title: Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside 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.
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Elissa M
authors.initials: EM
authors.lastname: Hudspeth
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Seid
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Hammond
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Junfei
authors.initials: J
authors.lastname: Wei
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Zhuyun
authors.initials: Z
authors.lastname: Liu
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhan
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Pollet
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Heffernan
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: C Patrick
authors.initials: CP
authors.lastname: McAtee
authors.affiliation: c Proteomics Programmatic Core Laboratory, Houston Methodist Hospital Research Institute , Houston , TX , USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Engler
authors.affiliation: c Proteomics Programmatic Core Laboratory, Houston Methodist Hospital Research Institute , Houston , TX , USA.
authors.forename: Risë K
authors.initials: RK
authors.lastname: Matsunami
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Strych
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.
authors.forename: Oluwatoyin A
authors.initials: OA
authors.lastname: Asojo
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.|d Department of Biology , Baylor University , Waco , TX , USA.|e James A. Baker III Institute for Public Policy, Rice University , Houston , TX , USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hotez
authors.affiliation: a Department of Pediatrics (Section of Tropical Medicine) , National School of Tropical Medicine, Baylor College of Medicine , Houston , TX , USA.|b Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development , Houston , TX , USA.|d Department of Biology , Baylor University , Waco , TX , USA.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Bottazzi
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D002850:Chromatography, Gel; D000067493:Dynamic Light Scattering; D005260:Female; D015870:Gene Expression; D007074:Immunoglobulin G / Q000097:blood; D007893:Leishmania donovani / Q000235:genetics / Q000276:immunology*; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000276:immunology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007074:Immunoglobulin G; D050505:Mutant Proteins; D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases
keywords: CpG Oligodeoxynucleotide; Leishmania donovani; Poly(lactic-co-glycolic acid) (PLGA); fermentation; purification
references.citation: Expert Rev Vaccines. 2010 Sep;9(9):1095-107
references.pmid: 20822351
references.citation: J Immunol. 1997 Jul 1;159(1):279-89
references.pmid: 9200464
references.citation: Nat Protoc. 2014 Jul;9(7):1621-33
references.pmid: 24922271
references.citation: J Pharm Sci. 1997 Nov;86(11):1315-8
references.pmid: 9383747
references.citation: Nucleic Acids Res. 2014 Jan;42(Database issue):D292-6
references.pmid: 24153109
references.citation: Lancet. 2015 Jan 10;385(9963):117-71
references.pmid: 25530442
references.citation: Adv Exp Med Biol. 2008;625:116-32
references.pmid: 18365663
references.citation: Adv Drug Deliv Rev. 1998 Jul 6;32(3):225-246
references.pmid: 10837646
references.citation: Infect Immun. 2005 Feb;73(2):812-9
references.pmid: 15664920
references.citation: Adv Drug Deliv Rev. 2009 Mar 28;61(3):248-55
references.pmid: 19272313
references.citation: Braz J Microbiol. 2013 Oct 30;44(2):351-6
references.pmid: 24294221
references.citation: Acta Trop. 1993 Mar;53(1):59-72
references.pmid: 8096110
references.citation: Am J Trop Med Hyg. 2012 Mar;86(3):417-25
references.pmid: 22403311
references.citation: Vaccine. 2001 Apr 30;19(23-24):3104-15
references.pmid: 11312005
references.citation: Iran J Allergy Asthma Immunol. 2013 Aug 28;12(4):361-7
references.pmid: 23996712
references.citation: Microb Cell Fact. 2009 Mar 24;8:17
references.pmid: 19317892
references.citation: Biotechnology (N Y). 1991 Nov;9(11):1067-72
references.pmid: 1367623
references.citation: Am J Trop Med Hyg. 2007 Dec;77(6):1060-5
references.pmid: 18165522
references.citation: J Biol Chem. 1999 Jul 23;274(30):21114-20
references.pmid: 10409664
references.citation: J Immunol Methods. 1998 Dec 1;221(1-2):35-41
references.pmid: 9894896
references.citation: Parasite. 2009 Sep;16(3):227-30
references.pmid: 19839269
references.citation: Trends Parasitol. 2012 Aug;28(8):345-52
references.pmid: 22726696
references.citation: Methods Mol Biol. 1998;103:107-20
references.pmid: 9680637
references.citation: Electrophoresis. 2009 Jun;30 Suppl 1:S162-73
references.pmid: 19517507
references.citation: Mol Oral Microbiol. 2010 Jun;25(3):178-89
references.pmid: 20502628
references.citation: Vaccine. 2013 Jan 7;31(3):480-6
references.pmid: 23176979
references.citation: Hum Vaccin Immunother. 2014;10(3):648-58
references.pmid: 24355931
references.citation: J Exp Med. 2002 Jun 17;195(12):1565-73
references.pmid: 12070284
references.citation: Eur J Pharm Biopharm. 2000 Jul;50(1):129-46
references.pmid: 10840197
references.citation: Vaccine. 2006 May 29;24(22):4863-73
references.pmid: 16635538
references.citation: Clin Exp Vaccine Res. 2015 Jan;4(1):1-10
references.pmid: 25648133
references.citation: PLoS Negl Trop Dis. 2010 Nov 09;4(11):e866
references.pmid: 21085470
references.citation: Biochim Biophys Acta. 1999 Jan 6;1426(2):227-37
references.pmid: 9878752
references.citation: Gene. 2001 Dec 12;280(1-2):153-62
references.pmid: 11738828
references.citation: Biotechnology (N Y). 1993 Aug;11(8):905-10
references.pmid: 7763913
references.citation: Vaccine. 2007 Sep 4;25(36):6572-80
references.pmid: 17675185
references.citation: Methods Mol Biol. 1999;112:531-52
references.pmid: 10027275
references.citation: J Biotechnol. 2012 Dec 31;162(2-3):171-82
references.pmid: 22981627
references.citation: Nat Rev Microbiol. 2011 Jul 11;9(8):604-15
references.pmid: 21747391
references.citation: Biomaterials. 2014 Sep;35(29):8385-93
references.pmid: 24986256
references.citation: J Biol Chem. 1991 Nov 5;266(31):20658-65
references.pmid: 1939115
references.citation: Vaccine. 2007 Aug 10;25(32):6107-17
references.pmid: 17629372
references.citation: Infect Immun. 2002 Aug;70(8):4471-6
references.pmid: 12117958
references.citation: J Pharm Sci. 2004 Oct;93(10):2573-84
references.pmid: 15349967
references.citation: Mol Cell Proteomics. 2006 Jan;5(1):144-56
references.pmid: 16219938
references.citation: J Exp Med. 1997 Nov 17;186(10):1623-31
references.pmid: 9362523
references.citation: J Virol. 2005 Aug;79(16):10386-96
references.pmid: 16051831
references.citation: Rev Soc Bras Med Trop. 1996 Mar-Apr;29(2):153-63
references.pmid: 8713607