Christine Andreoni

The serum albumin-binding region of streptococcal protein G (BB) potentiates the immunogenicity of the G130-230 RSV-A protein

BBG2Na is a protein comprising residues 130-230 of the respiratory syncytial virus subgroup A (RSV-A) G protein (G2Na) fused to the albumin-binding domain of streptococcal G protein (BB). BBG2Na was cloned, expressed in Escherichia coli and renaturated. In rodent models, this subunit RSV vaccine adjuvanted in Alhydrogel induced specific antibodies and conferred protection to RSV infection. Comparison of the antibody production in a BALB/c mouse model revealed that BBG2Na induced a stronger and earlier G2Na antibody response than G2Na alone, without altering the IgG subclass distribution. To address the role of the BB part, we explored its carrier properties and showed that it is a Th dependent antigen, generating a more potent G2Na-specific B cell memory response and able to generate Th cells that provide help for G2Na antibody production.

Challenge of BALB/c Mice with Respiratory Syncytial Virus Does Not Enhance the Th2 Pathway Induced after Immunization with a Recombinant G Fusion Protein, BBG2NA, in Aluminum Hydroxide

The polypeptide of aa 130-230 of the G protein (G2Na) of respiratory syncytial virus (RSV) was fused to BB, the albumin-binding region of streptococcal G protein, producing BBG2Na, which induced protective immune responses in rodent models. Evaluation of the immune response in mice immunized with BBG2Na in the adjuvant alhydrogel revealed high amounts of interleukin (IL)-5 and some IL-4 in splenocytes restimulated in vitro. This is compatible with a Th2 response. The activation of the Th2 pathway in such mice was further supported by the detection of IL-5 and G2Na-specific IgE in vivo. Of interest, in contrast to immunization with formalin-inactivated RSV, immunization of mice with BBG2Na followed by intranasal RSV challenge did not lead to increased production of IL-5- or G2Na-specific IgE. However, IgG1- and IgG2a-specific antibodies were boosted. These results demonstrate that the Th2 pathway is not enhanced by RSV challenge in BBG2Na-immunized mice.

Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, where coupling has been obtained either by genetic fusion or chemical conjugation. For genetically fused immunogens both N- and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impaired the biological functions of CTB, such as the ability to form pentamers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has been used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptococcal protein G, which is known to fold independently of N- or C-terminal fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) columns. The CTB fusion proteins were compared for their ability to form pentamers, by gel electrophoresis and size-exclusion chromatography, and it was concluded that all three fusion proteins were able to pentamerize. Interestingly, the C-terminal fusion to CTB showed most efficient pentamerization, while the dual fusion was much less efficient. Purified pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB. In addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM1, and it was found that the C-terminal fusion (CTB-BB) showed significant GM1-binding, but that also the N-terminal and dual CTB fusion proteins bound GM1, although less efficiently. The implications of the results for the design and use of CTB fusion proteins as subunit vaccines are discussed.

Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus

Protein engineering has been employed to investigate the effect of specific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus xylosus. Three different variants, corresponding to a 101 amino acid region of the major glycoprotein (G protein) of human respiratory syncytial virus (RSV), were generated in which multiple hydrophobic phenylalanine residues were either substituted or deleted. The different G protein fragments were expressed as one part of recombinant receptors designed for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engineered fragment, efficiently targeted to the outer cell surface of recombinant S. xylosus cells as determined by different methods, including fluorescence-activated cell sorting. In addition, immunization of mice with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The present strategy of hydrophobic engineering should be of general interest in surface-display applications and for secretion of proteins otherwise difficult to translocate through host cell membranes.

Surface display on staphylococci: a comparative study

Two different host‐vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence‐activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

Development of Non-Pathogenic Staphylococci as Vaccine Delivery Vehicles

Among the bacteria being considered as live recombinant vaccine vehicles, the most well studied during the past decade are attenuated Salmonella species1 and mycobacterial bacille Calmette-Guerin (BCG) due to their capacity to colonize mucosal surfaces and invade macrophages in the liver, spleen and lymph nodes of the host.2,3 Surface-display of the foreign antigens to be delivered, has in both these systems proven to be beneficial in eliciting an immune response.4–7 The risk of reversion to a virulent phenotype and the potential side-effects in immunocompromised individuals and infants have, however, raised concern of the use of Salmonella or BCG-based recombinant vaccines in humans.8