In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')(2) - ERIGs). According to international regulation, quality control of F(ab')(2) - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Securite du Medicament et des Produits de Sante where F(ab')(2) - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')(2) - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r = 0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice. (C) 2016 Elsevier Ltd. All rights reserved.

Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID(50)), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.

The possibility of improving the antibody responses to a model streptococcal antigen, administered by intranasal immunization as surface-displayed on the food-grade bacterium Staphylococcus carnosus, by co-exposure of a peptide (CTBp) comprising amino acids 50-75 of the cholera toxin B subunit, was investigated. It was found that the introduction of the CTBp into the chimeric surface proteins, containing a serum albumin binding protein (ABP) from streptococcal protein G as model antigen, significantly increased serum IgG responses upon intranasal immunization. Similarly, elicited local IgA responses were also found to be improved. Furthermore, it was demonstrated that live delivery of the staphylococci was required to obtain this effect, since UV-irradiated or heat-killed bacteria exposing the same chimeric surface proteins did not show increased anti-ABP IgG responses.

Surface display of recombinant proteins on bacteria and phages has become an important topic in bioscience. A system for the display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signal and propeptide from a Staphylococcus hyicus lipase for translocation and since the propeptide is of considerable size (207 amino acids) and not processed in S. carnosus, we have investigated the possibility to delete or substitute the propeptide for smaller protein domains, to thereby improve the surface display system. A set of new vectors was constructed and the surface expression of model proteins was investigated by various methods, including fluorescence-activated cell sorting. The results suggest that the propeptide region indeed can be deleted when proteins which are easily secretable are displayed. In contrast, the propeptide seems to be advantageous for translocation of inefficiently secreted proteins. Moreover, our study also presents a rational strategy for how to monitor the engineering efforts for the optimization of a surface display system.

The surface expression in Staphylococcus carnosus of three different fibronectin binding domains (FNBDs), derived from fibronectin binding proteins of Streptococcus dysgalactiae and Staphylococcus aureus, has been investigated. Surface localization of the chimeric proteins containing the FNBDs was demonstrated. All three surface-displayed FNBDs were demonstrated to bind fibronectin in whole-cell enzyme-linked binding assays. Furthermore, for one of the constructs, intranasal immunizations with the recombinant bacteria resulted in improved antibody responses to a model immunogen present within the chimeric surface proteins. The implications of the results for the design of live bacterial vaccine delivery systems are discussed.