Characterization of the Protein
The final product was tested for endotoxins, residual moisture, pH, concentration, physical appearance, identity, and quality
as measured by SDS-PAGE. These data are shown in Table 1.
Immunogenicity of the cGMP Material in a Murine Model
We compared the Leish-111f protein with the improved version Leish-110f in a murine potency assay. Mice were immunized with
a single dose of 10 µg of Leish-111f or Leish-110f adjuvanted with MPL-SE. Seven days post-injection, mice were bled and antigen-specific
IgG1 and IgG2a antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) (Figure 7). The two forms of the
protein induced comparable titers of both IgG1 and IgG2a antigen cross-reactive antibody. These results suggest that Leish-110f
made using our new process and construct induced antibody responses comparable to that obtained with Leish-111f in mice.
Table 1. Summary of in-process contaminant clearance using CEX for capture and HCI for polish
Researchers at IDRI have chosen to develop a Leishmania vaccine that is a fusion of three distinct, conserved Leishmania antigens. These three antigens—TSA, LmSTI1, and LeIF—were selected for the development of a subunit vaccine based on their
demonstrated abilities to induce at least partial protection in the BALB/c mouse model of L. major in either prophylactic
(TSA and LmSTI1) or therapeutic (LeIF) applications.13 All three antigens are present in the amastigote and the promastigote forms of the parasite and are highly conserved among
Leishmania species, a requisite for ensuring cross-species protection. Immunization with a single recombinant antigen, in addition to
having a simplified manufacturing process, may insure equivalent uptake of the components by individual antigen-presenting
cells; in turn, it may generate an immune response that is broadly specific for all the immunogenic epitopes spanning the
Creating a vaccine composed of three or more independently produced recombinant proteins would be prohibitively expensive
to manufacture and formulate, compared to the single polypeptide vaccine described here. The manufacturing cost of a vaccine
is particularly important in developing countries, and it was one of the considerations in producing Leish-110f. The original
Leish-111f fermentation was different from that used in the new process. Conditions were developed for Leish-110f to attain
a higher cell density before induction and thus, a higher product yield. This outcome was accomplished by using a richer,
semi-defined media, and a controlled feeding strategy to manage the growth of the culture. These two changes allowed for a
substantial increase in recombinant protein production per liter of culture.