Immunotoxin

The immunotoxin is composed of two main parts. The first is a single-chain antibody fragment (scFv) directed to Pbs2l, a surface membrane protein of P. berghei ookinetes. This fragment is linked to the second part, a lytic peptide, Shiva-1. Shiva-1 is a synthetic peptide with an amino acid sequence similar, in terms of length (38 amino acids, charge distribution as well as hydrophobicity, to Cecropin B which is an antibacterial peptide. The purpose of the immunotoxin is to specifically target and lyse the parasite.

Previous experiments conducted show that feeding mosquitoes with recombinant E.Coli that expressed this immunotoxin induced a significantly lower oocyst density. [1]

However E. Coli is not a natural symbiote of the mosquito. We would like to contribute to the fight against malaria by engineering Asaia in order to express the immunotoxin in the mosquito's intestinal tract, and run experiments to see if it significantly reduces the number of oocysts.

The sequence of the immunotoxin was taken from our reference paper [1], and the codons were optimized for E.Coli. The codon optimized sequence can be found here.

The "P-proteins"

The ookinete has been intensively studied by scientists, looking for an ideal transmission-blocking vaccine target. iGEM EPFL team's interest is focused on the ookinete surface membrane proteins Pfs25 and Pfs28. In fact, it has been shown that the binding of specific antibodies to these surface proteins was fatal to more than 99% of the parasites[2]. Indeed, P25 and P28 seem to be essential to the ookinete survival, the crossing of the midgut epithelium and its transformation into an oocyst.

The team decided to design Asaia to express a soluble form of these 2 P-proteins. It may seem contra-intuitive to provide more of these proteins (by expression in Asaia) that are essential for survival, migration, and development of the parasite. However, our hypothesis is based on the fact that these proteins over-expressed in a soluble form would compete with the parasite surface protein and prevent the interaction necessary to its transmission. Because the functions of P25 and P28 are redundant, both proteins need to be inhibited or outcompeted to efficiently block the malaria transmission.

References:

• [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T29-42JHDJD-9&_user=10&_coverDate=03%2F31%2F2001&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5f4b78b08a1846e241faaed33bc76cb3&searchtype=a 1. Shigeto Yoshida, Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes, Molecular and Biochemical Parasitology (2001)]
• [http://www.nature.com/emboj/journal/v20/n15/full/7593895a.html 2. Ana M. Tomas, Gabriele Margos, Robert E. Sinden, P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions, The EMBO Journal (2001)]
• [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951121/?tool=pubmed 3. Ajay K. Saxena, Yimin Wu, and David N. Garboczi, Plasmodium P25 and P28 Surface Proteins: Potential Transmission-Blocking Vaccines, Eukaryot Cell (2007)]