Female BALB/c (Bagg albino color locus genotype c/c) mice were purchased from Charles River Laboratories (Wilmington, MA)
and were maintained in specific-pathogen-free conditions. Eight- to 12-week-old mice at the beginning of experiments were
used. Three mice per group were immunized with 10 µg of Leish-110f or Leish-111f formulated with 20 µg of an adjuvant MPL-SE
(GlaxoSmithKline Biologicals, Rixensant, Belgium) in a volume of 0.1 mL. The mice were immunized subcutaneously once at the
base of the tail. Blood was taken from the mice one week after the immunization to determine immunoglobulin G1 (IgG1) and
immunoglobulin G2a (IgG2a) antibody titers. Plates were coated with 200 ng per well of Leish-110f or Leish-111f diluted in
coating buffer, followed by blocking with phosphate-buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin.
Mouse serum samples were diluted to 1:100 and applied to the plates in two-fold serial dilutions. The plates were incubated
with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG1 or IgG2a (SouthernBiotech, Birmingham, AL), were developed
with 3,3',5,5'-Tetramethylbenzidine (TMB) substrate, and were read by a microplate reader at a 450 nanometer wavelength. Endpoint
titers were calculated with GraphPad Prism software using an optical density (OD) value of 0.1 as a cutoff.
The fed-batch fermentation of the HMS-174/L110f recombinant system was consistent in its growth profile and production of
the vaccine product Leish-110f. The Leish-110f protein was produced in the inclusion bodies. A two-step orthogonal purification
procedure post-inclusion body washing achieved significant purification of the vaccine candidate. A chromatogram of the QFF
anion exchange column separation (Figure 3) shows a single major peak that contained the Leish-110f polyprotein. The second
step in the purification used ceramic hydroxyapatite, with the Leish-110f protein eluted as a single major peak (Figure 4)
at low concentrations of phosphate. The majority of the contaminants remained bound to the resins used. The in-process SDS-PAGE
gel shown in Figure 5 illustrates purification as additional purification steps were added. The utility of coupling these
methods is that the eluted protein from the first step need not be conditioned between chromatographies, as the protein binds
to the hydroxyapatite even in the presence of NaCl. SDS-PAGE of the eluted material illustrates the purity of the final Leish-110f
protein product (Figure 6). Yield of purified protein is more than 150 mg/L and is a tremendous improvement over the original
L-111f protein yield of 30 mg/L.
Following purification, urea was removed by diafiltration, and the protein was filled and lyophilized as described in the
materials and methods section, above. The protein formed a stable white cake in the vials after lyophilization, and that quickly
redissolved upon addition of water. Real-time stability studies at several temperatures are ongoing, but preliminary data
suggests that the purified bulk protein and the lyophilized cake are stable at all temperatures tested (data not shown).