Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model.

BACKGROUND/AIMS: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria. METHODS: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting. RESULTS: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed. CONCLUSION: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

Metabolic fluxes-oriented control of bioreactors: a novel approach to tune micro-aeration and substrate feeding in fermentations.

BACKGROUND: Fine-tuning the aeration for cultivations when oxygen-limited conditions are demanded (such as the production of vaccines, isobutanol, 2-3 butanediol, acetone, and bioethanol) is still a challenge in the area of bioreactor automation and advanced control. In this work, an innovative control strategy based on metabolic fluxes was implemented and evaluated in a case study: micro-aerated ethanol fermentation. RESULTS: The experiments were carried out in fed-batch mode, using commercial Saccharomyces cerevisiae, defined medium, and glucose as carbon source. Simulations of a genome-scale metabolic model for Saccharomyces cerevisiae were used to identify the range of oxygen and substrate fluxes that would maximize ethanol fluxes. Oxygen supply and feed flow rate were manipulated to control oxygen and substrate fluxes, as well as the respiratory quotient (RQ). The performance of the controlled cultivation was compared to two other fermentation strategies: a conventional "Brazilian fuel-ethanol plant" fermentation and a strictly anaerobic fermentation (with ultra-pure nitrogen used as the inlet gas). The cultivation carried out under the proposed control strategy showed the best average volumetric ethanol productivity (7.0 g L-1 h-1), with a final ethanol concentration of 87 g L-1 and yield of 0.46 gethanol g substrate -1 . The other fermentation strategies showed lower yields (close to 0.40 gethanol g substrate -1 ) and ethanol productivity around 4.0 g L-1 h-1. CONCLUSION: The control system based on fluxes was successfully implemented. The proposed approach could also be adapted to control several bioprocesses that require restrict aeration.

Recombinant Escherichia coli cultivation in a pressurized airlift bioreactor: assessment of the influence of temperature on oxygen transfer and uptake rates.

Temperature influences the rates of oxygen transfer (OTR) and uptake (q O2) in aerobic bioprocesses. Hence, joint analysis of q O2 and OTR at variable temperature is essential for bioprocess optimization and control. However, no such analyses have yet been reported for cultures of engineered E. coli producing recombinant proteins. E. coli cultivations at different temperatures (27-37 °C) were performed using a 5-L stirred tank bioreactor (STB), and a 5-L airlift bioreactor (ALB) was used to measure k L a and validate models of q O2 and OTR. The equations were then employed to evaluate the cultivation process in the ALB at different pressures (0.1-0.4 MPa) and temperatures (27-37 °C). The results showed that the positive effect of temperature on k L a was more pronounced than the negative influence on oxygen solubility, increasing the OTR in the ALB. The specific growth rate and temperature influenced q O2. In contrast to previous reports, the results showed that q O2 was not explicitly affected by recombinant protein synthesis. In addition, model predictions revealed that biomass concentration and productivity were greatly improved by pressurization of the system and use of a lower temperature.

Capsular polysaccharide production by Streptococcus pneumoniae serotype 1: from strain selection to fed-batch cultivation.

Streptococcus pneumoniae is a human pathogen largely transmitted by aerosols. Vaccines are the main strategy against this pathogen, and the capsular polysaccharide (PS) is its major antigen. S. pneumoniae serotype 1 is associated with large outbreaks and epidemics of invasive diseases. The aims of this work were to screen serotype 1 strains to identify the best PS1 producer, evaluate three peptones for PS1 production, investigate the effects of culture medium components using a design of experiments (DoE), a statistic tool for optimization, and propose a new medium/cultivation strategy. After flask cultivation of nine strains, two that produced high PS1 and biomass values were chosen for further evaluation in the bioreactor, and ST595/01 was chosen as the best PS1 producer strain. Among the peptones tested (Casamino acids, Soytone, and Phytone), the highest PS1 production (298 mg/L) was reached with Phytone. Next, DoE (2(4-1)) was performed to evaluate the effects of yeast extract (YE), Phytone, L-asparagine (Asn), and L-glutamine (Gln), yielding the following results: Phytone presented positive effects (p < 0.05) for maximum production of biomass, PS1, acetate, and lactate; YE showed positive effects for biomass and acid production (p < 0.05); Gln exerted a minor positive effect on PS1 yield factor on glucose (p < 0.1); and Asn presented only an effect on acetate production (p < 0.1). Hence, a new culture medium was formulated based on Phytone, YE, and glucose, and batch and fed-batch cultivations were evaluated. The fed-batch cultivation showed almost 2 times the biomass and 2.5 times the PS1 production as the batch culture, and 8-10 times higher PS1 production than has been previously reported.

Oxygen transfer in a pressurized airlift bioreactor.

Airlift bioreactors (ALBs) offer advantages over conventional systems, such as simplicity of construction, reduced risk of contamination, and efficient gas-liquid dispersion with low power consumption. ALBs are usually operated under atmospheric pressure. However, in bioprocesses with high oxygen demand, such as high cell density cultures, oxygen limitation may occur even when operating with high superficial gas velocity and air enriched with oxygen. One way of overcoming this drawback is to pressurize the reactor. In this configuration, it is important to assess the influence of bioreactor internal pressure on the gas hold-up, volumetric oxygen transfer coefficient (k(L)a), and volumetric oxygen transfer rate (OTR). Experiments were carried out in a concentric-tube airlift bioreactor with a 5 dm(3) working volume, equipped with a system for automatic monitoring and control of the pressure, temperature, and inlet gas flow rate. The results showed that, in disagreement with previous published results for bubble column and external loop airlift reactors, overpressure did not significantly affect k(L)a within the studied ranges of pressure (0.1-0.4 MPa) and superficial gas velocity in the riser (0.032-0.065 m s(-1)). Nevertheless, a positive effect on OTR was observed: it increased up to 5.4 times, surpassing by 2.3 times the oxygen transfer in a 4 dm(3) stirred tank reactor operated under standard cultivation conditions. These results contribute to the development of non-conventional reactors, especially pneumatic bioreactors operated using novel strategies for oxygen control.

High-throughput strategies for penicillin G acylase production in rE. coli fed-batch cultivations.

BACKGROUND: Penicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits. RESULTS: This work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively. CONCLUSIONS: To the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

Enhanced production of recombinant thermo-stable lipase in Escherichia coli at high induction temperature.

Thermostable microbial lipases are potential candidates for industrial applications such as specialty organic syntheses as well as hydrolysis of fats and oils. In this work, basic biochemical engineering tools were applied to enhance the production of BTL2 lipase cloned in Escherichia coli BL321 under control of the strong temperature-inducible λP(L) promoter. Initially, surface response analysis was used to assess the influence of growth and induction temperatures on enzyme production, in flask experiments. The results showed that temperatures of 30 and 45°C were the most suitable for growth and induction, respectively, and led to an enzyme specific activity of 706,000 U/gDCW. The most promising induction conditions previously identified were validated in fed-batch cultivation, carried out in a 2L bioreactor. Specific enzyme activity reached 770,000 U/gDCW, corresponding to 13,000 U/L of culture medium and a lipase protein concentration of 10.8 g/L. This superior performance on enzyme production was a consequence of the improved response of λP(L) promoter triggered by the high induction temperature applied (45°C). These results point out to the importance of taking into account protein structure and stability to adequately design the recombinant protein production strategy for thermally induced promoters.

Cloning, auto-induction expression, and purification of rSpaA swine erysipelas antigen.

This work reports the cloning, expression, and purification of a 42-kDa fragment of the SpaA protein from Erysipelothrix rhusiopathiae, the main antigenic candidate for a subunit vaccine against swine erysipelas. The use of an auto-induction protocol to improve heterologous protein expression in recombinant Escherichia coli cultures was also investigated. The cellular growth pattern and metabolite formation were evaluated under different induction conditions. The His-tagged protein was over-expressed as inclusion bodies, and was purified by a single chromatography step under denaturing conditions. Auto-induction conditions were shown to be an excellent process strategy, leading to a high level of rSpaA expression (about 25 % of total cellular protein content) in a short period of time.

Intensification of high cell-density cultivations of rE. coli for production of S. pneumoniae antigenic surface protein, PspA3, using model-based adaptive control.

This work proposes an innovative methodology to control high density fed-batch cultures of E. coli, based on measurements of the concentration of dissolved oxygen and on estimations of the cellular specific growth rate (µ), of the yield of biomass/limiting substrate (Y (xs)) and of the maintenance coefficient (m). The underlying idea is to allow cells to grow according to their metabolic capacity, without the constraints inherent to pre-set growth rates. Cellular concentration was assessed on-line through a capacitance probe. Three configurations of the control system were compared: (1) pre-set value for the three control parameters; (2) continuously updating µ; (3) updating µ, Y (xs) and m. Implementation of an efficient noise filter for the signal of the capacitance probe was essential for a good performance of the control system. The third control strategy, within the framework of an adaptive model-based control, led to the best results, with biomass productivity reaching 9.2 g(DCW)/L/h.

Improving 3-hydroxypropionic acid production in E. coli by in silico prediction of new metabolic targets.

3-Hydroxypropionic acid (3-HP) production from renewable feedstocks is of great interest in efforts to develop greener processes for obtaining this chemical platform. Here we report an engineered E. coli strain for 3-HP production through the ß-alanine pathway. To obtain a new strain capable of producing 3-HP, the pathway was established by overexpressing the enzymes pyruvate aminotransferase, 3-hydroxyacid dehydrogenase, and L-aspartate-1-decarboxylase. Further increase of the 3-HP titer was achieved using evolutionary optimizations of a genome-scale metabolic model of E. coli containing the adopted pathway. From these optimizations, three non-intuitive targets for in vivo assessment were identified: L-alanine aminotransferase and alanine racemase overexpression, and L-valine transaminase knock-out. The implementation of these targets in the production strain resulted in a 40% increase in 3-HP titer. The strain was further engineered to overexpress phosphoenolpyruvate carboxylase, reaching 0.79 ± 0.02 g/L of 3-HP when grown using glucose. Surprisingly, this strain produced 63% more of the desired product when grown using a mixture of glucose and xylose (1:1, C-mol), and gene expression analysis showed that the cellular adjustment to consume xylose had a positive impact on 3-HP accumulation. Fed-batch culture with xylose feeding led to a final titer of 29.1 g/L. These results reinforce the value of computational methods in strain engineering, enabling the design of more efficient strategies to be assessed. Moreover, higher production of 3-HP under a sugar mixture condition points towards the development of bioprocesses based on renewable resources, such as hemicellulose hydrolysates.

Robust artificial intelligence tool for automatic start-up of the supplementary medium feeding in recombinant E. coli cultivations.

One of the most important events in fed-batch fermentations is the definition of the moment to start the feeding. This paper presents a methodology for a rational selection of the architecture of an artificial intelligence (AI) system, based on a neural network committee (NNC), which identifies the end of the batch phase. The AI system was successfully used during high cell density cultivations of recombinant Escherichia coli. The AI algorithm was validated for different systems, expressing three antigens to be used in human and animal vaccines: fragments of surface proteins of Streptococcus pneumoniae (PspA), clades 1 and 3, and of Erysipelothrix rhusiopathiae (SpaA). Standard feed-forward neural networks (NNs), with a single hidden layer, were the basis for the NNC. The NN architecture with best performance had the following inputs: stirrer speed, inlet air, and oxygen flow rates, carbon dioxide evolution rate, and CO2 molar fraction in the exhaust gas.

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium.

Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017.

Characterization of polysaccharide production of haemophilus influenzae Type b and its relationship to bacterial cell growth.

Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached approximately 1.8 g of DCW/L, while polysaccharide concentration was about approximately 132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.

Live bacterial vaccine vectors: an overview.

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.

Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures.

In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose + glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L(-1).h(-1). Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW (-1)) and volumetric productivity (0.32 g.L(-1).h(-1)). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW (-1)) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L(-1), in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.

Live bacterial vaccine vectors: An overview

Genetically attenuated microorganisms, pathogens, and some commensal bacteria can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system, while still offering good levels of safety. A key feature of these live vectors is their capacity to stimulate mucosal as well as humoral and/or cellular systemic immunity. This enables the use of different forms of vaccination to prevent pathogen colonization of mucosal tissues, the front door for many infectious agents. Furthermore, delivery of DNA vaccines and immune system stimulatory molecules, such as cytokines, can be achieved using these special carriers, whose adjuvant properties and, sometimes, invasive capacities enhance the immune response. More recently, the unique features and versatility of these vectors have also been exploited to develop anti-cancer vaccines, where tumor-associated antigens, cytokines, and DNA or RNA molecules are delivered. Different strategies and genetic tools are constantly being developed, increasing the antigenic potential of agents delivered by these systems, opening fresh perspectives for the deployment of vehicles for new purposes. Here we summarize the main characteristics of the different types of live bacterial vectors and discuss new applications of these delivery systems in the field of vaccinology.

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up.

Haemophilus influenzae type b, an encapsulated bacterium, causes meningitis in infants worldwide. The capsular polysaccharide conjugated to a carrier protein is effective in the prevention of such infections. The traditional purification process of polysaccharide from bacterial cultures for vaccine production is based on several selective precipitations with solvents such as: ethanol, phenol, and cationic detergents. The separations of solid and liquid phases are based on continuous centrifugation in explosion proof installations. The lipopolysaccharides are separated by ultracentrifugation. A simple and efficient method that can easily be scaled-up was developed for purification of polysaccharides. The ethanol precipitation was reduced to only two steps. The phenol treatment was substituted by ultrafiltration and enzymatic digestion. Lipopolysaccharide was removed by ultrafiltration together with addition of detergent and chelating agent.

Bioreactor aeration conditions modulate growth and antigen expression during Erysipelothrix rhusiopathiae cultivation.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, was cultivated in a 5-L stirred and aerated bioreactor under different dissolved oxygen tensions (0%, 5%, and 30% of saturation) for evaluation of the influence of oxygen on cell growth as well as on the production of the main antigenic component of the vaccine against erysipelas, a 64-69 kDa protein (SpaA). The microorganism presented different growth profiles for different aeration conditions. However, at the end of the batch cultivations, similar cell concentrations were obtained under the studied conditions. In order to maximize biomass titers and antigen production, the microorganism was cultivated in fed-batch operation mode under aerobic conditions. Under this condition, there was a fivefold increase in biomass production in comparison to the results attained in batch cultivations. To follow up antigen expression, samples collected during batch cultivations were concentrated and treated with choline for antigen extraction. Antigen expression was then assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by murine immunization tests. It was observed a direct influence of oxygen availability upon antigen expression, which is favored in the presence of oxygen. Analysis of the samples collected throughout the fed-batch process also revealed that antigen production is growth associated.

Robust artificial intelligence tool for automatic start-up of the supplementary medium feeding in recombinant E. colicultivations

One of the most important events in fed-batch fermentations is the definition of the moment to start the feeding. This paper presents a methodology for a rational selection of the architecture of an artificial intelligence (AI)system, based on a neural network committee (NNC),which identifies the end of the batch phase. The AI systemwas successfully used during high cell density cultivations of recombinant Escherichia coli. The AI algorithm wasvalidated for different systems, expressing three antigens to be used in human and animal vaccines: fragments of surface proteins of Streptococcus pneumoniae (PspA), clades 1 and 3, and of Erysipelothrix rhusiopathiae (SpaA). Standard feed-forward neural networks (NNs), with a single hidden layer, were the basis for the NNC. The NN architecture with best performance had the following inputs: stirrer speed, inlet air, and oxygen flow rates, carbon dioxide evolution rate, and CO2 molar fraction in the exhaust gas.

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

Haemophilus inXuenzae type b, an encapsulatedbacterium, causes meningitis in infants worldwide. The capsularpolysaccharide conjugated to a carrier protein is eVectivein the prevention of such infections. The traditional puriWcationprocess of polysaccharide from bacterial cultures for vaccineproduction is based on several selective precipitationswith solvents such as: ethanol, phenol, and cationic detergents.The separations of solid and liquid phases are based oncontinuous centrifugation in explosion proof installations. Thelipopolysaccharides are separated by ultracentrifugation. Asimple and eYcient method that can easily be scaled-up wasdeveloped for puriWcation of polysaccharides. The ethanolprecipitation was reduced to only two steps. The phenol treatmentwas substituted by ultraWltration and enzymatic digestion.Lipopolysaccharide was removed by ultraWltrationtogether with addition of detergent and chelating agent.