Acquired immune responses to three malaria vaccine candidates and their relationship to invasion inhibition in two populations naturally exposed to malaria

Background: Malaria still represents a major cause of morbidity and mortality predominantly in several developing countries, and remains a priority in many public health programmes. Despite the enormous gains made in control and prevention the development of an effective vaccine represents a persisting challenge. Although several para site antigens including pre-erythrocytic antigens and blood stage antigens have been thoroughly investigated, the identification of solid immune correlates of protection against infection by Plasmodium falciparum or clinical malaria remains a major hurdle. In this study, an immuno-epidemiological survey was carried out between two populations naturally exposed to P. falciparum malaria to determine the immune correlates of protection. Methods: Plasma samples of immune adults from two countries (Ghana and Madagascar) were tested for their reactivity against the merozoite surface proteins MSP1-19, MSP3 and AMA1 by ELISA. The antigens had been selected on the basis of cumulative evidence of their role in anti-malarial immunity. Additionally, reactivity against crude P. falciparum lysate was investigated. Purified IgG from these samples were furthermore tested in an invasion inhibition assay for their antiparasitic activity. Results: Significant intra- and inter- population variation of the reactivity of the samples to the tested antigens were found, as well as a significant positive correlation between MSP1-19 reactivity and invasion inhibition (p < 0.05). Interestingly, male donors showed a significantly higher antibody response to all tested antigens than their female counterparts. In vitro invasion inhibition assays comparing the purified antibodies from the donors from Ghana and Madagascar did not show any statistically significant difference. Although in vitro invasion inhibition increased with breadth of antibody response, the increase was not statistically significant. Conclusions: The findings support the fact that the development of semi-immunity to malaria is probably con tingent on the development of antibodies to not only one, but a range of antigens and that invasion inhibition in immune adults may be a function of antibodies to various antigens. This supports strategies of vaccination including multicomponent vaccines as well as passive vaccination strategies with antibody cocktails

Generierung Plasmodium falciparum-spezifischer humaner Antikörperformate aus Epstein-Barr Virus-transformierten B Lymphozyten semi-immuner Individuen

Malaria tropica, caused by Plasmodium falciparum still poses a major challenge to global health care as well as to the economy in endemic countries. There is no efficient vaccine at hand, and multiple drug-resistant parasite strains are emerging. As yet, passive vaccination strategies with recombinant full-size human monoclonal antibodies (humAbs) are not pursued despite the fact that it has been shown early that these may confer passive immunity toward as well as effectively cure malaria infections by P. falciparum. The major goal of the present thesis was thus to generate anti-plasmodial human monoclonal antibodies (humAbs) which are highly specific, possess a high affinity, and are capable of inhibiting P. falciparum. To this end antigen-specific IgG+ / CD22+ B cells from semi-immune Ghanaian donors were enriched by flow-cytometric cell sorting and were then subjected to EBV transformation. VH-, Vkappa-, and Vlambda sequences of promising cultures were rescued and cloned into plant expression vectors. These V regions were then produced in thecontext of full-size IgG1:kappa and IgG1:lambda2 antibodies in Nicotiana benthamiana. Confocal immunofluorescence microscopy, surface plasmon resonance measurements and in vitro growth inhibition assays served to characterize the isolated humAbs. The flow cytometric enrichment of antigen-specific B cells prior to their virocrine transformation can be considered a novelty. This approach allows for a more efficient EBV transformation requiring less personnel and material. From this point of view, the work presented can also be regarded as a feasibility study as well as a process optimization. Four anti-plasmodial humAbs were isolated, cloned and characterized. Three of these humAbs recognized Merozoite Surface Protein 10 (MSP10). So far monoclonal anti-MSP10 antibodies have not been described yet. The fourth antibody was specific to Apical Membrane Antigen 1 (AMA1). To the best of our knowledge, this is the first human AMA1-specific antibody to be described. Those three antibodies which possessed the genuine pairing of heavy and light chains were highly maturated (at least 39-59 DNA mutations) and highly affine to their respective targets (KD = 5.46 x 10-9 M – 1.99 x 10-10 M). In growth inhibition assays the MSP10-specific humAbs inhibited P. falciparum in a strain-dependent fashion. Strain 3D7 showed EC50-values in the range of 4.1 mg/mL – 9.5 mg/mL. The maximal inhibition of strains HB3 and Dd2 was around 39% at 10 mg/mL IgG. In contrast, the AMA1-reactive humAb (humAbAMA1) was panspecific and (highly) inhibitory for all P. falciparum strains tested (EC50 = 34.9 μg/mL – 3.8 mg/mL). Regarding the possible mode of action humAbAMA1 likely binds in the hydrophobic trough of AMA1 and thus torpedoes its interaction with RON2. It is generally accepted that the formation of the AMA1:RON2 complex is vital to the invasion of merozoites into new red blood cells. Whereas the three MSP10-specific humAbs seem to be suitable primarily for basic research humAbAMA1 additionally appears to be an ideal candidate for passive immunization strategies for malaria prophylaxis and therapy

Generierung Plasmodium falciparum-spezifischer humaner Antikörperformate aus Epstein-Barr Virus-transformierten B Lymphozyten semi-immuner Individuen

Malaria tropica, caused by Plasmodium falciparum still poses a major challenge to global health care as well as to the economy in endemic countries. There is no efficient vaccine at hand, and multiple drug-resistant parasite strains are emerging. As yet, passive vaccination strategies with recombinant full-size human monoclonal antibodies (humAbs) are not pursued despite the fact that it has been shown early that these may confer passive immunity toward as well as effectively cure malaria infections by P. falciparum. The major goal of the present thesis was thus to generate anti-plasmodial human monoclonal antibodies (humAbs) which are highly specific, possess a high affinity, and are capable of inhibiting P. falciparum. To this end antigen-specific IgG+ / CD22+ B cells from semi-immune Ghanaian donors were enriched by flow-cytometric cell sorting and were then subjected to EBV transformation. VH-, Vkappa-, and Vlambda sequences of promising cultures were rescued and cloned into plant expression vectors. These V regions were then produced in thecontext of full-size IgG1:kappa and IgG1:lambda2 antibodies in Nicotiana benthamiana. Confocal immunofluorescence microscopy, surface plasmon resonance measurements and in vitro growth inhibition assays served to characterize the isolated humAbs. The flow cytometric enrichment of antigen-specific B cells prior to their virocrine transformation can be considered a novelty. This approach allows for a more efficient EBV transformation requiring less personnel and material. From this point of view, the work presented can also be regarded as a feasibility study as well as a process optimization. Four anti-plasmodial humAbs were isolated, cloned and characterized. Three of these humAbs recognized Merozoite Surface Protein 10 (MSP10). So far monoclonal anti-MSP10 antibodies have not been described yet. The fourth antibody was specific to Apical Membrane Antigen 1 (AMA1). To the best of our knowledge, this is the first human AMA1-specific antibody to be described. Those three antibodies which possessed the genuine pairing of heavy and light chains were highly maturated (at least 39-59 DNA mutations) and highly affine to their respective targets (KD = 5.46 x 10-9 M – 1.99 x 10-10 M). In growth inhibition assays the MSP10-specific humAbs inhibited P. falciparum in a strain-dependent fashion. Strain 3D7 showed EC50-values in the range of 4.1 mg/mL – 9.5 mg/mL. The maximal inhibition of strains HB3 and Dd2 was around 39% at 10 mg/mL IgG. In contrast, the AMA1-reactive humAb (humAbAMA1) was panspecific and (highly) inhibitory for all P. falciparum strains tested (EC50 = 34.9 μg/mL – 3.8 mg/mL). Regarding the possible mode of action humAbAMA1 likely binds in the hydrophobic trough of AMA1 and thus torpedoes its interaction with RON2. It is generally accepted that the formation of the AMA1:RON2 complex is vital to the invasion of merozoites into new red blood cells. Whereas the three MSP10-specific humAbs seem to be suitable primarily for basic research humAbAMA1 additionally appears to be an ideal candidate for passive immunization strategies for malaria prophylaxis and therapy