Download Slide 1

STUDY OF THE ROLE OF IgM IN
CSA BINDING PREGNANCYASSOCIATED 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
http://www.tulane.edu/~wiser/malaria/var1.JPG
Antigenic variation
 Only one gene is expressed at a time
 The parasite switches the expressed variant antigens in order to escape
the immune system
Miller LH et al. Nature. 2002
Cytoadherance
www.wehi.edu.au
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-5isothiocyanate) 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