Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools

Rodrigues, Rafael Rodrigues; Ferreira, Marcos Roberto Alves; Donassolo, Rafael Amaral; Alves, Mariliana Luiza Ferreira; Motta, Jaqueline Freitas; Moreira, Clóvis; Salvarani, Felipe Masiero; Moreira, Ângela Nunes; Conceição, Fabrício Rochedo

doi:10.1016/j.anaerobe.2021.102326

Cited by 9 publications

(3 citation statements)

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“…Tests involving other adjuvant molecules besides the aluminum hydroxide used in most commercial and recombinant vaccines can improve and prolong the efficacy of these vaccines [ 21 , 28 ] and bioinformatic tools can be used for the development of new immunogenic recombinant molecules. Rodrigues et al [ 29 ] reported that with the aid of such tools, they designed new versions of the rCPB protein and produced an unpurified protein (rCPB-C) with high productivity, solubility and antigenicity, which is another promising option for the development of recombinant vaccines that induce a more lasting humoral immune response. Probiotics can act as immunomodulators and enhancers of the immune response of animals after vaccination [ 30 , 31 , 32 ].…”

Section: Discussionmentioning

confidence: 99%

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens

Galvão

Barbosa

Oliveira

et al. 2021

Toxins

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The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.

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Section: Discussionmentioning

confidence: 99%

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens

Galvão

Barbosa

Oliveira

et al. 2021

Toxins

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“…Other strategies employed include the targeting of proteins to different cellular compartments, for example, the periplasmic space where an oxidising environment and lower protease concentration has been shown to increase protein production and stability (Malik 2016). Alongside the optimisation of bacterial systems, computational approaches have allowed the prediction of sequence and/or structural features that may impact efficient production and solubility (Chennamsetty et al 2009;Fernandez-Escamilla et al 2004;Hebditch et al 2017;Hebditch and Warwicker 2019;Rodrigues et al 2021;Sormanni et al 2015;Trainor et al 2017). Such tools have allowed the re-design of recombinant targets with increased expression and/or solubility (Courtois et al 2015;Perchiacca et al 2011;Rodrigues et al 2021;Sahin et al 2011;Sormanni et al 2015).…”

Section: Introductionmentioning

confidence: 99%

“…Alongside the optimisation of bacterial systems, computational approaches have allowed the prediction of sequence and/or structural features that may impact efficient production and solubility (Chennamsetty et al 2009;Fernandez-Escamilla et al 2004;Hebditch et al 2017;Hebditch and Warwicker 2019;Rodrigues et al 2021;Sormanni et al 2015;Trainor et al 2017). Such tools have allowed the re-design of recombinant targets with increased expression and/or solubility (Courtois et al 2015;Perchiacca et al 2011;Rodrigues et al 2021;Sahin et al 2011;Sormanni et al 2015). Together, the optimisation of recombinant protein production tends to be tailored to each specific recombinant target, and therefore more generic predictive tools and/or guides to aid efficient recombinant protein production would be beneficial.…”

Section: Introductionmentioning

confidence: 99%

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

Hussain

McKenzie

Robinson

et al. 2021

Appl Microbiol Biotechnol

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Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

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Large Clostridial Toxins: A Brief Review and Insights into Antigen Design for Veterinary Vaccine Development

Rodrigues Rodrigues,

Alves,

Bilhalva

et al. 2024

Mol Biotechnol

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Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools

Cited by 9 publications

References 49 publications

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

Large Clostridial Toxins: A Brief Review and Insights into Antigen Design for Veterinary Vaccine Development

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