Pichia pastoris yeast as a vehicle for oral vaccination of larval and adult teleosts.

Oral vaccination is of major interest because it can be used for mass vaccination of fish of various size and age. Given that their administration is relatively easy and stress-free, oral vaccines have both economic and animal welfare benefits. Yet, mostly due to their limited efficacy, only very few oral vaccines are available to aquaculture industry. Here we present a method for oral vaccine delivery based on the yeast Pichia pastoris. We could express a model antigen, green fluorescent protein (GFP), in this yeast and subsequently show delivery of the GFP protein to the intestine of juvenile flounder or adult carp and trout. We tested this approach in several commercially-relevant fish species, from juvenile to adult stage. To test the oral delivery of antigen to larval fish, the GFP-expressing Pichia pastoris was first fed to planktonic crustacean Daphnia or rotifers that served as 'bioencapsulation vehicles' and afterwards, fed to flounder larvae. Again, we could show delivery of intact GFP protein to the intestine. In rainbow trout, the orally-administered GFP-expressing yeast elicited a rapid local innate immune response in the intestine and a subsequent systemic response in the spleen. Our results show that Pichia pastoris is a good vehicle for oral antigen delivery and that it can be used in non-encapsulated form for older fish or in bioencapsulated form for larval fish. We discuss the immunomodulatory properties of the yeast itself, and its potential to enhance local immune responses and act as an adjuvant.

Nervous Necrosis Virus-like Particle (VLP) Vaccine Stimulates European Sea Bass Innate and Adaptive Immune Responses and Induces Long-Term Protection against Disease.

The rapidly increasing Mediterranean aquaculture production of European sea bass is compromised by outbreaks of viral nervous necrosis, which can be recurrent and detrimental. In this study, we evaluated the duration of protection and immune response in sea bass given a single dose of a virus-like particle (VLP)-based vaccine. Examinations included experimental challenge with nervous necrosis virus (NNV), serological assays for NNV-specific antibody reactivity, and immune gene expression analysis. VLP-vaccinated fish showed high and superior survival in challenge both 3 and 7.5 months (1800 and 4500 dd) post-vaccination (RPS 87 and 88, OR (surviving) = 16.5 and 31.5, respectively, p < 0.01). Although not providing sterile immunity, VLP vaccination seemed to control the viral infection, as indicated by low prevalence of virus in the VLP-vaccinated survivors. High titers of neutralizing and specific antibodies were produced in VLP-vaccinated fish and persisted for at least ~9 months post-vaccination as well as after challenge. However, failure of immune sera to protect recipient fish in a passive immunization trial suggested that other immune mechanisms were important for protection. Accordingly, gene expression analysis revealed that VLP-vaccination induced a mechanistically broad immune response including upregulation of both innate and adaptive humoral and cellular components (mx, isg12, mhc I, mhc II, igm, and igt). No clinical side effects of the VLP vaccination at either tissue or performance levels were observed. The results altogether suggested the VLP-based vaccine to be suitable for clinical testing under farming conditions.

Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

New milliliter-scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms.

A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k(L)a) > 0.15 s(-1) were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically.

New insights into rifamycin B biosynthesis: isolation of proansamycin B and 34a-deoxy-rifamycin W as early macrocyclic intermediates indicating two separated biosynthetic pathways.

Proansamycin B, the formerly postulated intermediate of rifamycin B biosynthesis, was isolated from cultures of the Amycolatopsis mediterranei mutant F1/24. The structure was determined using UV, IR, NMR and MS techniques. Biotransformation studies demonstrate that proansamycin B is an intermediate of a shunt pathway, a 8-deoxy variant, of rifamycin B biosynthesis leading to 8-deoxy-rifamycin B as the final product. In addition, 34a-deoxy-rifamycin W, the direct precursor of rifamycin W, could be isolated representing the earliest macrocyclic intermediate obtained so far in the biosynthetic route to rifamycin B. Furthermore, the new rifamycin W-28-desmethyl-28-carboxy and rifamycin W-hemiacetal, intermediates in the transformation sequence of rifamycin W to rifamycin S, were isolated. Application of proton NMR measurements (double resonance and ROESY experiments) on the latter compound indicated that the stereochemistry at the chiral center C-28 is R.

Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene.

Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.

Biosynthesis of the C(7)-cyclitol moiety of acarbose in Actinoplanes species SE50/110. 7-O-phosphorylation of the initial cyclitol precursor leads to proposal of a new biosynthetic pathway.

We have previously demonstrated that the biosynthesis of the C(7)-cyclitol, called valienol (or valienamine), of the alpha-glucosidase inhibitor acarbose starts from the cyclization of sedo-heptulose 7-phosphate to 2-epi-5-epi-valiolone (Stratmann, A., Mahmud, T., Lee, S., Distler, J., Floss, H. G., and Piepersberg, W. (1999) J. Biol. Chem. 274, 10889-10896). Synthesis of the intermediate 2-epi-5-epi-valiolone is catalyzed by the cyclase AcbC encoded in the biosynthetic (acb) gene cluster of Actinoplanes sp. SE50/110. The acbC gene lies in a possible transcription unit, acbKLMNOC, cluster encompassing putative biosynthetic genes for cyclitol conversion. All genes were heterologously expressed in strains of Streptomyces lividans 66 strains 1326, TK23, and TK64. The AcbK protein was identified as the acarbose 7-kinase, which had been described earlier (Drepper, A., and Pape, H. (1996) J. Antibiot. (Tokyo) 49, 664-668). The multistep conversion of 2-epi-5-epi-valiolone to the final cyclitol moiety was studied by testing enzymatic mechanisms such as dehydration, reduction, epimerization, and phosphorylation. Thus, a phosphotransferase activity was identified modifying 2-epi-5-epi-valiolone by ATP-dependent phosphorylation. This activity could be attributed to the AcbM protein by verifying this activity in S. lividans strain TK64/pCW4123M, expressing His-tagged AcbM. The His-tagged AcbM protein was purified and subsequently characterized as a 2-epi-5-epi-valiolone 7-kinase, presumably catalyzing the first enzyme reaction in the biosynthetic route, leading to an activated form of the intermediate 1-epi-valienol. The AcbK protein could not catalyze the same reaction nor convert any of the other C(7)-cyclitol monomers tested. The 2-epi-5-epi-valiolone 7-phosphate was further converted by the AcbO protein to another isomeric and phosphorylated intermediate, which was likely to be the 2-epimer 5-epi-valiolone 7-phosphate. The products of both enzyme reactions were characterized by mass spectrometric methods. The product of the AcbM-catalyzed reaction, 2-epi-5-epi-valiolone 7-phosphate, was purified on a preparative scale and identified by NMR spectroscopy. A biosynthetic pathway for the pseudodisaccharidic acarviosyl moiety of acarbose is proposed on the basis of these data.