The Infectious Disease Research Institute (IDRI) has developed a prophylactic vaccine for cutaneous leishmaniasis by constructing
a recombinant fusion protein consisting of portions of three Leishmania proteins: thiol-specific antioxidant (TSA),15
major (L. major) stress-inducible protein 1 (LmSTI1),16 and Leishmania elongation initiation factor (LeIF).17 The schematic of this construct is shown in Figure 1. The recombinant approach was preferable to live vaccine or a killed
parasite vaccine. Live vaccination is being discontinued because of undesirable side effects,18 and the manufacturing process of a killed parasite vaccine presents many difficulties relating to reproducibility, the need
for animal-derived products in the culture medium, and variability in potency. The recombinant approach lends itself to the
production of an inexpensive and effective vaccine at large scale, and if efficacious, would drastically reduce the morbidity
and mortality caused by Leishmania throughout the world.
The original form of this vaccine was manufactured under current good manufacturing practice (cGMP) conditions, is currently
undergoing human clinical testing, and will ideally provide a lasting protective immunological response. To simplify manufacturing,
several subtle process improvements included removal of a 6-histidine sequence near the amino terminus, and the mutation of
a proteolytic hot spot. The improved manufacturing method consists of fermenting an engineered version of the HMS-174 strain
of Escherichia coli (E. coli) following cGMP regulations at the 30-L scale. A purification scheme combining anion exchange chromatography with hydroxyapatite
chromatography was developed that yielded purified protein product at greater than 100 mg/L. The purified protein was formulated
and lyophilized to yield a stable cake that can safely be sent to clinical trials worldwide. The cGMP-manufactured product
was tested with the adjuvant monophosphoryl lipid A in stable emulsion (MPL-SE) in a murine potency assay. The Leish-110f
recombinant protein product reported here is an improved version of the original leishmaniasis vaccine.
Materials and Methods
Master and Working Cell Banks
Master and working cell banks were manufactured under cGMP conditions at Charles River Laboratories (Malvern, PA). The cell
banks were tested for purity, viability, and identity and are stored as frozen stocks in glycerol at –80° C. The flow chart
of fermentation and purification is shown in Figure 2.
Both the inoculums and the fermentation used 2XYS as the growth media, with dextrorotatory glucose (D-glucose) as the primary
carbon source, to grow the recombinant E. coli in a fed-batch fermentation. The fermentor was programmed to control the pH at 7.0 and dissolved oxygen at 30%. When the optical
density at 600 nanometers had increased to 8 + 1, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the fermentor to
a final concentration of 1 mM to induce expression of the recombinant gene encoding the Leish-110f protein. At three hours
post-induction, the culture was harvested by centrifugation.