age and transmission season. High levels of anti-(NANP)3 antibodies raised under field conditions might offer protection against small inocula of sporozoites.

2. New methods in epidemiology and diagnosis of malaria and babesiosis. lmmunotechniques for epidemiology of malaria: appropriate tools for integration of primary health care with malaria research and control

Abstract Community-based malaria control with integrated primary health care appears to be the most feasible approach for endemic countries in their struggle against malaria. To plan and implement personal protection and vector control measures, there is the need for comprehensive information about local modes of transmission. Experience with insecticide-based vector control programmes and entomological data accumulated over the years has revealed vector systems of extraordinary heterogeneity, creating multifaceted transmission situations. The primary health care-system offers an appropriate structure to collect and evaluate microepidemiological information countrywide. Community and health workers trained and supervised by qualified personnel could be involved in the assessment of clinical, parasitological and entomological indices. Community participation is facilitated if personnel are taught the use of immunotechniques. Tests can be performed on dried material which allows samples to be stored for months without refrigeration, so that transport to and processing in a central laboratory are not subject to time constraints. This paper describes and discusses enzyme-linked immunosorbent assays to determine if antibodies to sporozoites are present in blood collected as dried spots and to identify the origin of bloodmeals using dried mosquito abdomens.Community-based malaria control with integrated primary health care appears to be the most feasible approach for endemic countries in their struggle against malaria. To plan and implement personal protection and vector control measures, there is the need for comprehensive information about local modes of transmission. Experience with insecticide-based vector control programmes and entomological data accumulated over the years has revealed vector systems of extraordinary heterogeneity, creating multifaceted transmission situations. The primary health care-system offers an appropriate structure to collect and evaluate microepidemiological information countrywide. Community and health workers trained and supervised by qualified personnel could be involved in the assessment of clinical, parasitological and entomological indices. Community participation is facilitated if personnel are taught the use of immunotechniques. Tests can be performed on dried material which allows samples to be stored for months without refrigeration, so that transport to and processing in a central laboratory are not subject to time constraints. This paper describes and discusses enzyme-linked immunosorbent assays to determine if antibodies to sporozoites are present in blood collected as dried spots and to identify the origin of bloodmeals using dried mosquito abdomens.

Evaluation of an ELISA kit for epidemiological detection of antibodies to Plasmodium falciparum sporozoites in human sera and bloodspot eluates

Antibodies to Plasmodium falciparum sporozoites represent a serological transmission indicator, which can be applied in epidemiological studies to estimate the intensity of malaria transmission. An ELISA method has been developed as an industrial kit to detect these antibodies, using a chemically synthesized (NANP)40 peptide as antigen. The results obtained with this kit are compared in the present paper with those obtained by an ELISA test already applied in epidemiological studies. In testing sera from individuals living in endemic areas, a high diagnostic concordance (92.1%) was obtained between the two assays. The absorbances of these sera correlated well, as shown by a correlation coefficient r = 0.877. Sera from individuals never exposed to malaria gave very low absorbances with the kit. This minimum non-specific binding increases the probability of comparable results in different studies. When the two ELISAs were evaluated for analytical sensitivity and precision, similar satisfactory results were achieved. The test can be performed not only with sera but also with eluates from filterpaper bloodspots. Modifications of the kit to reduce its cost and suggestions regarding distribution and funding are also proposed.

In vitro immune recognition of synthetic peptides from the Plasmodium falciparum CS protein by individuals naturally exposed to different sporozoite challenge

The impact of duration and intensity of sporozoite challenge on the in vitro cell immune response to synthetic peptides of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated in residents of a malaria endemic area in Burkina Faso (West Africa). Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used to assess immune recognition of synthetic peptides corresponding to the polymorphic Th2R and Th3R regions, to the conserved CS.T3 sequence and to NANP and degenerate NVDP repeats. Immune responses were measured in adults and children from a village where they received more than 100 sporozoite inoculations per year and in adults living in a town, exposed to a 10-100 times lower challenge. A lifetime intense exposure apparently increased the ability to proliferate in response to most peptides in the rural adults, who all produced antibodies to NANP repeats. Surprisingly, cell cultures from these subjects seldom contained appreciable levels of IFN-gamma. In the urban adults, possibly due to the moderate challenge they are exposed to, significant differences in the proliferative potentials of the peptides could be detected. The highest stimulation indices were obtained with the genetically unrestricted CS.T3 peptide. Remarkably, proliferative responses to Th2R and Th3R appeared to be correlated with the humoral response to the CS protein, indicating a T helper significance of the epitopes. The differing proliferative potential of the polymorphic epitopes in the urban adults suggests that polymorphism might delay the development of immune responsiveness under conditions of sporadic transmission. The children from the highly malarious village displayed the lowest proliferative scores, accompanied by a high prevalence of antibodies to NANP repeats. On the basis of these findings, the hypothesis is proposed that a pure B cell reactivity to NANP repeats could ontogenetically precede the mounting of a conventional T-B cooperative immune response.

The in vitro adherence of murine eosinophils, neutrophils and non-induced and induced macrophages to infective larvae of Toxocara canis (Nematoda, ascarididae)

Infective larvae of the parasite nematode Toxocara canis were incubated in vitro with murine eosinophils, neutrophils and non-induced and induced macrophages. The interactions between the different types of cells and the worms were observed in the presence or absence of immune mouse serum and/or complement. Cells showed considerable differences in the manner, duration and outcome of this interaction. Despite the adhesion of cells to the larvae of T. canis, there was no evidence of damage to the worms. Scanning and transmission electron microscopic observations suggest that the cells adhere to the cuticular surface via an electron-dense material. This material might play a protective role against the helmintotoxic capacity of the inflammatory cells.

A new method for identification of the animal origin of mosquito bloodmeals by the immunobinding of peroxidase-anti-peroxidase complexes on nitrocellulose

A method is described for identification of the animal origin of mosquito bloodmeals (MBM). The immunoassay is named DOT-PAP, it makes use of nitrocellulose as solid phase and of peroxidase-anti-peroxidase soluble complexes as detectors. DOT-PAP includes a built-in absorbing system to remove possible cross-reactivities. The sensitivity of the assay is higher than that of precipitin test or ELISA, and it dispenses with machineries for the reading of results. The method requires nanogram amounts of antigen, therefore it lends itself to the identification of incomplete MBM and of 28 h digested MBM as well. The assay can be applied to different hematophagous arthropods and the small volumes of antigen used allow to scan a vast array of possible animal sources on the same bloodmeal.

Intraerythrocytic administration of a synthetic Plasmodium antigen elicits antibody response in mice, without carrier molecules or adjuvants.

The synthetic peptide (NANP)40, reproducing the tandem-repeated epitope of the circumsporozoite protein of Plasmodium (Laverania) falciparum, was entrapped into murine, autologous erythrocytes by a hypotonic dialysis method. Mice immunized intravenously with minute amounts of encapsulated peptide produced considerable antibody titres. This result indicates that intraerythrocytic antigen administration may have a potential as an immunization system for humans, since it dispenses with adjuvants and carrier molecules.