Figures
Abstract
Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.
Citation: Arnold M, Durairaj V, Mundt E, Schulze K, Breunig KD, Behrens S-E (2012) Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit. PLoS ONE 7(9): e42870. https://doi.org/10.1371/journal.pone.0042870
Editor: Man-Seong Park, Hallym University, Republic of Korea
Received: March 5, 2012; Accepted: July 12, 2012; Published: September 14, 2012
Copyright: © Arnold et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the ForMaT initiative of the Bundesministerium für Bildung und Forschung (BMBF; project VAKZiNOVA, 03FO1232), Germany. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. Dr. Mundt is now employed by a pharmaceutical company; however, this does not does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Introduction
Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease (IBD), a highly contagious immunosuppressive disease in young chickens. IBDV is a non-enveloped virus; it consists of the bi-segmented double-strand RNA genome and the genome-enclosing viral capsid that is mainly formed by the viral protein VP2 [1], [2]. IBDV infects premature B-lymphocytes [3]–[5], and the primary effect of IBD is caused by the depletion of B-lymphocytes [6] that impairs the animal’s ability to develop antibodies [7]. In the poultry industry, IBD is controlled by administration of live, inactivated or recombinant IBDV vaccines [8]–[13]. Repeated vaccination of breeder hens leads to an enduring high serum antibody response [14], and progeny chickens receive maternal-derived antibodies (MDA) via the yolk sac that provide protection for the first few weeks after hatching [15]. The appearance of IBDV variants in the US [16], [17] and very virulent strains in Europe [18], [19] that may escape from MDA resulted in recent changes of vaccination regimes with broilers getting vaccinated with more virulent vaccines at 2–3 weeks of age when the MDA have declined [20], [21].
The presently used inactivated vaccines are effective against IBD but include certain disadvantages. While the obtained titers of antibody may not considerably vary from hen to hen in one flock of similar age, the offspring of various vaccinated flocks may show different IBDV antibody titers. When raised together, this may result in different levels of MDA in the offspring and compartmentalizes the herd into individuals with low or high susceptibility to virulent IBDV. Moreover, due to a poor efficiency, the available inactivated vaccines need to be applied at least twice, which is labor intensive and costly. Live attenuated vaccines entail the risk of not taking as they may be neutralized by the MDA. To overcome the latter problem, more virulent live vaccines have been used in the field, which may cause bursal damage themselves [22]. Accordingly, there is a need to develop new vaccines that combine a straight forward administration with high efficacy and few side-effects. An attractive approach involves the development of effective subunit-based vaccines as this would eliminate the use of embryonated eggs or live birds for bursal vaccine production. In addition, the use of material that exhibits a high level of biosafety and a low environmental risk would be of great benefit.
Along this line, the IBDV VP2 is in the focus of interest because this viral protein folds epitopes that induce protective immunity. That is, neutralizing antibodies against VP2 mediate a complete protection against lethal IBDV challenge [23]–[27].
Various heterologous viral vectors expressing VP2 were successfully tested in vaccination approaches [28]–[38]. Also the individual VP2 protein was shown to be highly immunogenic and protective when expressed in and purified from Escherichia coli [39], Arabidopsis thaliana [40] or from the yeasts Saccharomyces cerevisiae (S. cerevisiae) or Pichia pastoris (P. pastoris) [41]–[43]. Still, all these approaches involve either elaborate, longsome procedures to recover and grow recombinant viruses or plants, and/or expensive biochemical procedures to purify the recombinant VP2 or VP2 composed virus-like particles (vlps).
Here we describe vaccination procedures against IBD that base on fast generable recombinants of the yeast species Kluyveromyces lactis (K. lactis). Importantly, inoculation is performed directly with the complete heat-inactivated yeast.
Results
Generation and Optimization of K. lactis Strains Expressing IBDV VP2
Following the rationale of this study to inoculate animals against IBD with entire, non-fractionated yeast material, it was of central importance to generate stable K. lactis strains that expressed the IBDV VP2 antigen at high and reproducible amounts and to use a procedure that avoided antibiotic resistance genes for selection. For this purpose, we took advantage of a recently developed K. lactis variant, VAK367-D4, which permits the defined chromosomal integration of a foreign gene in a one-step procedure based on selection of transformants for growth on lactose [44]. In VAK367-D4, the 5′ end of the K. lactis specific LAC4 open reading frame (ORF) is replaced by the S. cerevisiae URA3 gene. LAC4 encodes β-galactosidase that permits K. lactis to use lactose as carbon source; URA3 encodes orotidine-5′-phosphate decarboxylase (OMP decase) that is essential for the synthesis of uracil. For the integration of a foreign gene, VAK367-D4 is transformed with a plasmid that contains an expression cassette where the gene of choice is 5′-flanked by the 5′UTR and upstream region of LAC4 including the strong LAC4 promoter, and 3′-flanked by the TEF1 terminator, the KlGAL80 promoter and the 5′-region of the LAC4 ORF (see scheme in Fig. 1). Following homologous recombination, the LAC4 ORF is restored and the URA3 gene is lost. Thus, foreign gene expression is mediated by the LAC4 promoter while LAC4 expression is driven by the KlGAL80 promoter. Importantly, the LAC4 and KlGAL80 promoters are co-regulated by the transcription activator KlGal4 (Lac9; [45]) and inducible by lactose or galactose in the yeast’s growth medium. Accordingly, the desired recombinants can be selected on lactose plates and screened for the loss of the URA3 gene (uracil auxotrophy) and foreign gene expression is inducible by shifting glucose grown cultures to lactose containing media. The expression and activity of β-galactosidase may serve as additional indicators of gene-induction (Fig. 1).
Upper panel: Schematic representation of the genomic organization of the LAC genes of K. lactis strain VAK367. The localization of the LAC4 and LAC12 genes and the directions of transcription are indicated by arrows. Lower panel: Schematic representation of the genomic organization of the mutated LAC4 gene of K. lactis strain VAK367-D4 and of the recombination procedure. As described in the text, in the genome of VAK367-D4, a portion of the LAC4 gene (indicated as :lac4) was substituted by the URA3 gene (boxed). Homologous recombination with the plasmid Klp3 replaces the URA3 gene by the gene of choice (boxed), the latter which is under control of the PLAC4 promoter and the TEF1 terminator. The recombination leads to the restoration of the LAC4 ORF under the control of the PGAL80 promoter. Selection of recombinant clones was performed on Lac+ Ura – medium.
Using this system, the VP2-encoding genetic unit of the IBDV strain D78 was inserted into the K. lactis LAC4 locus. The correct orientation of the insert was confirmed by PCR, and gene expression initially determined by measuring VP2 mRNA synthesis via Northern blot (not shown). Western blot analysis of yeast cell lysates further confirmed the expression of VP2 though our initially generated strain VAK887 (VP2) produced only moderate amounts of the viral protein (Fig. 2). However, in analogy to earlier findings of Jagadish et al. (1991) [46] in S. cerevisiae, VP2 protein expression could be markedly improved in strain VAK888 (VP2-T2S), where threonine at amino acid position 2 of VP2 was exchanged by serine resulting in increased protein stability (Fig. 2). A further significant optimization was achieved with strain VAK890 (VP2-T2S_GAL4). This strain contained an additional tandem integration of the KlGAL4 trans-activator gene, which resulted in increased transcription rates of the VP2 gene (Fig. 2; transcription data not shown; details of the procedure are described by Krijger et al. [44]). The integration of additional KlGAL4 genes also correlated with a higher growth rate of this strain (not shown).
Total protein of extracts of naïve and IBDV-infected chicken fibroblasts (negative and positive controls) and of the various K. lactis strains that were generated in the course of this study (described in the text) were analyzed by SDS PAGE and Western blot using an anti IBDV antiserum. The positions of the different IBDV VP proteins in the gel are marked by arrows. As revealed by the negative control performed with protein of strain VAK367, the antiserum also reacted non-specifically with a yeast protein that migrated shortly above the heterologously expressed VP2 protein. Note that the yeast-expressed VP2 protein contained fifteen additional amino acids, which explains its slightly different migration behavior in the SDS-PAGE.
Production of VP2-expressing K. lactis for Subcutaneous and Oral Applications in Mice and Chickens
For the intended vaccination experiments it was important to establish protocols that enabled high density fermentation of yeast cells with high expression levels of VP2. For this purpose, the growth conditions were optimized in a bioreactor by modifying additives, pH, and an applied fed batch feeding scheme. Thus, besides increasing the amount of biomass significantly (up to 7 times in comparison to conventional flask cultures) also higher expression levels of VP2 were achieved. The applied fermentation protocol for strain VAK890 is provided as supporting information (Table S1).
After production, the yeast cells were freeze dried and heat-inactivated by incubation for 2 hrs at 90°C. For the subcutaneous immunization, the dried and powdered yeast (100 µg per vaccination in mice; 1 mg per vaccination in chickens) was mixed and applied with adjuvant. For the oral inoculation of mice, dried yeast nuggets were directly mixed with the feed resulting in a 5% (w/w) end-concentration of the recombinant, inactivated yeast. For the oral vaccination of chickens we generated and applied pellets that consisted of chicken feed and 5% (w/w) of the recombinant, inactivated yeast (see Materials and methods and below for further details).
PAGE analysis of the protein content of the heat-inactivated and feed-supplemented yeast revealed that the heterologously expressed VP2 protein stayed intact and that also its concentration per total amount of yeast protein remained constant throughout the heat-treatment and pelleting procedures (Fig. 3A). With strain VAK890, which was used in all subsequent inoculation experiments, a semi-quantitative calculation of the intracellular VP2 level indicated a concentration of ca. 0.7 fg of heterologous protein/per yeast cell (Fig. 3B).
VP2 is expressed at similar rates in high density fermentations. (A) Western blot analysis of total protein of VAK890 yeast after heat inactivation and after encapsulation (5% w/w) into feed pellets. Comparable amounts of yeast protein were tested for the presence of VP2 using the same procedures and controls as described in Fig. 2. The VP2 protein is indicated. (B) Upper panel: Western blot of defined (ng) amounts of purified VP2. The applied amounts of purified VP2 were estimated in comparison with a standard (data not shown). The blot of purified VP2 was performed side-by-side with protein of total lysates of ca. 6×106 cells (determined by platting and colony counting) of 3 independent fermentations (samples 1–3) of K. lactis strain VAK890. Lower panel: Densitometric measurements of the VP2 bands. The average amount of VP2 protein was calculated to correspond to ca. 0.7 ng of VP2 protein per cell.
Accordingly, in the applied immunization schemes with mice, we estimated the total amount of VP2 that was applied per subcutaneous vaccination to approximately 18 ng, while a total of approximately 340 µg of VP2 was used per oral immunization and animal. With the chickens, approximately 180 ng of VP2 was applied during subcutaneous immunization, while an amount of approximately 2.7 mg of VP2 was administered per animal for oral immunization.
Subcutaneous and Oral Immunization of Mice and Chickens with VP2-expressing K. lactis
To gain initial hints on the applicability of entire heat-inactivated yeast in vaccination approaches, we decided to test first for the induction of neutralizing antibodies against VP2 in mice. For the vaccination, we applied immunization schemes that were established during pilot-studies with other recombinant K. lactis strains (data not shown). Thus, within a time interval of six weeks, groups of mice were inoculated three times subcutaneously with a yeast emulsion together with a strong adjuvant or, following a 2/2/2 scheme (two weeks feeding, two weeks break, two weeks feeding), vaccinated orally with 5% yeast-supplemented feed (see Materials and methods and immunization scheme in Fig. 4A). Two weeks after the last application, the sera were analyzed for the presence of anti-VP2 antibodies with an indirect ELISA that was originally established for the detection of IBDV-specific chicken antibodies but modified such that it enabled the detection of IBDV-specific antibodies in mice. The presence of neutralizing antibodies was tested in an IBDV-neutralizing assay (VN; Fig. 4B, 4C). As summarized in Table 1, in comparison to animals that were fed with non-VP2 expressing K. lactis (strain VAK367), 2 of 5 mice that were orally vaccinated with VP2-expressing K. lactis (strain VAK890; conditions as explained) showed increased titers of IBDV specific antibodies in the ELISA and VN assay, respectively. With the subcutaneously vaccinated animals, all vaccinees revealed titers of neutralizing antibodies that were significantly higher with respect to the controls (Fig. 4, Table 1).
(A) Immunization scheme. Six weeks old mice were immunized at the indicated times/time-intervals (schematized as bars) by either subcutaneous injection (sc) or by feeding (oral) of the yeast K. lactis VAK890. Injection or feeding of the yeast K. lactis VAK367 was performed as a negative control. Arrows indicate the time points where the animals were euthanized. (B) IDEXX Elisa assay that was performed with an anti-mouse secondary antibody indicating the titers of anti IBDV antibodies in the sera of the vaccinated mice. (C) Serum neutralization (VN) assay indicating the titers of IBDV neutralizing antibodies in the sera of the vaccinated mice. A serum of a one-day-old specific-pathogen-free (SPF) chicken and a serum of an IBDV-infected chicken were used as controls.
The apparent trend that was obtained in the experiments with mice encouraged us to next perform vaccination experiments with chickens. For this purpose, groups of leghorn chickens were immunized subcutaneously by using the same vaccination design as earlier with the mice. However, in the chickens, we used a ca. tenfold higher amount of recombinant yeast (Fig. 5A) per injection thus adapting the quantity of inoculated yeast per bodyweight to a ratio comparable with that used in mice. Alternatively, the chickens were fed with pellets that contained 5% VP2-expressing (strain VAK890) or naïve (strain VAK367) K. lactis applying the aforementioned 2/2/2 scheme (Fig. 5A). In addition, we performed oral vaccination with yeast after pre-treatment of the animals with saponin, an oral adjuvant that is described to generally induce Th1 and Th2 responses [47]. Importantly, after a break of one or two weeks post vaccination, all vaccinees were challenged with the classical IBDV-strain Edgar using 100 EID50 per chicken (Fig. 5A). This virus titer of IBDV was earlier established causing a significant IBD in the used chicken type (E. Mundt, data not shown). Employing the same assays as described above to test for serum antibody titers we obtained similar results as in the mice experiments. That is, with the oral vaccination procedure, we found that 4 of the 19 vaccinated animals displayed increased titers of virus-neutralizing antibodies. This was observed irrespective of whether the animals had been treated with saponin or not. Subcutaneous immunization with the recombinant K. lactis yielded high titers of neutralizing antibodies in all vaccinated animals (Fig. 5B, C). Interestingly, with both vaccination regiments, none of the vaccinees died after IBDV challenge. This was different with the control animals where the rate of mortality was 10–35% (Table 2). Determining the bursal lesion score in the collected bursae, we found that 1 bird of 19 of the orally vaccinated animals showed no signs of depletion of B lymphocytes in the bursal follicles after challenge indicating full protection against IBDV while one bird was partially protected with a bursal lesion score of 2. In the case of the subcutaneous vaccination approach, 4 of 5 animals (80%) showed a full protection while one chicken was partially protected (Table 2).
(A) Immunization scheme. Two weeks old chickens were immunized at the indicated times/time-intervals (schematized as bars) by either subcutaneous injection (sc) or by feeding (oral or oral with saponin (sap)) of the yeast K. lactis VAK890. The yeast K. lactis VAK367 was applied as a negative control. The black bars indicate the time intervals that followed virus challenge; arrows indicate the time point where the animals were euthanized. (B) IDEXX Elisa assay indicating the titers of anti IBDV antibodies in the sera of the vaccinated chickens. (C) Serum neutralization (VN) assay indicating the titers of IBDV neutralizing antibodies in the sera of the vaccinated chickens. Sera of a one-day-old specific-pathogen-free (SPF) chicken, of an IBDV-infected chicken, of a mock-fed and of a phosphate-buffered saline (PBS) injected chicken were used as controls.
Discussion
Yeast cells are capable of expressing large quantities of foreign proteins at a relatively low cost. Accordingly, for decades S. cerevisiae, P. pastoris and K. lactis are in use as eukaryotic protein expression systems [48], [49]. More recently, particularly S. cerevisiae (“bakers yeast”) has been also adopted as an antigen delivery vehicle for vaccination. When subcutaneously injected, whole antigen-expressing S. cerevisiae was shown to activate Dendritic cells, to elicit antigen-specific cytotoxic T lymphocytes (CTL) responses and to confer protective cell-mediated immunity against tumor challenge in mice [50], [51]. Recombinant S. cerevisiae was also implicated as a tracer for the oral application of vaccines and drugs because the yeast cells are comparably stable, noninvasive and nonpathogenic, and they are believed to be well absorbed by M-cells in the Peyer’s patches of the gut [52]–[55].
In this study, we applied recombinants of the “milk yeast” K. lactis in both, subcutaneous and oral vaccination approaches. As S. cerevisiae, also K. lactis is ‘generally regarded as safe’ (“GRAS status”) for animal and human consumption [56]. However, both yeasts differ in certain physiological properties that considerably affect their usability. Thus, K. lactis and S. cerevisiae display significant differences in protein glycosylation, cell wall biosynthesis and protein secretion (reviewed by Backhaus et al., 2011 [57]), which may have important implications for the intracellular localization, folding, stability and immunogenicity of heterologously expressed proteins [58].
In biotechnological applications, K. lactis has evident advantages versus S. cerevisiae. Thus, glucose repression of respiratory genes and ethanol formation (“Crabtree effect”) are less dominant in K. lactis under aerobic conditions, which enables higher biomass yields during fermentation at high growth rates [59]–[61]. The here applied strain VAK367-D4 is particularly advantageous for fermentation purposes: as its parent strain VAK367, which was generated by mutagenesis [44], VAK367-D4 and derivatives hereof show no significant cell lysis during high-density fermentation. Moreover, K. lactis is one of the few yeast species that can utilize lactose, a cheap and abundant sugar, as a sole source of carbon energy. Thus, VP2 expression was controlled via the LAC4 promoter and could be effectively induced by the addition of lactose to glucose grown cultures (Fig. 1 and 2).
As demonstrated here for the first time, K. lactis not only represents a useful system for the production and preparation of foreign proteins but also is a valuable vehicle for vaccination. That is, entire heat-inactivated material of a K. lactis strain (VAK890) where heterologous expression of the IBDV VP2 was improved by moderate overproduction of the KlGal4 transactivator [62], [44] (Fig. 2), induced immune responses against IBDV, the trends which were comparable in mice and chickens (Fig. 4 and 5; Tables 1 and 2). As outlined, in chickens, this immune response was at ca. 80% protective against IBDV infections in subcutaneous and partially (at ca. 10%) protective in oral approaches (Table 2); further investigations to determine if the birds without disease were still able to be infected were not conducted. Thus, within these limits and as a main result of this report, the subcutaneously applied VP2-expressing K. lactis turned out to be an adequate and effective anti-IBD subunit vaccine. Importantly, subcutaneous vaccination with K. lactis and a strong adjuvant produced a humoral response with high titers of anti VP2 and IBDV-neutralizing antibodies. In keeping with the current knowledge, we believe that these antibodies represent the major functional element of the protective immune response against IBD. In this respect our data markedly differ from subcutaneous vaccination regiments with recombinant S. cerevisiae that induced primarily T-cell responses [50], [63]. Hence, an important future project involves a detailed comparison of the immune responses induced by oral and subcutaneous applications of whole recombinant K. lactis and S. cerevisiae, respectively.
Another aspect worth emphasizing concerns the surprising small quantities of VP2 that turned out to be sufficient for subcutaneous vaccination with K. lactis against IBD. Following our estimations of the amounts of protein per yeast cell (Fig. 3), full protection of an individual chicken was achieved with approximately 180 ng of heterologously expressed VP2 only. This amount of antigen may be even further reduced since our subcutaneous vaccination scheme involved three applications of yeast; however, similar titers of neutralizing antibodies were already detectable after two applications (data provided as supporting information, Fig. S1). Considering that comparable experiments with partially purified and intramuscularly injected VP2 from P. pastoris applied ca. 100 µg of antigen per chicken for protective vaccination [41], it may be speculated that the adjuvant character of K. lactis itself plays a major role in mediating this remarkable vaccination efficiency.
Since injections are laborious, time-consuming, cause discomfort to the vaccinee and may lead to inflammations at the injection sites [64]–[68], oral applications are considered the “silver bullet” of vaccination, particularly against fecal-orally transmitted pathogens as IBDV. In mice, K. lactis was earlier observed to function as an oral immunogen itself (data not shown), which was explained by the high content of immunogenic β-glucans in the yeast’s cell wall and by a lack of immunotolerance to milk yeast ingredients [69]. Our data, in particular the finding that all vaccinees survived challenge and showed no clinical signs as ruffled feathers, lassitude and prostration, suggest that also oral vaccination with recombinant K. lactis is a promising option. However, it is obvious that a protection rate of 10% (as indicated by the lack of B-cell depletion after IBDV challenge) requires further improvement. A major issue here may concern the concentration of applied VP2. Thus, studies with transgenic rice seeds, the only example where oral vaccination of chickens against IBD turned out to be fully protective [40] applied ca. 5–10 mg of VP2 per animal versus 1–3 mg in our case. Using codon–optimization, the concentration of VP2 per yeast cell could be considerably increased (strain VAK910, Fig. 2). Other ways of optimization involve the generation of native IBDV vlps to further augment the immunogenicity of VP2. For this purpose, the commonly applied protocol for the preparation of IBDV vlp [70], which generated yet neither vlps nor subviral particles (slp) but VP2 containing particles (Fig. S2), is currently adapted to a recently established procedure that enabled the generation of vlps of murine polyomavirus in K. lactis (Simon et al., manuscript submitted). Despite that saponin had no detectable effect (Fig. 5) the systematic testing of oral adjuvants represents another promising avenue to further increase the efficiency of the oral immune response.
Taken together, our study recommends the use of non-fractionated yeast, particularly of the here-established K. lactis system as a powerful tool for vaccination projects. This is particularly evident considering that new vaccine strains can be generated and applied within a time frame of two to three weeks.
Materials and Methods
Generation of Recombinant Yeast Strains
The cDNA encoding the IBDV D78 VP2 was amplified with the following primer pair on plasmid pD78A [71]: IBDV_AscI_fwd (5′-GGCGCGCCGATGACAAACCTGCAAGATC-3′) containing an AscI site and VP2_NotI_rev (5′-ATAAGAATGCGGCCGCTCACACAGCTATCCTCCTTATG-3′) containing a NotI site. The primer pair used to generate VP2-T2S was IBDV_S:T_AscI_fwd (5′-GGCGCGCCGATGTCTAACCTGCAAGATCAAACCCA-3`) and VP2_NotI_rev. After sequence confirmation, the amplified DNA fragments were cloned via AscI and NotI into the vector KIp3-MCS (detail of construction of this plasmid provided on request). Genomic integration was performed as described in the text (Fig. 1). Specifically, the integrative plasmid was cut with EcoRI and the digested fragments transformed into competent VAK367-D4 cells. The transformed cells were platted on YPD medium [72] and incubated at 30°C overnight. For the screening of positive colonies, the transformation plate was replicate-plated on SD media (0,67% yeast nitrogen base w/o amino acid supplemented amino acids and nucleotides [11.2 mg/l adenine; 14.4 mg/l tyrosine; 38.4 mg/l of uracil, histidin, tryptophan, arginine, methionine; 48 mg/l phenylalanine; 57.7 mg/l of leucine, isoleucine, valine, threonine]) containing lactose as carbon source and incubated for 2 days at 30°C. Lactose-positive clones were further analyzed as described in the text.
The genomic integration of additional GAL4 loci was performed as described by Kuger et al. (1990) [62]. The codon optimization was performed with the S. cerevisiae algorithm (mr.gene.com) [73]. The codon-optimized DNA fragments were synthesized with 5′ AscI and 3′ NotI restriction sites (mr.gene, Regensburg, Germany) and cloned into the Klp3-MCS vector.
Western Blot Analysis
Pellets of yeast cells or chicken fibroblasts (DF1) that had been earlier infected with IBDV strain D78 were re-suspended in B60 buffer (50 mM Hepes-KOH pH 7.3; 60 mM potassium acetate; 5 mM magnesium acetate; 0,1% Triton X100; 10% glycerol; 1 mM sodium fluoride; 20 mM glycerophosphate; 10 mM MgCl2; 1 mM DTT; complete protease inhibitor (1 tablet/50 ml; Roche)) and disrupted by vortexing in the presence of glass beads. The extract was centrifuged (14000 rpm, 20 min at 4°C) and the protein concentration determined by a standard procedure. 40 µg of protein extract were separated by 12% SDS-PAGE, the proteins transferred and Western blot analyses carried out with a rabbit anti-IBDV antiserum (1∶15,000; [74]) and a goat α-rabbit HRP-coupled antibody (1∶3000; Santa Cruz Biotechnology, Inc.) using standard procedures.
Northern Blot Analysis
For total RNA extraction, yeast cells of a 5 ml culture were pelleted and immediately chilled on ice. Cell lysis was performed in ProtK Buffer (100 mM Tris/HCl pH 7.9, 150 mM NaCl, 25 mM EDTA, 1% (w/v) SDS) and 50 mg Proteinase K (Fermentas) by vigorous vortexing in the presence of glass beads. The samples were incubated for 1 h at 35°C and the RNA was extracted, precipitated and re-suspended in RNAse-free water. Northern blotting was performed as previously described by Engler-Blum et al. with some modifications [75]. Briefly, 5 µg of total RNA were separated on a 1% (w/v) agarose gel (containing 1.85% (v/v) formaldehyde) and the gel blotted onto a nylon membrane (Amersham HybondTM-N+, GE Healthcare). The membrane was hybridized at 68°C with a DIG-labeled RNA probe that had been generated by in vitro transcription from PCR fragments in the presence of DIG-NTPs (Roche). The blot was treated with a blocking solution and incubated with an anti-DIG alkaline-phosphatase-conjugated antibody (Roche). The detection of alkaline-phosphatase activity was performed using standard procedures.
Concentration and Purification of Heterologously Produced VP2
For the purification of IBDV VP2, we applied the protocol of Saugar et al., 2005 [70] that was developed for the purification of IBDV VP2 vlps from infected chicken cells. 2000 odu (optic density units) of a yeast culture, the cells that had been transformed with an episomal VP2 plasmid (pADH1-P_VP2-T2S; construction detail provided on request), were grown on auxotrophic SD-uracil media with 2% glucose overnight. After harvesting and washing with distilled water, the cells were disrupted with glass beads in lysis buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 20 mM CaCl2, 1 mM EDTA, complete protease inhibitor (1 tablet/50 ml; Roche). The resulting protein extract was centrifuged (10,000 x g for 1 h at 4°C) and the soluble fraction applied to a 20% (w/v) sucrose cushion in sucrose buffer (10 mM Tris pH 8.0, 150 mM NaCl, 20 mM CaCl2) containing complete protease inhibitor. After centrifugation at 170,000×g for 3 h at 4°C, the pellet was solved in 200 µl sucrose buffer and further centrifuged (17 h 114.000×g) on a 20–53% (w/v) sucrose gradient in sucrose buffer. The gradient was harvested in 700 µl fractions and the heterologously expressed VP2 which formed neither vlps nor slps but protein particles under the applied conditions was thus purified and concentrated. The amount of protein in the peak fraction was estimated by SDS-PAGE and Coomassie staining in comparison to a standard protein (BSA; Fig. S2) and the estimated amounts correlated with signals obtained in parallel-performed Western blots (Fig. 3).
Yeast Production and Heat killing
All experimental fermentations and optimizations were carried out in a DasGip parallel bioreactor system (DasGip AG, Jülich, Germany) with four 2L fully equipped fermenters. Fermentations for production purposes were performed by Organobalance GmbH (Berlin, Germany) or in a Biostat ED bioreactor with a 10L working volume (B. Braun Biotech, Melsungen, Germany). All production processes were operated in a fed-batch mode. A complex culture medium with some defined additives was used; the compositions of finally used media and feed solutions will be supplied on request. The temperature of the yeast culture was maintained at 30°C and the pO2 controlled to 30%sat. The pH during culturing was set to 5.0 by the addition of 2M NaOH and 2M H3PO4, respectively. For the feeding experiments with mice and chickens, the yeast was freeze dried and subsequently heat-killed for 2h at 90°C. Using this procedure, less than 10 cells per gram dry cell weight remained viable.
Vaccination of Mice
Ethics Statement I: all experiments with mice were performed along the guidelines and by approval of the Animal Care and Use Committee of the Landesverwaltungsamt Sachsen-Anhalt, Germany and in accordance with the EU Directive 2010/63/EU for animal experiments (Permit Numbers 42502-2-1055MLUÄ and 42502-2-1088MLUG). During injection and for euthanasia the mice were anesthetized with 1.5% isoflurane and all efforts were made to minimize suffering.
The immunization experiments were performed with six weeks old female mice (CL57BL/6; Harlan Laboratories, The Netherlands) in groups of five individuals. For subcutaneous immunization, the dried, powdered yeast was mixed with complete Freund’s adjuvant (CFA; Sigma) in the first application and with incomplete Freund’s adjuvant (IFA; Sigma) in the subsequent booster vaccinations. Per immunization/boost step, 200 µl emulsion (containing 100 µg of yeast) was injected per mouse. During the injection procedure (number of application as indicated in Fig. 4A), the mice were anesthetized with 1.5% isoflurane. After initial injection, the mice were boosted twice in two weeks intervals (i.e., at day 14 and 28, respectively; Fig. 4). For the oral immunization, dried yeast nuggets were mixed with mice feed to an end concentration of 5% (w/w). Each mouse was fed twice with 2 g of total feed per day. The yeast-containing feed was applied for four weeks with a two weeks break (Fig. 4A). Two weeks after the last subcutaneous or oral application, the mice were euthanized with 1.5% isoflurane and blood samples taken from vena cava.
Vaccination of Chickens
Ethics Statement II: all studies with chickens were conducted in BSL-2 approved animal facilities at the Poultry Diagnostic and Research Center at the University of Georgia. The experiments were approved by the Institutional Animal Care and Use Committee of the University of Georgia (IACUC number A2010 12-017-Y1-A0). For euthanasia, the birds were anesthetized with CO2 and all efforts were made to minimize suffering.
The immunization experiments were performed with fourteen-days-old specific pathogen-free (SPF) leghorn chickens in groups of up to 10 individuals. For subcutaneous vaccination, 5 mg of dried, powdered yeast were solved in 750 µl PBS and 500 µl of sterile distilled water and an emulsion was prepared with 1.25 ml IFA. 500 µl of this emulsion (containing 1 mg of yeast or only PBS) was injected subcutaneously at day 0, 14 and 28, respectively (see Fig. 5). For oral immunization, the dried and heat-killed yeast was mixed with chicken feed and pellets with a yeast concentration of 5% (w/w) were formed. The birds were fed once a day with increasing amounts of feed depending on their age. The immunization scheme was performed as described in the text and shown in Fig. 5. The treatment with saponin (Quillaja saponaria Molina, Sigma) was performed prior to the initiation of the oral immunization. That is, 5 mg of saponin were dissolved in PBS containing 30 mg/ml NaHCO3, and 1 mg of saponin solution was applied per bird by esophageal intubation [76]. The animals were bled prior to each application; the last bleed was done two weeks after the last application. Using the oral route, the birds were challenged with 100 EID50 of the classical IBDV Edgar strain (kindly provided by Dr. Holly Sellers, University of Georgia, Athens). Seven days post challenge the birds were euthanized with CO2, the bursae of Fabricius were collected, fixed for 24 h in 10% neutral-buffered formalin and embedded in paraffin.
Enzyme-linked Immunosorbent Assay (ELISA)
IBDV specific antibody titers in the chickens sera were determined with the IDEXX FlockChek® IBD ELISA kit (IDEXX Laboratories, Inc.) using the procedure recommended by the manufacturer. The same ELISA was applied to the mice sera; in the latter case, an anti-mouse HRP-coupled secondary antibody was used (Sigma Aldrich, 1∶5000). Since this ELISA was established for the detection of IBDV-specific chicken antibodies, the IBDV VP2-specifc antibody R63 was used as a positive control [77].
Virus Neutralization Assay
The assay to determine IBDV neutralization by mice and chickens sera was performed essentially as described by Schröder et al., 2000 [78]. Virus strain D78 was applied. As a negative control for the virus neutralization assay, a serum pool obtained from SPF one-day old chickens (Merial, Gainesville, GA, USA) was used. The positive control serum was obtained after vaccination of ten three-week-old SPF chickens (Merial) with the live IBDV vaccine NOBILIS GUMBORO D78 (Intervet, Millsboro, DE, USA) according to the manufacturer’s recommendation. Three weeks after vaccination, the chickens were exsanguinated and the serum pool was used for the assays.
Histopathology
Four micrometer thin tissue sections were obtained from the parafinned bursae of Fabricius. These were de-parafinned and stained with hematoxylin and eosin following standard procedures. The samples were microscopically evaluated and the lesion score [79], [80] was determined on a scale from 1 to 4 (1 = normal to 10% follicular atrophy; 2 = 10–30% follicular atrophy; 3 = 30–70% follicular atrophy; 4 = >70% atrophy). The presence of up to 10% follicular atrophy was attributed to individual apoptotic cells that affect each follicle during the normal development of the bursa. This can only be attributed to sections of the bursae of Fabricius obtained from SPF animal under controlled experimental conditions.
Supporting Information
Figure S1.
Serum neutralization assay indicating the titers of IBDV neutralizing antibodies in the sera of the subcutaneously vaccinated chickens after each application. The sera were collected 13 days after each shot (Fig. 5A). Serum of a one-day-old specific-pathogen-free (SPF) chicken and serum from an IBDV-infected chicken were used as controls.
https://doi.org/10.1371/journal.pone.0042870.s001
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Figure S2.
Purification and semi-quantification of IBDV VP2. Protein extracts of VAK890 and VAK367 cells were supplied to two centrifugations via a sucrose cushion (20%) and a subsequent sucrose gradient (20–53%) following the protocol of Saugar et al., 2005 [68] (A). Fractions of the sucrose gradient (F1 (20%)–F18 (53%)) were analyzed by western blot for the presence of VP2. Electron Microscopy (not shown) revealed that fractions F6–F8 contained higher-sedimenting particles of VP2 that contained the protein highly enriched. (B) The amount of VP2 contained in fraction F7 (pVP2) was compared side-by-side with standard amounts of bovine serum albumin (BSA) in a Coomassie stained SDS-PAGE using densitometry of the individual protein bands. Fraction F7 that was prepared from protein samples of strain VAK367 served as a negative control (contr.).
https://doi.org/10.1371/journal.pone.0042870.s002
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Table S1.
Conditions under which high density fermentation of K. lactis strain VAK890 was performed.
https://doi.org/10.1371/journal.pone.0042870.s003
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Acknowledgments
We are particularly thankful to Sebastian Dücker for the coordination of these studies. Jenny Wieczorek is acknowledged for technical assistance and Dr. Michael Bulang for producing feed pellets.
Author Contributions
Conceived and designed the experiments: MA VD EM KS KB SEB. Performed the experiments: MA VD EM KS. Analyzed the data: MA EM KB SEB. Contributed reagents/materials/analysis tools: EM KB SEB. Wrote the paper: EM KB SEB. Generated and characterized the applied yeast strains, performed vaccination experiments in mice and some of the vaccination experiments in chickens: MA. Performed some of the vaccination experiments in chickens, performed VN assays. VD Performed some of the vaccination experiments in chickens, performed VN assays, analyzed bursal lesions: EM. Established the cultivation conditions of K. lactis in fermenter; cultivated K. lactis strains in fermenter: KS. Co-guided the project, developed the VAK367-D4 strain: KB. Guided the project: SEB.
References
- 1. Kibenge FS, Dhillon S, Russell RG (1988) Biochemistry and immunology of infectious bursal disease virus. J Gen Virol 69: 1757–1775.
- 2. Coulibaly F, Chevalier C, Gutsche I, Pous J, Navaza J, et al. (2005) The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120: 761–772.
- 3. Müller H (1986) Replication of infectious bursal disease virus in lymphoid cells. Arch Virol 87: 191–203.
- 4. Petkov DI, Linnemann EG, Kapczynski DR, Sellers HS (2009) Identification and characterization of two distinct bursal B-cell subpopulations following infectious bursal disease virus infection of White Leghorn chickens. Avian Dis 53: 347–355.
- 5. Rodríguez-Lecompte JC, Niño-Fong R, Lopez A, Frederick Markham RJ, Kibenge FS (2005) Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells. Comp Immunol Microbiol Infect Dis 28: 321–337.
- 6. Hirai K, Calnek BW (1979) In vitro replication of infectious bursal disease virus in established lymphoid cell lines and chicken B lymphocytes. Infect Immun 25: 964–970.
- 7. Okoye JO, Shoyinka SV (1983) Newcastle disease in a vaccinated flock which had experienced subclinical infectious bursal disease. Trop Anim Health Prod 15: 221–225.
- 8. Bublot M, Pritchard N, Le Gros FX, Goutebroze S (2007) Use of a vectored vaccine against infectious bursal disease of chickens in the face of high-titred maternally derived antibody. J Comp Pathol 137: 81–84.
- 9. Lai SY (2004) Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein, rVP2H, in insect larvae. Process Biochem 39: 571.
- 10. Li L, Fang W, Li J, Huang Y, Yu L (2006) Oral DNA vaccination with the polyprotein gene of infectious bursal disease virus (IBDV) delivered by the attenuated Salmonella elicits protective immune responses in chickens. Vaccine 24: 5919–5927.
- 11. Lucio B, Hitchner SB (1979) Response of susceptible versus immune chicks to killed, live-modified, and wild infectious bursal disease virus vaccines. Avian Dis 23: 1037–1050.
- 12. Wood GW, Muskett JC, Thornton DH (1983) Use of inactivated oil emulsion infectious bursal disease vaccines in breeder chickens to prevent immunosuppression in progeny chicks. Res Vet Sci 35: 114–115.
- 13. Müller H, Mundt E, Eterradossi N, Islam MR (2012) Current status of vaccines against infectious bursal disease. Avian Pathol 41: 133–139.
- 14. Lucio B, Hitchner SB (1979) Infectious Bursal Disease Emulsified Vaccine: Effect upon Neutralizing-Antibody Levels in the Dam and Subsequent Protection of the Progeny. Avian Dis 23: 1037–1050.
- 15. Wyeth PJ, Chettle N (1982) Comparison of the efficacy of four inactivated infectious bursal disease oil emulsion vaccines. Vet Rec 110: 359–361.
- 16.
Rosenberger JK, Cloud SS (1986) Isolation and characterization of variant infectious bursal disease viruses. Abstracts of the 123rd Meeting of the American Veterinary Medical Association Jul 20–24, Atlanta, Georgia, USA, Abstr. 181.
- 17. Snyder DB, Vakharia VN, Savage PK (1992) Naturally occurring-neutralizing monoclonal antibody escape variants define the epidemiology of infectious bursal disease viruses in the United States. Arch Virol 127: 89–101.
- 18. Box P (1989) High maternal antibodies help chicks beat virulent strains. World Poultry 53: 17–19.
- 19. Chettle N, Stuart JC, Wyeth PJ (1989) Outbreak of virulent infectious bursal disease in East Anglia. Vet Rec 125: 271–272.
- 20. Kumar K, Singh KCP, Prasad CB (2000) Immune Responses to Intermediate Strain IBD Vaccine at Different Levels of Maternal Antibody in Broiler Chickens. Trop Anim Health Prod 32: 357–360.
- 21. Rautenschlein S, Yeh HY, Sharma JM (2002) The role of T cells in protection by an inactivated infectious bursal disease virus vaccine. Vet Immunol Immunopath 89: 159–167.
- 22. Tsukamoto K, Tanimura N, Kakita S, Ota K, Mase M, et al. (1995) Efficacy of three live vaccines against highly virulent infectious bursal disease virus in chickens with or without maternal antibodies. Avian Dis 39: 218–229.
- 23. Azad AA, McKern NM, Macreadie IG, Failla P, Heine HG, et al. (1991) Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus. Vaccine 9: 715–722.
- 24. Becht H, Müller H, Müller HK (1988) Comparative studies on structural and antigenic properties of two serotypes of infectious bursal disease virus. J Gen Virol 69: 631–640.
- 25. Fahey KJ, Erny K, Crooks J (1989) A conformational immunogen on VP-2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens. J Gen Virol 70: 1473–1481.
- 26. Fahey KJ, McWaters P, Brown MA, Erny K, Murphy VJ, Hewish DR (1991) Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens. Avian Dis 35: 365–373.
- 27. Heine HG, Boyle DB (1993) Infectious bursal disease virus structural protein VP2 expressed by a fowlpox virus recombinant confers protection against disease in chickens. Archives Virol 131: 277–292. XXX.
- 28. Vakharia VN, Snyder DB, Lütticken D, Mengel-Whereat SA, Savage PK, et al. (1994) Active and passive protection against variant and classic infectious bursal disease virus strains induced by baculovirus-expressed structural proteins. Vaccine 12: 452–456.
- 29. Pitcovski J, Di-Castro D, Shaaltiel Y, Azriel A, Gutter B, et al. (1996) Insect cell-derived VP2 of infectious bursal disease virus confers protection against the disease in chickens. Avian Diseases 40: 753–761.
- 30. Xu XG, Tong DW, Wang Z S, Zhang Q, Li ZC, et al. (2011) Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection. Avian Dis 55: 223–229.
- 31. Darteil R, Bublot M, Laplace E, Bouquet JF, Audonnet JC, et al. (1995) Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens. Virology 211: 481–490.
- 32. Tsukamoto K, Kojima C, Komori Y, Tanimura N, Mase M, et al. (1999) Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek’s disease virus (MDV) with a recombinant MDV expressing IBDV VP2. Virology 257: 352–362.
- 33. Tsukamoto K, Saito S, Saeki S, Sato T, Tanimura N, et al. (2002) Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. J Virol 76: 5637–5645.
- 34. Sheppard M, Werner W, Tsatas E, McCoy R, Prowse S, et al. (1998) Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease. Archives Virol 143: 915–930.
- 35. Francois A, Chevalier C, Delmas B, Eterradossi N, Toquin D, et al. (2004) Avian adenovirus CELO recombinants expressing VP2 of infectious bursal disease virus induce protection against bursal disease in chickens. Vaccine 22: 2351–2360.
- 36. Shaw I, Davison TF (2000) Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine, fpIBD1, is dependent on the titre of challenge virus and chicken genotype. Vaccine 18: 3230–3241.
- 37. Zhou X, Wang D, Xiong J, Zhang P, Li Y, et al. (2010) Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein. Vaccine 28: 3990–3996.
- 38. Wu J, Yu L, Li L, Hu J, Zhou J, et al. (2007) Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnology Journal 5: 570–578.
- 39. Rong J, Jiang T, Cheng T, Shen M, Du Y, et al. (2007) Large-scale manufacture and use of recombinant VP2 vaccine against infectious bursal disease in chickens. Vaccine 25: 7900–7908.
- 40. Wu H, Singh NK, Locy RD, Scissum-Gunn K, Giambrone JJ (2004) Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana. Avian Dis 48: 663–668.
- 41. Pitcovski J, Gutter B, Gallili G, Goldway M, Perelman B, et al. (2003) Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens. Vaccine 21: 4736–4743.
- 42. Villegas P, Hamoud M, Purvis LB, Perozo F (2008) Infectious bursal disease subunit vaccination. Avian Dis 52: 670–674.
- 43. Dey S, Upadhyay C, Madhan MC, Kataria JM, Vakharia VN (2009) Formation of subviral particles of the capsid protein VP2 of infectious bursal disease virus and its application in serological diagnosis. J Virol Methods 157: 84–89.
- 44.
Krijger JJ, Baumann J, Wagner M, Schulze K, Reinsch C, et al. (2012) A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis. Microb Cell Fact: in press.
- 45. Zenke FT, Zachariae W, Lunkes A, Breunig KD (1993) Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon. Mol Cell Biol 13: 7566–7576.
- 46. Jagadish MN, Laughton DL, Azad AA, Macreadie IG (1991) Stable synthesis of viral protein 2 of infectious bursal disease virus in Saccharomyces cerevisiae. Gene 108: 275–279.
- 47. Cox JC, Coulter AR (1997) Adjuvants-a classification and review of their modes of action. Vaccine 15: 248–256.
- 48. Bathurst IC (1994) Protein expression in yeast as an approach to production of recombinant malaria antigens. Am J Trop Med Hyg 50: 20–26.
- 49. Gellissen G, Hollenberg CP (1997) Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis – a review. Gene 190: 87–97.
- 50. Stubbs AC, Martin KS, Coeshott C, Skaates SV, Kuritzkes DR, et al. (2001) Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity. Nature Med 7: 625–629.
- 51. Lu Y, Bellgrau D, Dwyer-Nield LD, Malkinson AM, Duke RC, et al. (2004) Mutation-selective tumor remission with Ras-targeted, whole yeast-based immunotherapy. Cancer Res 64: 5084–5088.
- 52. Schreuder MP, Deen C, Boersma WJ, Pouwels PH, Klis FM (1996) Yeast expressing hepatitis B virus surface antigen determinants on its surface: implications for a possible oral vaccine. Vaccine 14: 383–388.
- 53. Blanquet S, Antonelli R, Laforet L, Denis S, Marol-Bonnin S, et al. (2004) Recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery system in the gut. J Biotechnol 110: 37–49.
- 54. Beier R, Gebert A (1998) Kinetics of particle uptake in the domes of Peyer’s patches. Am J Physiol 275: 130–137.
- 55. Shin SJ, Bae JL, Cho YW, Lee DY, Kim DH, et al. (2005) Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobacillus pleuropneumoniae ApxIIA. FEMS Immunol Med Microbiol 43: 155–164.
- 56. van Ooyen AJJ, Dekker P, Huang M, Olsthoorn MMA, Jacobs DI, et al. (2006) Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res 6: 381–392.
- 57. Backhaus K, Buchwald U, Heppeler N, Schmitz HP, Rodicio R, et al. (2011) Milk and sugar: regulation of cell wall synthesis in the milk yeast Kluyveromyces lactis. Eur J Cell Biol 90: 745–750.
- 58. Uccelletti D, Farina F, Mancini P, Palleschi C (2004) KlPMR1 inactivation and calcium addition enhance secretion of non-hyperglycosylated heterologous proteins in Kluyveromyces lactis. J Biotechnol 109: 93–101.
- 59. Mulder W, Scholten IHJM, Grivell LA (1995) Carbon catabolite regulation of transcription of nuclear genes coding for mitochondrial proteins in the yeast Kluyveromyces lactis. Curr Genet 28: 267–273.
- 60. Breunig KD, Bolotin-Fukuhara M, Bianchi MM, Bourgarel D, Falcone C, et al. (2000) Regulation of primary carbon metabolism in Kluyveromyces lactis. Enzyme and Microbial Technology 26: 771–780.
- 61. Donnini C, Farina F, Neglia B, Compagno MC, Uccelletti D, et al. (2004) Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis. Appl Environ Microbiol 70: 2632–2638.
- 62. Kuger P, Gödecke A, Breunig KD (1990) A mutation in the Zn-finger of the GAL4 homolog LAC9 results in glucose repression of its target genes. Nucl Acids Res 18: 745–751.
- 63. Ardiani A, Higgins JP, Hodge JW (2010) Vaccines based on whole recombinant Saccharomyces cerevisiae cells. FEMS Yeast Res 10: 1060–1069.
- 64. Fuller TE, Martin S, Teel JF, Alaniz GR, Kennedy MJ, et al. (2000) Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model. Microb Pathog 29: 39–51.
- 65. Hensel A, van Leengoed LA, Szostak M, Windt H, Weissenböck H, et al. (1996) Induction of protective immunity by aerosol or oral application of candidate vaccines in a dose-controlled pig aerosol infection model. J Biotechnol 44: 171–181.
- 66. Prideaux CT, Lenghaus C, Krywult J, Hodgson AL (1999) Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxII operon. Infect Immun 67: 1962–1966.
- 67. Droual R, Bickford AA, Charlton BR, Kuney DR (1990) Investigation of problems associated with intramuscular breast injection of oil-adjuvanted killed vaccines in chickens. Avian Dis 34: 473–478.
- 68. Burns KE, Ruiz J, Glisson JR (2003) Evaluation of the effect of heating an oil-emulsion Pasteurella multocida bacterin on tissue reaction and immunity. Avian Dis 47: 54–58.
- 69. Cox E, Verdonck F, Vanrompay D, Goddeeris B (2006) Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa. Veterinary Res 37: 511–539.
- 70. Saugar I, Luque D, Oña A, Rodríguez JF, Carrascosa JL, et al. (2005) Structural polymorphism of the major capsid protein of a double-stranded RNA virus: an amphipathic alpha helix as a molecular switch. Structure 13: 1007–1017.
- 71. Icard AH, Sellers HS, Mundt E (2008) Detection of infectious bursal disease virus isolates with unknown antigenic properties by reverse genetics. Avian Dis 52: 590–598.
- 72.
Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics: A laboratory course manual. New York: Cold Spring Harbor Press.
- 73. Raab D, Graf M, Notka F, Schödl T, Wagner R (2010) The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst Synth Biol 4: 215–225.
- 74. Granzow H, Birghan C, Mettenleiter TC, Beyer J, Köllner B, et al. (1997) A second form of infectious bursal disease virus-associated tubule contains VP4. J Virol 71: 8879–8885.
- 75. Engler-Blum G, Meier M, Frank J, Müller GA (1993) Reduction of background problems in nonradioactive northern and Southern blot analyses enables higher sensitivity than 32P-based hybridizations. Anal Biochem 210: 235–244.
- 76. Hoshi S, Uchino A, Saito N, Kusanagi KI, Ihara T, et al. (1999) Comparison of adjuvants with respect to serum IgG antibody response in orally immunized chickens. Comp Immunol Microbiol Infect Dis 22: 63–69.
- 77. Snyder DB, Lana DP, Cho BR, Marquardt WW (1988) Group and strain-specific neutralization sites of infectious bursal disease virus defined with monoclonal antibodies. Avian Dis 32: 527–534.
- 78. Schröder A, van Loon AA, Goovaerts D, Mundt E (2000) Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens. J Gen Virol 81: 533–540.
- 79. Perozo F, Villegas P, Estevez C, Alvarado IR, Purvis LB, et al. (2008) Protection against infectious bursal disease virulent challenge conferred by a recombinant avian adeno-associated virus vaccine. Avian Dis 52: 315–319.
- 80. Rosales AG, Villegas P, Lukert PD, Fletcher OJ, Mohamed MA, et al. (1989) Pathogenicity of recent isolates of infectious bursal disease virus in specific-pathogen-free chickens: protection conferred by an intermediate vaccine strain. Avian Dis 33: 729–734.