Intranasal delivery of Salmonella OMVs decorated with Chlamydia trachomatis antigens induces specific local and systemic immune responses.

Chlamydia trachomatis is an obligate intracellular pathogen responsible for the most prevalent bacterial sexually transmitted disease globally. The high prevalence of chlamydial infections underscores the urgent need for licensed and effective vaccines to prevent transmission in populations. Bacterial outer membrane vesicles (OMVs) have emerged as promising mucosal vaccine carriers due to their inherent adjuvant properties and the ability to display heterologous antigens. In this proof-of-concept study, we evaluated the immunogenicity of Salmonella OMVs decorated with C. trachomatis MOMP-derived CTH522 or HtrA antigens in mice. Following a prime-boost intranasal vaccination approach, two OMV-based C. trachomatis vaccines elicited significant humoral responses specific to the antigens in both systemic and vaginal compartments. Furthermore, we demonstrated strong antigen-specific IFN-γ and IL17a responses in splenocytes and cervical lymph node cells of vaccinated mice, indicating CD4+ Th1 and Th17 biased immune responses. Notably, the OMV-CTH522 vaccine also induced the production of spleen-derived CD8+ T cells expressing IFN-γ. In conclusion, these results highlight the potential of OMV-based C. trachomatis vaccines for successful use in future challenge studies and demonstrate the suitability of our modular OMV platform for intranasal vaccine applications.

Heterologous Display of Chlamydia trachomatis PmpD Passenger at the Surface of Salmonella OMVs.

Chlamydia trachomatis is the bacterial pathogen that causes most cases of sexually transmitted diseases annually. To combat the global spread of asymptomatic infection, development of effective (mucosal) vaccines that offer both systemic and local immune responses is considered a high priority. In this study, we explored the expression of C. trachomatis full-length (FL) PmpD, as well as truncated PmpD passenger constructs fused to a "display" autotransporter (AT) hemoglobin protease (HbpD) and studied their inclusion into outer membrane vesicles (OMVs) of Escherichia coli and Salmonella Typhimurium. OMVs are considered safe vaccine vectors well-suited for mucosal delivery. By using E. coli AT HbpD-fusions of chimeric constructs we improved surface display and successfully generated Salmonella OMVs decorated with a secreted and immunogenic PmpD passenger fragment (aa68-629) to 13% of the total protein content. Next, we investigated whether a similar chimeric surface display strategy could be applied to other AT antigens, i.e., secreted fragments of Prn (aa35-350) of Bordetella pertussis and VacA (aa65-377) of Helicobacter pylori. The data provided information on the complexity of heterologous expression of AT antigens at the OMV surface and suggested that optimal expression strategies should be developed on an antigen-to-antigen basis.

Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane.

A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein (Ct-MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive candidate for subunit-based vaccine formulations. Unfortunately, Ct-MOMP is difficult to express in its native structure in the E. coli outer membrane (OM). Here, by co-expression of the Bam complex, we improved the expression and localization of recombinant Ct-MOMP in the E. coli OM. Under these conditions, recombinant Ct-MOMP appeared to assemble into a ß-barrel conformation and express domains at the cell surface indicative of correct folding. The data indicate that limited availability of the Bam complex can be a bottleneck for the production of heterologous OM vaccine antigens, information that is also relevant for strategies aimed at producing recombinant OMV-based vaccines.

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type and Delta variants.

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titres of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titres in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type and Delta variants.

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster ( Mesocricetus auratus ) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

Overproducing the BAM complex improves secretion of difficult-to-secrete recombinant autotransporter chimeras.

Monomeric autotransporters have been used extensively to transport recombinant proteins or protein domains to the cell surface of Gram-negative bacteria amongst others for antigen display. Genetic fusion of such antigens into autotransporters has yielded chimeras that can be used for vaccination purposes. However, not every fusion construct is transported efficiently across the cell envelope. Problems occur in particular when the fused antigen attains a relatively complex structure in the periplasm, prior to its translocation across the outer membrane. The latter step requires the interaction with periplasmic chaperones and the BAM (ß-barrel assembly machinery) complex in the outer membrane. This complex catalyzes insertion and folding of ß-barrel outer membrane proteins, including the ß-barrel domain of autotransporters. Here, we investigated whether the availability of periplasmic chaperones or the BAM complex is a limiting factor for the surface localization of difficult-to-secrete chimeric autotransporter constructs. Indeed, we found that overproduction of in particular the BAM complex, increases surface display of difficult-to-secrete chimeras. Importantly, this beneficial effect appeared to be generic not only for a number of monomeric autotransporter fusions but also for fusions to trimeric autotransporters. Therefore, overproduction of BAM might be an attractive strategy to improve the production of recombinant autotransporter constructs.

Exploring metal availability in the natural niche of Streptococcus pneumoniae to discover potential vaccine antigens.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.

Combining Protein Ligation Systems to Expand the Functionality of Semi-Synthetic Outer Membrane Vesicle Nanoparticles.

Bacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple SpyCatcher-equipped fusion proteins. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics.

Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8+ T cells.

Outer membrane vesicles (OMVs) are vesicular nano-particles produced by Gram-negative bacteria that are recently being explored as vaccine vector. The fact that OMVs can be efficiently produced by a hypervesiculating Salmonella typhimurium strain, are packed with naturally-occurring adjuvants like lipopolysaccharides (LPS), and can be engineered to express any antigen of choice, makes them ideal candidates for vaccinology. However, it is unclear whether OMVs induce dendritic cell (DC)-mediated antigen-specific T cell responses and how immune activation is coordinated. Here, we show that OMVs induce maturation of human monocyte-derived DCs, murine bone marrow-derived DCs and CD11c+ splenic DCs. OMV-induced DC maturation was dependent on the presence of LPS and the myeloid differentiation primary response 88 (MyD88) adapter protein downstream of toll-like receptor signaling. Importantly, OMVs did not induce pyroptosis/cell death, but instead provided a significant survival benefit in DCs over non-stimulated DCs. OMVs displaying a sizeable ovalbumin fragment at the vesicle surface induce potent cross-presentation in BMDCs and splenic CD11c+ DCs to OTI CD8+ T cells, dependent on MyD88. Interestingly, the OMV-induced preference to cross-presentation was only partly dependent on the BATF3-dependent CD8a+ professional cross-presenting DC subset. Hence, an OMV-specific programming of DCs that induces maturation and provides a survival benefit for antigen presentation to T cells is identified. Additionally, for the first time, antigen-specific and potent cross-presentation of antigen-loaded OMVs to CD8+ T cells is demonstrated. These data provide mechanistical insight into the processes needed for the DC-mediated cross-presentation of OMV-derived antigens to CD8+ T cells with implications for therapeutic strategies. STATEMENT OF SIGNIFICANCE: Bacteria are primarily known to cause disease. However, recent research has focused on using engineered bacteria and its byproducts as vaccine agents. In particular, outer membrane vesicles (OMVs) have shown promise in eliciting potent immunity against a variety of pathogens. While most vaccines rely on the generation of antibodies, the control of viral replication and tumor growth is driven by cytotoxic CD8+ T cells induced by dendritic cells (DCs). As such, there is a dire need for vaccines that use DCs to elicit CD8+ T cell responses. Studying OMVs as engineered biomaterial and its interaction with DCs allows tailored induction of immunity. This study includes important findings on OMV-dendritic cell interactions and for the first time supports OMVs as vehicles for the induction of antigen-specific CD8+ T cell responses. Additionally, important mechanistical insight into the molecular pathways needed for the cross-presentation of OMV-derived antigens to CD8+ T cells is provided.

Inhibition of autotransporter biogenesis by small molecules.

Disarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram-negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane-based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence-based high-throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen-43. VUF15259 also impairs ß-barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam-complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug-like compounds.

Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag.

Whereas, bacterial inclusion bodies (IBs) for long were regarded as undesirable aggregates emerging during recombinant protein production, they currently receive attention as promising nanoparticulate biomaterials with diverse applications in biotechnology and biomedicine. We previously identified ssTorA, a signal sequence that normally directs protein export via the Tat pathway in E. coli, as a tag that induces the accumulation of fused proteins into IBs under overexpression conditions. Here, we used targeted mutagenesis to identify features and motifs being either critical or dispensable for IB formation. We found that IB formation is neither related to the function of ssTorA as a Tat-signal sequence nor is it a general feature of this family of signal sequences. IB formation was inhibited by co-overexpression of ssTorA binding chaperones TorD and DnaK and by amino acid substitutions that affect the propensity of ssTorA to form an α-helix. Systematic deletion experiments identified a minimal region of ssTorA required for IB formation in the center of the signal sequence. Unbiased genetic screening of a library of randomly mutagenized ssTorA sequences for reduced aggregation properties allowed us to pinpoint residues that are critical to sustain insoluble expression. Together, the data point to possible mechanisms for the aggregation of ssTorA fusions. Additionally, they led to the design of a tag with superior IB-formation properties compared to the original ssTorA sequence.

On display: autotransporter secretion and application.

The classical monomeric autotransporters are ubiquitously used by Gram-negative bacteria to export virulence and colonization factors to their cell surface or into their surroundings. They are expressed as monomeric proteins that pass the inner and outer membrane in two consecutive steps facilitated by the Sec translocon and the Bam complex, respectively. In this mini-review we discuss how autotransporters translocate their secreted functional domains across the outer membrane. We highlight the interactions with the Bam complex and discuss how specific features of the recently solved structure of Bam lead to a mechanistic model for autotransporter secretion. Furthermore, the autotransporter secretion pathway is the system of choice for surface display of heterologous proteins for biotechnical and biomedical purposes. We summarize recent advances in the application of autotransporters with a focus on outer membrane vesicle vaccine development and discuss its limitations in secreting more complex heterologous proteins. Finally, we present an exciting new technology to circumvent secretion limitations by ligating heterologous proteins of interest to autotransporters that are displayed on the cell surface.

Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation.

The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

Comparing autotransporter ß-domain configurations for their capacity to secrete heterologous proteins to the cell surface.

Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the ß-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight ß-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight ß-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the ß-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp ß-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

Th17-Mediated Cross Protection against Pneumococcal Carriage by Vaccination with a Variable Antigen.

Serotype-specific protection against Streptococcus pneumoniae is an important limitation of the current polysaccharide-based vaccines. To prevent serotype replacement, reduce transmission, and limit the emergence of new variants, it is essential to induce broad protection and restrict pneumococcal colonization. In this study, we used a prototype vaccine formulation consisting of lipopolysaccharide (LPS)-detoxified outer membrane vesicles (OMVs) from Salmonella enterica serovar Typhimurium displaying the variable N terminus of PspA (α1α2) for intranasal vaccination, which induced strong Th17 immunity associated with a substantial reduction of pneumococcal colonization. Despite the variable nature of this protein, a common major histocompatibility complex class (MHC-II) epitope was identified, based on in silico prediction combined with ex vivo screening, and was essential for interleukin-17 A (IL-17A)-mediated cross-reactivity and associated with cross protection. Based on 1,352 PspA sequences derived from a pneumococcal carriage cohort, this OMV-based vaccine formulation containing a single α1α2 type was estimated to cover 19.1% of strains, illustrating the potential of Th17-mediated cross protection.

Application of an E. coli signal sequence as a versatile inclusion body tag.

BACKGROUND: Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. RESULTS: When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. CONCLUSIONS: We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

Highly conserved nucleotide phosphatase essential for membrane lipid homeostasis in Streptococcus pneumoniae.

Proteins belonging to the DHH family, a member of the phosphoesterase superfamily, are produced by most bacterial species. While some of these proteins are well studied in Bacillus subtilis and Escherichia coli, their functions in Streptococcus pneumoniae remain unclear. Recently, the highly conserved DHH subfamily 1 protein PapP (SP1298) has been reported to play an important role in virulence. Here, we provide a plausible explanation for the attenuated virulence of the papP mutant. Recombinant PapP specifically hydrolyzed nucleotides 3'-phosphoadenosine-5'-phosphate (pAp) and 5'-phosphoadenylyl-(3'->5')-adenosine (pApA). Deletion of papP, potentially leading to pAp/pApA accumulation, resulted in morphological defects and mis-localization of several cell division proteins. Incubation with both polar solvent and detergent led to robust killing of the papP mutant, indicating that membrane integrity is strongly affected. This is in line with previous studies showing that pAp inhibits the ACP synthase, an essential enzyme involved in lipid precursor production. Remarkably, partial inactivation of the lipid biosynthesis pathway, by inhibition of FabF or depletion of FabH, phenocopied the papP mutant. We conclude that pAp and pApA phosphatase activity of PapP is required for maintenance of membrane lipid homeostasis providing an explanation how inactivation of this protein may attenuate pneumococcal virulence.

Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization.

Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if presented at the vesicle surface and thus optimally exposed to the immune system. In this study, we explored the feasibility of our recently developed autotransporter Hbp platform, designed to efficiently and simultaneously display multiple antigens at the surface of bacterial OMVs, for vaccine development. Using two Streptococcus pneumoniae proteins as model antigens, we showed that intranasally administered Salmonella OMVs displaying high levels of antigens at the surface induced strong protection in a murine model of pneumococcal colonization, without the need for a mucosal adjuvant. Importantly, reduction in bacterial recovery from the nasal cavity was correlated with local production of antigen-specific IL-17A. Furthermore, the protective efficacy and the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at increased concentrations of the displayed antigen. This discovery highlights the importance of an adequate antigen expression system for development of recombinant OMV vaccines. In conclusion, our findings demonstrate the suitability of the Hbp platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to develop a broadly protective mucosal pneumococcal vaccine.

Autotransporter-based antigen display in bacterial ghosts.

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.