Recombinant S-layer Research Articles

Bacterial Surface Layer Proteins: A Promising Nano-Technological Tool for Bio-Sensing Applications

The phenomenal rise in the demand of biosensors accelerated their rapid development and immersive applications in the myriads of fields. The essential requirement of developing efficient bio-sensing platform is to find stable well organized interfacial architecture that can serve as an excellent matrix for binding and recognizing biomolecules. In this context, the enormous potential has been envisaged in surface layer proteins that represented themselves as most primitive and simplest self-assembled system with repetitive physicochemical properties for the molecular functionalization of surfaces and various interfaces. The prominence of S-layer proteins has been broadened by integrating genetic engineering approaches for the fine tuning of functional groups and protein domains in geometrically well-defined manner. The efficient and stable binding of various nanomaterials with S-layers in regular arrays has led to paradigmatic shift in their nano-biotechnological sensing applications. More recently, functional S-layer supported lipid membranes have been generated through covalent binding of lipid molecules either with native or recombinant S-layer proteins at nano-scale dimensions serving as “proof of concept” for the development of bio-sensing platform. Thus, in the light of benefits conferred by surface layer proteins for the development of highly efficient biosensors, an exciting path has been opened for broadening their translational applications in drug delivery, disease diagnosis, vaccines development, lab-on-chip devices etc. Therefore, this review intends to describe about the importance of surface layer proteins in the development of biosensors.

COMPLIANCE OF ANTHRAX RECOMBINANT VACCINE PROTOTYPE WITH THE REQUIREMENTS TO IMMUNE-BIOLOGICAL PREPARATIONS

Current areal of anthrax among live stock and wild animals covers almost all the continents. The cause of human infection is conventionally considered to be the contact with diseased animals in the process of carrying for an animal, forced slaughter or further trimming of carcasses, as well as contact with infected raw materials of animal origin. Licensed vaccines has made an invaluable contribution to the improvement of epidemiological situation on anthrax, however, development of the vaccines complying with current scientific progress remains relevant. We have constructed a vaccine prototype containing recombinant protective antigen and S-layer EA1 protein and added state-of-the-art CpG 2006 adjuvant to the formulation. We have demonstrated the advantage of the lyophilized form of the preparation. Objective — obtainment of anthrax vaccine prototype in lyophilized state and assessment of the prototype compliance with the requirements to vaccine preparations. Materials and methods. Isolation of rPA and EA1 protein was carried out using producer-strain B. anthracis 55ΔTПА-1Spo– on an integrated end-to-end manufacturing line, including concentration, diafiltration and two-phase chromatography. CpG 2006 adjuvant was synthesized according to known sequences. Components were mixed and lyophilized in sublimation unit. Combination of 1% sucrose and 3% glycine was used as cryoprotector. Effectiveness and safety of the preparation were evaluated on the model of BALB/c mice and guinea pigs applying immunological, morphometric, and histological assays. Antibody titers in sera of immunized animals were evaluated using standard ELISA procedures. Protective properties were investigated through LD50 values for the test-strain used on immunized and control animals and immunity index. Results. We have performed complex investigation of the anthrax vaccine prototype, containing B. anthracis 55ΔTПА-1Spo– proteins as main and supplementary antigens, as well as CpG 2006 adjuvant, stabilizers and preservative agent. The prototype meets the requirements to immunobiological medicinal drugs by all physical-chemical properties. Our preparation does not have a toxic effect on the organism of laboratory animals in case of subcutaneous and intraperitoneal administration of single human dose. Pathomorphological study of guinea pigs’ organs immunized with double dose of the vaccine prototype has not revealed any evidence of damaging effect on the cells and tissues of macroorganism. The prototype protects BALB/c mice from the infection with B. anthracis 71/12 test-strain.

Acoustic and hybrid 3D-printed electrochemical biosensors for the real-time immunodetection of liver cancer cells (HepG2)

This study presents an efficient acoustic and hybrid three-dimensional (3D)-printed electrochemical biosensors for the detection of liver cancer cells. The biosensors function by recognizing the highly expressed tumor marker CD133, which is located on the surface of liver cancer cells. Detection was achieved by recrystallizing a recombinant S-layer fusion protein (rSbpA/ZZ) on the surface of the sensors. The fused ZZ-domain enables immobilization of the anti-CD133 antibody in a defined manner. These highly accessible anti-CD133 antibodies were employed as a sensing layer, thereby enabling the efficient detection of liver cancer cells (HepG2). The recognition of HepG2 cells was investigated in situ using a quartz crystal microbalance with dissipation monitoring (QCM-D), which enabled the label-free, real-time detection of living cells on the modified sensor surface under controlled conditions. Furthermore, the hybrid 3D additive printing strategy for biosensors facilitates both rapid development and small-scale manufacturing. The hybrid strategy of combining 3D-printed parts and more traditionally fabricated parts enables the use of optimal materials: a ceramic substrate with noble metals for the sensing element and 3D-printed capillary channels to guide and constrain the clinical sample. Cyclic voltammetry (CV) measurements confirmed the efficiency of the fabricated sensors. Most importantly, these sensors offer low-cost and disposable detection platforms for real-world applications. Thus, as demonstrated in this study, both fabricated acoustic and electrochemical sensing platforms can detect cancer cells and therefore may have further potential in other clinical applications and drug-screening studies.

S‐layer Coated Emulsomes as Potential Nanocarriers

The present study introduces a novel nanocarrier system comprising lipidic emulsomes and S-layer (fusion) proteins as functionalizing tools coating the surface. Emulsomes composed of a solid tripalmitin core and a phospholipid shell are created reproducibly with an average diameter of approximately 300 nm using temperature-controlled extrusion steps. Both wildtype (wt) and recombinant (r) S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2 are capable of forming coherent crystalline envelope structures with oblique (p1) lattice symmetry, as evidenced by transmission electron microscopy. Upon coating with wtSbsB, positive charge of emulsomes shifts to a highly negative zeta potential, whereas those coated with rSbsB become charge neutral. This observation is attributed to the presence of a negatively charged glycan, the secondary cell wall polymer (SCWP), which is associated only with wtSbsB. The present study shows for the first time the ability of recombinant and wildtype S-layer proteins to cover the entire surface of emulsomes with its characteristic crystalline lattice. Furthermore, in vitro cell culture studies reveal that S-layer coated emulsomes can be uptaken by human liver carcinoma cells (HepG2) without showing any significant cytotoxicity over a wide range of concentrations. The utilization of S-layer fusion proteins equipped in a nanopatterned fashion by identical or diverse functions may lead to further development of emulsomes in nanomedicine, especially for drug delivery and targeting.

Nanoscale mono- and multi-layer cylinder structures formed by recombinant S-layer proteins of mosquitocidal Bacillus sphaericus C3-41

The mature surface layer (S-layer) protein SlpC of mosquitocidal Bacillus sphaericus C3-41 comprises amino acids 31-1,176 and could recrystallize in vitro. The N-terminal SLH domain is responsible for binding function. Deletion of this part, S-layer proteins could not bind to the cell wall sacculi. To investigate the self-assembly ability of SlpC from B. sphaericus, nine truncations were constructed and their self-assembly properties were compared with the recombinant mature S-layer protein rSlpC₃₁₋₁,₁₇₆. The results showed that rSbsC₃₁₋₁,₁₇₆ and truncations rSlpC₂₁₁₋₁,₁₇₆, rSlpC₂₇₈₋₁,₁₇₆, rSlpC₃₁₋₁,₁₀₀, and rSlpC₃₁₋₁,₀₅₀ could assemble into multilayer cylinder structures, while N-terminal truncations rSlpC₃₃₈₋₁,₁₇₆, rSlpC₄₃₈₋₁,₁₇₆, and rSlpC₄₉₈₋₁,₁₇₆ mainly showed monolayer cylinders in recombinant Escherichia coli BL21 (DE3) cells. Growth phase analysis of the self-assembly process revealed that rSlpC₄₉₈₋₁,₁₇₆ mainly formed monolayer cylinders in the early stage (0.5 and 1 h induction of expression), but few double-layer or multilayer cylinders were also found with the cells growing, while rSlpC₃₁₋₁,₁₇₆ could formed multilayer cylinders in all the growth stage in the E. coli cells. It is concluded that the deletion of the C-terminal 126 aa or the N-terminal 497 aa did not interfere with the self-assembly process, the fragment (amino acids 278 to 337) is essential for the multilayer cylinder formation in E. coli BL21 (DE3) cells in the early stage and the fragment (amino acids 338 to 497) is related to monolayer cylinder formation. The information is important for further studies on the assembly mechanism of S-layer proteins and forms a basis for further studies concerning surface display and nanobiotechnology.

Fast and easy protocol for the purification of recombinant S‐layer protein for synthetic biology applications

A goal of synthetic biology is to make biological systems easier to engineer. One of the aims is to design, with nanometer-scale precision, biomaterials with well-defined properties. The surface-layer protein SbpA forms 2D arrays naturally on the cell surface of Lysinibacillus sphaericus, but also as the purified protein in solution upon the addition of divalent cations. The high propensity of SbpA to form crystalline arrays, which can be simply controlled by divalent cations, and the possibility to genetically alter the protein, make SbpA an attractive molecule for synthetic biology. To be a useful tool, however, it is important that a simple protocol can be used to produce recombinant wild-type and modified SbpA in large quantities and in a biologically active form. The present study addresses this requirement by introducing a mild and non-denaturing purification protocol to produce milligram quantities of recombinant, active SbpA.

E. coli filament formation induced by heterologous S-layer expression

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 µm x 2.0-6.0 µm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of >100 µm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM® 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process.

Calcium dependent formation of tubular assemblies by recombinant S-layer proteins in vivoand in vitro

Surface layer proteins have the appealing property to self-assemble in nanosizedarrays in solution and on solid substrates. In this work, we characterize theformation of assembly structures of the recombinant surface layer protein SbsC ofGeobacillus stearothermophilus ATTC 12980, which was tagged with enhancedgreen fluorescent protein and expressed in the yeast Saccharomyces cerevisiae.The tubular structures formed by the protein in vivo are retained upon burstingthe cells by osmotic shock; however, their average length is decreased. Duringdialysis, monomers obtained by treatment with chaotropic chemicals recrystallizeagain to form tube-like structures. This process is strictly dependent on calcium(Ca2 + ) ions, with an optimal concentration of 10 mM. Further increase of theCa2 + concentration results in multiple non-productive nucleation points. Wefurther show that the lengths of the S-layer assemblies increase with timeand can be controlled by pH. After 48 h, the average length at pH 9.0 is4.13 µm comparedto 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential ofrecrystallized mSbsC-eGFP structures for nanobiotechnological applications.

English

The S-layer, crystalline arrays of proteinaceous subunits, seems to be a typical surface structure in several lactobacilli species. Due their self-assembly ability to recrystallize into isoporous monolayers in suspension, at liquid-surface interfaces, lipid structures and on solid supports, S-layers were demonstrated to possess a great potential for nanobiotechnological applications. Interest in lactobacilli S-layer has been reinforced by claimed and demonstrated probiotic properties for human and animal consumers. Several lactobacillar S-layer have been found to be involved in adherence to intestinal epithelial cells and to the mammalian extracellular matrix. Due to these observed adhesive properties, the possible therapeutic applications of lactobacillar S-layers have become increasingly of interest, e.g. as targeted antigen delivery vehicles to host tissues. In addition, S-layers may provide superior expression levels and surface density of antigens when compared to other bacterial antigen presentation systems. It has already been demonstrated that S-layer protein subunits can be modified to carry foreign epitopes as a uniform recombinant S-layer on the Lactobacillus cell surface. The adhesion and immunogenic functions of S-layer proteins, combined with the properties of Lactobacillus spp., could lead to new, safe, and stable liposomal particles for drug delivery.

Heterologous expression of the surface-layer-like protein SllB induces the formation of long filaments of Escherichia coli consisting of protein-stabilized outer membrane

Escherichia coli is one of the best studied micro-organisms and is the most widely used host in genetic engineering. The Gram-negative single cells are rod-shaped, and filaments are usually not found. Here, we describe the reproducible formation of elongated E. coli cells. During heterologous expression of the silent surface (S)-layer protein gene sllB from Lysinibacillus sphaericus JG-A12 in E. coli BL21(DE3), the cells were arranged as long chains which were surrounded by highly stable sheaths. These filaments had a length of >100 μm. In the stationary growth phase, microscopic analyses demonstrated the formation of unusually long transparent tube-like structures which were enclosing separate single cells. The tube-like structures were isolated and analysed by SDS-PAGE, infrared-spectroscopy and different microscopic methods in order to identify their unusual composition and structure. The tube-like structures were found to be like outer membranes, containing high levels of proteins and to which the recombinant S-layer proteins were attached. Despite the entire structure being indicative of a disordered cell division, the bacterial cells were highly viable and stable. To our knowledge, this is the first time that the induction of drastic morphological changes in E. coli by the expression of a foreign protein has been reported.

Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish

A recombinant protein for the S-layer protein of Aeromonas hydrophila was produced and its ability to protect common carp Cyprinus carpio L. against six virulent isolates of A. hydrophila was assessed. A group of 120 carp (30–40 g) were vaccinated intra-peritoneally with 0.1 ml of adjuvanted vaccine (30 μg protein per fish). Another group of 120 carp were injected with 0.1 ml of PBS-adjuvant mixture to serve as controls. Twenty fish from each group were challenged with each one of six virulent isolates of A. hydrophila 35 days post-vaccination. The fish were maintained in 12 separate tanks before terminating the experiment at 16 days post-challenge. The relative percentage survival (RPS) for the six isolates of A. hydrophila ranged from 56 to 87%. The difference in survival rate of fish challenged with four of the isolates was statistically significant in vaccinated fish compared to control fish, when analysed using a Chi-square test. The results of the study suggest that the recombinant S-layer protein of A. hydrophila could be useful as a vaccine antigen to protect fish against different isolates of this pathogenic bacterium.

The high‐molecular‐mass amylase (HMMA) of Geobacillus stearothermophilus ATCC 12980 interacts with the cell wall components by virtue of three specific binding regions

The complete nucleotide sequence encoding the high-molecular-mass amylase (HMMA) of Geobacillus stearothermophilus ATCC 12980 was established by PCR techniques. Based on the hmma gene sequence, the full-length rHMMA, four N- or C-terminal rHMMA truncations as well as three C-terminal rHMMA fragments were cloned and heterologously expressed in Escherichia coli. Purified rHMMA forms were used either for affinity studies with the recombinant (r) S-layer protein SbsC (rSbsC), peptidoglycan-containing sacculi (PGS) and pure peptidoglycan (PG) devoid of the secondary cell wall polymer (SCWP), or for surface plasmon resonance (SPR) studies using rSbsC and isolated SCWP. In the C-terminal part of the HMMA, three specific binding regions, one for each cell wall component (rSbsC, SCWP and PG), could be identified. The functionality of the PG-binding domain could be confirmed by replacing the main part of the SCWP-binding domain of an S-layer protein by the PG-binding domain of the HMMA. The present work describes a completely new and highly economic strategy for cell adhesion of an exoenzyme.

The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC

Surface layers (S-layers) comprise the outermost cell envelope component of most archaea and many bacteria. Here we present the structure of the bacterial S-layer protein SbsC from Geobacillus stearothermophilus, showing a very elongated and flexible molecule, with strong and specific binding to the secondary cell wall polymer (SCWP). The crystal structure of rSbsC((31-844)) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. The N-terminal domain exhibits positively charged residues regularly spaced along the putative ligand binding site matching the distance of the negative charges on the extended SCWP. Upon SCWP binding, a considerable stabilization of the N-terminal domain occurs. These findings provide insight into the processes of S-layer attachment to the underlying cell wall and self-assembly, and also accommodate the observed mechanical strength, the polarity of the S-layer, and the pronounced requirement for surface flexibility inherent to cell growth and division.

Surfaces functionalized with self-assembling S-layer fusion proteins for nanobiotechnological applications

The fabrication of supramolecular structures and devices requires molecules that are capable of interlocking in a predictable well defined manner on surfaces required for nanobiotechnological applications. Thus, molecular self-assembly systems which exploit the molecular scale manufacturing precision of biological systems are prime candidates for supramolecular engineering. In this context, crystalline bacterial cell surface layer (S-layer) proteins of prokaryotic organisms represent a unique self-assembly system which can be exploited as patterning element for a biomolecular construction kit involving all major species of biological molecules, for example, glycans such as S-layer-specific heteropolysaccharides, lipids, and nucleic acids. One of the most fascinating properties of native or recombinant S-layer proteins is their capability to self-assemble in suspension (as flat sheets or cylinders), into monomolecular protein lattices on artificial surfaces (e.g. silicon wafers, noble metals, plastics) or on Langmuir lipid films and liposomes. Functional groups (e.g. carboxyl groups, amino or hydroxyl groups) or genetically incorporated functional domains (e.g. streptavidin) are repeated with the periodicity of the S-layer lattice at a distance resembling the lattice constants, leading to regular arrays of bound functional molecules or nanoparticles. Thus, genetically and/or chemically modified S-layer proteins can be exploited as building blocks and templates for generating functional nanostructures at meso- and macroscopic scale for both, life and non-life science applications.

Construction of an S-layer protein exhibiting modified self-assembling properties and enhanced metal binding capacities

The functional S-layer protein gene slfB of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 was cloned as a polymerase chain reaction product into the expression vector pET Lic/Ek 30 and heterologously expressed in Escherichia coli Bl21(DE3). The addition of His tags to the N and C termini enabled the purification of the recombinant protein by Ni-chelating chromatography. The Ni binding capacity of the His-tagged recombinant S-layer protein was compared with that of the wild-type S layer. The inductively coupled plasma mass spectrometry analyses demonstrate a significantly enhanced Ni binding capability of the recombinant protein. In addition, the self-assembling properties of the purified modified S-layer proteins were studied by light microscopy and scanning electron microscopy. Whereas the wild-type S-layer proteins re-assembled into regular cylindric structures, the recombinant S-layer proteins reassembled into regular sheets that formed globular agglomerating structures. The nanoporous structure of the protein meshwork, together with its enhanced Ni binding capacity, makes the recombinant S-layer attractive as a novel self-assembling biological template for the fabrication of metal nanoclusters and construction of nanomaterials that are of technical interest.

Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts – a delivery system for the nontypeable Haemophilus influenzae antigen Omp26

This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.

Surface display of foreign epitopes on the Lactobacillus brevis S-layer.

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.

Molecular Characterization of the S-Layer Gene,sbpA, ofBacillus sphaericusCCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.

Extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new stategy for adjuvant free combination vaccines.

New strategies for combination vaccines based on the extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.