From Genomes to Vaccines for Leishmaniasis
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A total of 2183 clones derived from four life cycle stage-specific, spliced-leader cDNA libraries of Leishmania major LV39 Neal strain were randomly picked and sequenced to generate expressed sequence tags (ESTs). Then 1094 unique genes were identified, with 18.2% having BLAST hits with known genes/proteins and 81.8% failing to match genes currently deposited in public databases. Approximately 250 unique genes were obtained from a lesion-derived amastigote complementary DNA (cDNA) library, the form of the parasite that is infective to the mammalian host. Polymerase chain reaction (PCR)-amplified ESTs were spotted onto glass slides, and DNA microarray used to identify a further approx 100 unique cDNAs highly expressed in amastigotes.
One hundred unique, randomly selected amastigote-expressed genes were PCR amplified to exclude the 5′-spliced leader, and the full-length genes subcloned into the TOPO-TA cloning vector. The genes were sequence-verified, excised using restriction enzyme digestion, and cloned upstream of the eukaryotic cytomegalovirus promoter into the expression vector pcDNA3. Expression plasmids were sequence-verified and large-scale, endotoxin-free plasmids prepared. Then 100 �g of each expression plasmid (DNA vaccine) was delivered subcutaneously to the rump of susceptible BALB/c mice, the mice boosted 4 wk later and then challenged 2 wk postboost with 2 � 106 L. major LV39 parasites to the hind footpad. Infection was monitored on a weekly basis by measuring footpad depth with digital calipers. Protection was scored by comparing the footpad depth of mice receiving empty vector DNA to those immunized with DNA containing L. major amastigote-expressed genes.
