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Studies on Plasmodium falciparum asexual blood stage antigens: RAP-2/RSP-2 and Pf332 in focus
Stockholm University, Faculty of Science, The Wenner-Gren Institute . (Klavs Berzins)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The life cycle of the malaria parasite is very complex and provides a number of potential targets for vaccination. In this thesis, data on two plasmodial asexual blood stage antigens (RAP-2 and Pf332) are presented.

A partial aim of the work presented herein was to investigate the mechanisms responsible for the destruction of erythroid cells in anaemia, and more specifically to define the role of the rhoptry associated protein (RAP)-2 and other members of the RAP complex, RAP-1 and -3 in processes resulting in anaemia. Antibodies to the RAP complex were shown to have the potential to mediate the destruction of RAP-2-tagged erythroid cells by phagocytosis or by complement activation and lysis. In addition, antibodies to RAP-1 and RAP-2 could induce the apoptotic death of RAP-2- tagged erythroblasts. The frequency and functionality of naturally occurring RAP-2 antibodies in the sera of anaemic and non-anaemic Cameroonian children were also investigated. All sera tested contained RAP-2-reactive antibodies by both immunofluorescence and flow cytometry. The anaemic group of children had higher levels of IgG than the non-anaemic ones, while the levels of IgM were similar. With respect to IgG subclasses, higher levels of IgG3 were seen in the non-anaemic individuals as compared to anaemic subjects. The non-anaemic individuals recognised a greater proportion of RAP-2-tagged RBCs and activated complement to a greater extent than the anaemic ones.

Earlier studies observed that humans continuously exposed to malaria, recognised Pf332 extensively. Further studies revealed that Pf332 antibodies were able to inhibit parasite growth and cytoadherence in vitro. Making use of Pf332-C231, a sub-fragment of Pf332, we studied the effects/mode of action of C231-specific antibodies on P. falciparum parasite growth and development in vitro. The antibodies appeared to act mainly on late stage parasites by two main mechanisms: 1) through the induction of abnormal/pyknotic parasites, and, 2) RBC lysis (disintegration of RBCs), thus limiting parasite growth and development. The antibody isotype in this context was IgG. Following the removal of immune pressure, parasites resumed growth, albeit at a much slower rate. The results suggest that during natural infections, antibodies to C231 could play a role in parasite control.

In summary, these data suggest that antibodies to both antigens could be instrumental in immune responses leading to disease control, but could also mediate pathology.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University , 2011. , 94 p.
Keyword [en]
immunity, malaria, RBCs, anaemia
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
URN: urn:nbn:se:su:diva-57255ISBN: 978-91-7447-284-4OAI: oai:DiVA.org:su-57255DiVA: diva2:414871
Public defence
2011-06-14, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following publication was unpublished and had a status as follows: Paper 3: Manuscript.Available from: 2011-05-12 Created: 2011-05-04 Last updated: 2011-08-22Bibliographically approved
List of papers
1. Mechanisms of malarial anaemia: potential involvement of the Plasmodium falciparum low molecular weight rhoptry-associated proteins.
Open this publication in new window or tab >>Mechanisms of malarial anaemia: potential involvement of the Plasmodium falciparum low molecular weight rhoptry-associated proteins.
2009 (English)In: Acta Tropica, ISSN 0001-706X, E-ISSN 1873-6254, Vol. 112, no 3, 295-302 p.Article in journal (Refereed) Published
Abstract [en]

Plasmodium falciparum malaria is a major cause of morbidity and mortality throughout the tropics. Anaemia is a constant feature of the disease. Pregnant women mostly primigravidae and children below the age of 5 years are the most afflicted. Its pathogenesis is multifactorial and incompletely understood. Among several factors, the destruction of erythrocytes (RBCs) is the most frequently observed cause of severe malarial anaemia and the removal of non-parasitized RBCs (nEs) is thought to be the most important, accounting for approximately 90% of the reduction in haematocrit in acute malaria. Previous studies demonstrated that the tagging of nEs with the parasite antigen RAP-2 (rhoptry-associated protein-2; also designated RSP-2) due to either failed or aborted invasion by merozoites resulted in the destruction of these cells. In this study we further investigated the mechanisms mediating the destruction of nEs in the development of severe malarial anaemia and the possible involvement of RAP-2/RSP-2 and other members of the low molecular weight rhoptry complex (RAP-1: rhoptry-associated protein-1 and RAP-3: rhoptry-associated protein-3). Antibodies to the rhoptry-associated proteins were found to recognise the surface of nEs in a parasitaemia-dependent manner after merozoite release in P. falciparumin vitro cultures. These cells, as well as erythroblasts co-cultured with infected RBCs (IEs), could then be destroyed by either phagocytosis or lysis after complement activation. The ability of anti-rhoptry antibodies to mediate the destruction of RAP-2/RSP-2-tagged erythroblasts in the presence of effector cells was also investigated. Data obtained suggest that mouse monoclonal antibodies to the low molecular weight RAP proteins mediate the death of RAP-2/RSP-2-tagged erythroblasts on interaction with adherent monocytes. The mechanism of cell death is not yet fully known, but seems to involve primarily apoptosis. The above observations suggest that the antibody response against RAP-2/RSP-2 and other members of the complex could trigger the destruction of RAP-2/RSP-2-tagged host cells. Taken together it appears that during severe anaemia a defective bone marrow or dyserythropoiesis possibly due to erythroblast cell death, may overlap with the accelerated destruction of normal erythroid cells, either by opsonisation or complement activation further aggravating the anaemia which may become fatal. These observations could therefore have implications in the design, development and deployment of future therapeutic interventions against malaria.

Keyword
Malaria, Anaemia, Plasmodium falciparum, Rhoptry-associated protein complex, Complement, Phagocytosis, Apoptosis
National Category
Immunology
Identifiers
urn:nbn:se:su:diva-32901 (URN)10.1016/j.actatropica.2009.08.017 (DOI)000271797300009 ()19695213 (PubMedID)
Available from: 2009-12-17 Created: 2009-12-17 Last updated: 2011-06-15Bibliographically approved
2. Antibodies to the Plasmodium falciparum rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children
Open this publication in new window or tab >>Antibodies to the Plasmodium falciparum rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children
Show others...
2011 (English)In: Parasite immunology (Print), ISSN 0141-9838, E-ISSN 1365-3024, Vol. 33, no 2, 104-115 p.Article in journal (Refereed) Published
Abstract [en]

Previous studies have implicated reactive antibodies to the low molecular weight rhoptry-associated proteins (RAP-1, RAP-2/RSP-2 and RAP-3) in erythroid cell destruction during Plasmodium falciparum infection. In this pilot study, the frequency, specificity and functional capacity of naturally acquired anti-RAP-2/RSP-2 antibodies were investigated in the sera of anaemic and nonanaemic malaria-infected Cameroonian children. All sera recognized RAP-2/RSP-2 by FACS, irrespective of the clinical status of the subjects. However, the anaemic children showed higher levels of IgG antibodies than the nonanaemic group, while both groups showed similar levels of IgM antibodies. Only few individuals had detectable levels of RAP-2/RSP-2-specific IgG1 and IgG3 subclass antibodies, while no IgG2 and IgG4 subclass antibodies were detected in these subjects. By ELISA, the anaemic group tended to show higher levels of antibodies to RAP-2/RSP-2 regarding all antibody classes tested, except for IgG4 and IgE. Unexpectedly, sera from the nonanaemic group activated complement to a greater extent than those from the anaemic group. These results need to be confirmed in extended studies but indicate that the effector functions of the RAP-2/RSP-2-reactive antibodies may be more important than their amounts. Such antibodies could play a role in both immunity and pathogenesis during P. falciparum infection.

Keyword
antibodies, complement, malarial anaemia; Rhoptry-associated protein-2 (RAP-2/RSP-2), Plasmodium falciparum
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:su:diva-57253 (URN)10.1111/j.1365-3024.2010.01259.x (DOI)
Available from: 2011-05-05 Created: 2011-05-04 Last updated: 2011-12-14Bibliographically approved
3. Pf332-C231- reactive antibodies affect growth and development of intraerythrocytic Plasmodium falciparum parasites
Open this publication in new window or tab >>Pf332-C231- reactive antibodies affect growth and development of intraerythrocytic Plasmodium falciparum parasites
(English)Article in journal (Refereed) Submitted
Keyword
Malaria, Pf332-C231, antibodies, growth inhibition
National Category
Infectious Medicine Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:su:diva-60515 (URN)
Available from: 2011-08-19 Created: 2011-08-18 Last updated: 2011-08-22Bibliographically approved

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