Study of Factors Involved in Resistance to Severe Malaria

Malaria remains an important public health threat, responsible for over two million deaths annually, the majority among African children. The mechanisms underlying resistance to the most severe manifestations of malaria remain elusive. Severe malaria has been associated with high levels of pro-inflammatory cytokines that may contribute to the pathogenesis of cerebral malaria, severe anemia and acidosis. This response is thought to be driven primarily by glycosylphosphatidylinositol (GPI) anchors derived from erythrocyte-stage Plasmodium falciparum. It has been suggested that antibodies against GPI are responsible for resistance to severe malaria, which has led to efforts to develop a GPI-based vaccine. An alternative explanation for the development of resistance to severe malaria is down regulation of the innate pro-inflammatory response by repeated ligation of toll-like receptors (TLR) by GPI, or other putative P. falciparum-derived TLR ligands, such as hemozoin. This cross-sectional study at l'Hopital Gabriel Toure in Bamako, Mali, proposes to test the hypothesis that resistance to severe malaria is associated with an attenuation of the TLR-mediated pro-inflammatory cytokine response. After informed consent is obtained from the participant's parent or guardian, 2-3 mL of venous blood will be drawn from each of 60 children: 40 children presenting with acute P. falciparum infection (20 severe and 20 uncomplicated) and 20 healthy, P. falciparum uninfected controls. The blood will be tested for antibody titers against GPI and Merozoite Surface Protein-1 (MSP-1). Peripheral blood mononuclear cells (PBMCs) will be isolated and cultured with GPI, hemozoin, lipoteichoic acid (LTA), lipopolysaccharide (LPS), and cytosine-guanine dinucleotide (CpG). At 24 hours, supernatant will be collected and the following cytokines measured: IL-1, IL-6, IL-8, IL-10, IL-12, and TNF-alpha. A clearer understanding of the responsiveness to TLR ligands in children from malaria endemic areas may provide information on potential intervention strategies such as GPI-based vaccines or the use of TLR agonists as vaccine adjuvants.

• INCLUSION CRITERIA -MALARIA PATIENTS:

• Males or females ages 3 to 10.
• Severe malaria as defined by positive blood smear for P. falciparum and need for hospitalization in accordance with the WHO definition of severe malaria (group I), or mild malaria as defined by positive blood smear for P. falciparum and triage to outpatient treatment (group II).
• Willingness of parent or guardian to have his or her child participate in the study as evidenced by the completed informed consent document.

Inclusion criteria

-HEALTHY VOLUNTEERS:

• Males or females ages 3 to 10.
• No clinical evidence of malaria and negative blood smear.
• No acute febrile or systemic illness.
• Willingness of parent or guardian to have his or her child participate in the study as evidenced by the completed informed consent document.

Exclusion criteria

-MALARIA PATIENTS:

• Active bleeding or hematocrit less than or equal to 15%.
• Participation in a vaccine or drug trial within 30 days of starting this study.
• Use of corticosteroids or immunosuppressive drugs within 30 days of starting this study.
• Receipt of a live vaccine within past 4 weeks or killed vaccine within past 2 weeks prior to entry into the study.
• Known history of HIV infection.

Exclusion criteria

-HEALTHY VOLUNTEERS:

• Positive malaria smear.
• Sibling of malaria patient.
• Active bleeding or hematocrit less than or equal to 15%.
• Participation in a vaccine or drug trial within 30 days of starting this study.
• Use of corticosteroids or immunosuppressive drugs within 30 days of starting this study.
• Receipt of a live vaccine within past 4 weeks or killed vaccine within past 2 weeks prior to entry into the study.
• Known history of HIV infection.