Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

Multi-antigenic recombinant subunit vaccine against Lawsonia intracellularis: The etiological agent of porcine proliferative enteropathy.

Proliferative enteropathy, caused by Lawsonia intracellularis, represents a threat for swine industry. Current vaccines are effective but difficult to obtain and scaled up, because of demanding bacterial culture conditions. In this work, a subunit vaccine candidate against L. intracellularis was developed and its efficacy was evaluated in vivo, alone or co-formulated with pig recombinant IFN-α. The vaccine formulation contains three chimeric antigens: two outer membrane proteins and a secreted one, which were engineered by adding T epitopes using bioinformatics tools. After simultaneously expressing the three antigens in E. coli, its immunogenicity was tested in mice and pigs. Antigens co-formulated with porcine IFN-α were also assayed in the last species. Immune response was assessed by ELISA and qPCR, and histopathological studies of intestinal epithelial tissue were performed after challenge. Mice and pigs showed an increased IgG response against chimeric antigens. Particularly, there were significant differences in the antibody response when porcine IFN-α was co-administrated with L. intracellularis antigens. Besides, mRNAs from il12 and cd4 marker were detected during the first week after immunization of pigs, suggesting a Th1-type cellular immune response. The significant enhancement of oas2 gene expression indicates the effect exerted by porcine IFN-α. Post-mortem histopathological analysis post-challenge revealed damage only into epithelial cells of the gastrointestinal tract from animals of the negative control group. Injuries were related to atrophy of the intestinal villi, where a decrease of globet cells and a greater migration of lymphocytes were observed. Overall, our results demonstrated that the vaccine candidate elicited significant humoral and cellular immune responses. Besides, histopathological analysis suggested that vaccinated animals were protected against experimental L. intracellularis infection. This research constitutes a step forward to the generation of the first recombinant chimeric vaccine against L. intracellularis, representing a faster, easier and cost effective approach to counteract the porcine proliferative enteropathy.

Neuroprotective effect of a new variant of Epo nonhematopoietic against oxidative stress.

Human erythropoietin is mainly recognized for its hematopoietic function; however, by binding to its receptor (EpoR), it can activate different signaling pathways as STAT, PI3K, MAPK and RAS to increase cellular differentiation or provide neuroprotective effects, among others. A recombinant human erythropoietin variant with low glycosylation and without hematopoietic effect (EpoL) was purified from skimmed goat milk. Recombinant human erythropoietin (Epo) was obtained from CHO cell line and used as control to compare EpoL effects. Neuroprotection studies were performed in PC12 cells and rat hippocampal slices. Cells were pretreated during 1h with EpoL or Epo and exposed to oxidative agents (H2O2 or FCCP); cell viability was assayed at the end of the experiment by the MTT method. Hippocampal slices were exposed to 15min of oxygen and glucose deprivation (OGD) and the neuroprotective drugs EpoL or Epo were incubated for 2h post-OGD in re-oxygenated medium. Cell cultures stressed with oxidative agents, and pretreated with EpoL, showed neuroprotective effects of 30% at a concentration 10 times lower than that of Epo. Moreover, similar differences were observed in OGD ex vivo assays. Neuroprotection elicited by EpoL was lost when an antibody against EpoR was present, indicating that its effect is EpoR-dependent. In conclusion, our results suggest that EpoL has a more potent neuroprotective profile than Epo against oxidative stress, mediated by activation of EpoR, thus EpoL represents an important target to develop a potential biopharmaceutical to treat different central nervous system pathologies related to oxidative stress such as stroke or neurodegenerative diseases.

Monosialylated biantennary N-glycoforms containing GalNAc-GlcNAc antennae predominate when human EPO is expressed in goat milk.

Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-labeled enzymatically released N-glycan pool from rhEPO-goat milk, combined with MALDI, ESI-MS and LC/MS, revealed that low branched, core-fucosylated, N-glycans predominate. The labeled N-glycans were separated into neutral and charged fractions by anion exchange chromatography and the charged N-glycans were found to be mostly alpha2,6-monosialylated with Neu5Ac or Neu5Gc in a ratio of 1:1. Unlike the N-glycans from rhEPO produced in CHO cells, where the glycans are multiantennary highly sialylated, core-fucosylated oligosaccahrides, or even in the goat mammary gland epithelial cell line cultured in vitro in which multiantennary, core- and outer-arm fucosylated, monosialylated N-glycans are the most abundant species, a large proportion of the N-glycans from rhEPO-milk were monosialylated, biantennary, antennae mostly terminating with the more unusual GalNAc-GlcNAc motive and without outer-arm fucosylation. These findings, emphasizing the difference in the N-glycan repertoire between the rhEPO-milk and EPO-GMGE, are consistent with the principle that glycosylation is cell-type dependent and that the cell environment is crucial as well.

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin.

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

Hypogalactosylation of serum IgG in patients with coeliac disease.

Coeliac disease (CD) is described as an autoimmune enteropathy associated with the presence of IgG and IgA antigliadin and antitransglutaminase autoantibodies. While of diagnostic significance, the role of these autoantibodies in the immunopathogenesis of CD is elucidated. An inappropriate T cell immune response to gluten is also involved in the pathogenesis of CD, as evidenced by autoantibody switching. The N-glycans released from serum IgG of CD patients and three groups of healthy controls, of differing age ranges, were analysed by NH2-high performance liquid chromatography (HPLC). The fucosylated biantennary N- glycans were the most abundant neutral oligosaccharides; in particular, the agalacto form (G0F) showed a mean value of 42% (s.d. +/- 7.4), 30% (s.d. +/- 5.9), 26% (s.d. +/- 4.2) and 35% (s.d. +/- 6.8) for CD patients, healthy children, healthy adults under 40 and healthy adults over 40 years old, respectively. The ratio of asialo agalacto fucosylated biantenna to asialo monogalacto fucosylated biantenna (G0F)/(G1F) for CD patients showed a significant increase compared to healthy children (P < 0.0002), healthy adults under 40 (P < 0.0002) and healthy adults over 40 years old (P < 0.01). Hypogalactosylation was more pronounced for CD patients than for the patients with other autoimmune diseases such as rheumatoid arthritis or psoriatic arthritis.

Characterization of protein glycoforms with N-linked neutral and phosphorylated oligosaccharides: studies on the glycosylation of endoglucanase 1 (Cel7B) from Trichoderma reesei.

Using anion-exchange chromatography the catalyticdomain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glycoforms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N-glycans were analysed by fluorophore-assisted carbohydrate electrophoresis and amine-adsorption HPLC. The results show that this particular preparation contained at least 14 different glycoforms. The major isoform contained only one GlcNAc, presumably N-linked, and one mannose, most probably O-linked to serine/threonine at a separate site. Except for a small population containing Man(5)GlcNAc(2)+1-2 Man, the rest of the protein had negatively charged phosphate-containing N-glycans. All glycoforms contained at least one O-linked mannose residue. The increased negative charge of the protein, introduced by oligosaccharide phosphorylation, is the most probable reason for the different isoelectric points and the occurrence of multiple peaks during purification.

Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation.

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.

Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease.

Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.

Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris.

We report the characterization of N-linked oligosaccharides on six foreign glycoproteins secreted from the methylotrophic yeast Pichia pastoris. These proteins included: a bacterial enzyme, Bacillus licheniformis alpha-amylase; three fungal enzymes, Saccharomyces cerevisiae invertase, Penicillium minioluteum dextranase, and Mucor pusillus aspartic protease; and two higher eukaryotic proteins, Boophilus microplus (tick) gut antigen and bovine enterokinase catalytic subunit. The carbohydrates on these proteins were observed to vary in size, with Man8GlcNAc2 and Man9GlcNAc2 structures being the most frequently observed species. Substantial amounts of shorter oligomannoside structures were present only on invertase, and longer structures (up to Man18GlcNAc2) were common on aspartic protease and enterokinase. Phosphorylated oligosaccharides were observed on one protein, aspartic protease. Unlike oligosaccharides on glycoproteins secreted from S. cerevisiae, no terminal alpha1,3-linked mannosylation was observed on any of the six P. pastoris-secreted proteins. Changing the growth and induction medium from a minimal salt-based medium to a molasses-based medium had little effect on the size of the oligomannosides. From these results, it is apparent that most foreign proteins secreted from P. pastoris are not subjected to the extensive mannosylation (hyperglycosylation) that commonly occurs in proteins secreted from S. cerevisiae.

Two-dimensional mapping of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives of N-linked neutral and sialyloligosaccharides.

We describe a simple and sensitive two-dimensional sugar-mapping technique of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives (ANTS derivatives) of neutral and sialyloligosaccharides for structure analysis and characterization of N-linked oligosaccharides using picomoles of samples. The method includes: (1) reductive amination with ANTS of enzymatically released oligosaccharides, (2) simultaneous separation of oligosaccharide derivatives in a fluorophore-assisted carbohydrate electrophoresis and NH2-HPLC column under ion suppression conditions, (3) plotting of the relative migration indexes (X axis) and relative retention times (Y axis), and (4) when necessary, additional exoglycosidase digestion. As illustrated by the glycosylation profiling and structural analysis of alpha 1 anti-trypsin and murine IgG 2a, this methodology fulfills most of the requirements for a complete characterization of neutral and charged oligosaccharides released from N-glycosylated glycoprotein.

Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed Pichia pastoris cells.

In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris. The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm. The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein.

Concanavalin A- and wheat germ agglutinin-conjugated lectins as a tool for the identification of multiple N-glycosylation sites in heterologous protein expressed in yeast.

We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein. Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry. This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris.

Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium.

The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.

Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences.

We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.

The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.