Expression and purification of an engineered, yeast-expressedLeishmania donovaninucleoside hydrolase with immunogenic properties

Abstract

Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.

Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

“…NHs have been considered as attractive drug targets in these organisms, taking into account their central role in the purine salvage pathway while many of these parasitic protozoa lack a de novo purine biosynthesis pathway . More recently, it was also proposed that vaccine development against NHs would prevent the replication of several pathogens during their early stages of life . Interestingly, in many other organisms NHs are present in parallel to a de novo purine biosynthetic pathway and nucleoside phosphorylases.…”

“…[17][18][19][20][21] More recently, it was also proposed that vaccine development against NHs would prevent the replication of several pathogens during their early stages of life. 22,23 Interestingly, in many other organisms NHs are present in parallel to a de novo purine biosynthetic pathway and nucleoside phosphorylases. Often in these organisms the specificity constants (k cat /K M ) of the NHs for the canonical nucleosides are substantially lower compared to the NHs of protozoa.…”

Process Characterization and Biophysical Analysis for a Yeast-Expressed Phlebotomus papatasi Salivary Protein (PpSP15) as a Leishmania Vaccine Candidate

“…With 79% of high overall recovery, we expect to collect approximately 785 mg of purified PpSP15 per litter of FS. When comparing with the purification process for several other reported recombinant protein vaccine antigens at similar fermentation scale, 24,27,[29][30][31][32] one can find that the process for PpSP15 requires much less volume of resins; in addition, size exclusion chromatography, a process not ideal for large-scale production, had been included in the purification process for some of these recombinant protein vaccine antigens to increase their purity. 24,27,30,31 However, the current purification scheme for PpSP15 does not require an size exclusion chromatography step to improve the purity to >95%, and all the chosen purification steps are scalable, except that dialysis was used to exchange PpSP15 into an appropriate buffer due to the small volumes at this development phase.…”

“…22 In collaboration with the National Institutes of Health and the Uniformed Services University of the Health Sciences, we are now developing a leishmaniasis vaccine consisting of the sandfly antigen, PpSP15, and a well-characterized Leishmania parasite antigen, LdNH36. 24,25 Although the immunogenicity and efficacy studies of PpSP15 are ongoing, in parallel, we have been developing a scalable production process for this protein, thus allowing its rapid advancement to the clinic. During molecular cloning, the Escherichia coli expression platform was first explored since a protein homolog with close to 70% sequence similarity, PdSP15, from Phlebotomus duboscqi, had already been expressed in E. coli and then crystallized.…”

Not All Antigens Are Created Equally: Progress, Challenges, and Lessons Associated with Developing a Vaccine for Leishmaniasis

“…An E. coli-expressed L. donovani nucleoside hydrolase (LdNH36) surface protein has provided partial protection against L. donovani infection in mice (33). Expression of LdNH36 in P. pastoris produced LdNH36-Y-WT that was of significantly higher molecular weight than the E. coli expressed protein (34). Gel analyses suggested that expression in yeast resulted in several high-mannose glycoforms, whose presence was first reduced by mutating N-linked glycosylation sites located outside the major immunogenic domain from asparagines to serines (LdNH36-dg) and then removed by mutating asparagines to glutamines.…”