The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein. In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coli are described. The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods. In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E. coli but not the denatured form or the same protein submitted to a different refolding condition. Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods.

Bacterial carriers for the mucosal delivery of antigens, particularly those capable of colonizing or invading through the mucosal epithelia, have been intensively investigated (1). Live bacterial vectors can be generated with attenuated pathogens, such as Salmonella and Listeria, or nonpathogenic commensal species, such as Lactococcus and Lactobacillus. Bacteria in the former group of are capable of inducing strong and long-lasting immune responses to passenger antigens but present serious safety concerns, whereas bacteria in the latter group are safer but typically induce much lower immune responses to the passenger antigens (1-3). As a safe nonpathogenic live bacterial vector, Bacillus subtilis, a spore-forming soil Gram-positive bacterial species, has been engineered to express antigens as either vegetative cells or spores (4, 5). As antigen carriers, B. subtilis spores have several attractive features, including a safe record of human and animal use as both probiotic and food additives, remarkable heat resistance, and rather easy genetic and bacteriological manipulation.Currently, two major genetic approaches have been proposed to generate recombinant B. subtilis spores as vaccine delivery vectors. The first approach relies on the expression of a heterologous protein genetically fused to surface-exposed spore coat proteins, such as CotB, CotC, or CotG (6, 7). Such a strategy would allow a better presentation of the passenger antigen to the mucosa-associated lymphoid tissue (MALT) afferent sites, leading to the induction of adaptive immune responses, such as mucosal secretory (IgA) or systemic (IgG) antigen-specific antibody responses (6-8). The second approach is based on a distinct rationale and has employed episomal vectors in which the target antigen is expressed under the control of a promoter (PgsiB) active only at the vegetative cell stage, which means immediately after spore germination (9-11). This antigen delivery approach relies on the fact that B. subtilis spores germinate during transit through the gastrointestinal tract and produce the target antigen at the intestinal lumen or inside the phagocytes of antigen-presenting cells (APCs), leading to the induction of antibody responses in the serum (IgG) and mucosa (fecal IgA) (9-11).However, in both cases, the administration of recombinant spores via mucosal routes typically confers immune responses to the passenger antigen lower than those achieved with delivery systems based on attenuated bacterial strains capable of colonizing the mammalian gastrointestinal tract. The reduced mucosal adjuvant effects of B. subtilis spores have been attributed to several factors, such as a previously established immunity generated by the frequent ingestion of spores, the reduced amount of expressed antigens, and the rapid transit through the gastrointestinal tract, which reduces the chances of a productive interaction with the gut-associated lymphoid tissue (GALT), such as M cells and APCs at Peyer's patches (PPs) (12, 13).In an attempt to improve the performance o...

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P139–512) derived from the S. mutans strain UA159. Purified P139–512 reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P139–512 induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P139–512 antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P139–512 eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P139–512, expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Bacillus subtilis endospores have applications in different fields including their use as probiotics and antigen delivery vectors. Such specialized applications frequently require highly purified spore preparations. Nonetheless, quantitative data regarding both yields and purity of B. subtilis endospores after application of different growth conditions and purification methods are scarce or poorly reported. In the present study, we conducted several quantitative and qualitative analyses of growth conditions and purification procedures aiming generation of purified B. subtilis spores. Based on two growth media and different incubations conditions, sporulation frequencies up to 74.2 % and spore concentrations up to 7 × 10(9) spores/ml were achieved. Application of a simplified spore isolation method, in which samples were incubated with lysozyme and a detergent, resulted in preparations with highly purified spores at the highest yields. The present study represents, therefore, an important contribution for those working with B. subtilis endospores for different biotechnological purposes.