STUDY OF THE ROLE OF IgM IN CSA BINDING PREGNANCY- ASSOCIATED MALARIA PROTEINS Victoire Ndong ‘09 Tenaya Vallery ‘10 Amy Springer Biology Department Mount Holyoke College

What is Malaria?  Infectious disease caused by the Plasmodium falciparum  Transmitted by a vector: the female anopheles  The parasites genome has between 50 and 60 var genes  Very difficult to find a vaccine for malaria because of antigenic variation

Global incidence of malaria Sachs

The life cycle of the parasite Inst. Pasteur

The variant antigens PfEMP1s  Transmembrane proteins  They have a variable extracellular region and a conserved intracellular region  They are encoded by var genes  Influenced by two factors: need to avoid immune system and the need to keep cytoadherance properties

Antigenic variation Only one gene is expressed at a time The parasite switches the expressed variant antigens in order to escape the immune system

Cytoadherance

PREGNANCY-ASSOCIATED MALARIA (PAM) Gamain B., S. Gratepanche, L.H. Miller

Var2CSA and Var1CSA’s Conservation Trimnell et al 2006

Immunoglobulin M There are five classes of antibodies Ig (A, G, M, D, E) They exist mostly in pentamers but they can be found in hexamers too They are the first to respond in case of an infection Previous studies have suggested that IgM acts as a bridge between healthy and infected red blood cells in rosettes IgM does not cross into the placenta only IgG does

OUR EXPERIMENT We selected domains that bind CSA, var1DBL7ε and var2DBL6ε We amplified the domains using PCR; we cloned it and transformed it into E-coli along with an HA amino acid sequence We transfected the plasmid into the mammalian cells We will incubate the cells in IgM. Afterwards we will incubate with a cy3 fluor (orange) labeled goat anti human IgM We will make a series of washes to remove the non binding IgM Then we will perform an Immunofluorescence assay (IFA) using a fluorescent anti HA antibody (Alexa fluor) to detect the cells that are expressing the domain (green) and the cells that are binding IgM (orange) We will also have a negative control

Immunofluorescence Assay (IFA) View of the mammalian cells at 400X phase 2 Nuclei seen using the DAPI (4,6 diamidino-2-phenylindole dihydrochloride)

FITC fluorescence Phase contrast The FITC ( Fluorescein-5- isothiocyanate) only shows cells expressing the var domains

A Special Thank You to Our Lab In order from left to right: Kathryn, Tenaya, Amy, Danielle, Victoire