Characterisation of new potential vaccine candidates against infections caused by Staphylococcus aureus

Due to the rapid emergence of S. aureus strains resistant to multiple antibiotics and the therewith increased mortality rates, the development of alternative strategies to prevent and treat S. aureus infections is of great clinical and economical importance. Based on the results concerning both monovalent active and passive immunisation, it is getting obvious that only multivalent vaccine strategies might confer full protection from S. aureus related infections. Furthermore, due to their short term applicability and potential composition of immunoglobulins of different isotypes and functionalities, strategies based on passive immunisation are particularly advantageous. Using an intravenous immunoglobulin preparation (IVIG) as source of naturally occurring S. aureus specific IgGs, a significant inhibition of staphylococcal growth was observed in vitro. Thus, confirming the bacteriostatic effect on S. aureus as observed using human serum in the 1970s. Since this inhibitory effect was not observed upon treatment with IVIG depleted of S. aureus - specific IgGs (dSaIVIG), bacteriostasis is triggered solely by S. aureus specific IgGs. In order to analyse the underlying mechanism, gene expression profiling was conducted, using a S. aureus-seven genome PCR-product microarray. Comparison of IVIG to dSaIVIG treated samples led to the identification of 236 differentially expressed genes over the course of bacteriostasis. In contrast, IVIG compared to PBS treated samples as additional control resulted in 78 genes with altered expression. Only 13 genes were identified by both sets of microarrays, indicating a strong difference between the two applied controls. Moreover, the most prominent signature representing genes related to iron uptake and metabolism was only identified by comparison of IVIG to dSaIVIG samples. qPCR on iron related genes not only verified the microarray results, but also indicated that the iron signature was derived from dSaIVIG, thus not representing the mechanism underlying bacteriostasis. Due to the lack of a reliable signature the mechanism underlying bacteriostasis could not be characterised. Additionally, we aimed to enlarge the repertoire of potential candidates for a polyvalent vaccine. For this purpose a novel subtractive proteomic approach (SUPRA) on anchorless cell wall (ACW) proteins of S. aureus was developed. This method is based on immunodetection of in vivo expressed, immunogenic proteins separated by 2D gelelectrophoresis with either complete IVIG or dSaIVIG. Proteins immunoreactive with IVIG but not, or to a lesserextent using dSaIVIG were identified by MALDI-TOF analysis. SUPRA led to the identification of 37 new potential vaccine candidates among ACW proteins. Three of these, BT1, BT2 and BT3 were characterised in this study. The surface localisation of these antigens was confirmed by flow cytometry using specific antibodies enriched from IVIG. Purified IgGs for each antigen mediated opsonophagocytosis and subsequent opsonophagocytic killing by human neutrophils. However, when used for monovalent immunisation of BalbC mice only BT1 and BT3 conferred significant protection against lethal S. aureus challenge in a murine model of sepsis. Despite the protective potential upon monovalent immunisation a bivalent vaccination using BT1 and BT3 did not exhibit a synergistic protective effect, most likely due to the reduced amount of antigen used for immunisation. Among the six so far investigated vaccine candidates identified by SUPRA, three conferred protection against lethal challenge with S. aureus (hp2160, BT1 and BT3) and two led to a reduction of bacterial load in organs (eno and oxo). Therefore, SUPRA represents a valuable tool for the identification of promising vaccine candidates for subsequent use in a multicomponent vaccine against S. aureus

Proteomics-Based Identification of Anchorless Cell Wall Proteins as Vaccine Candidates against Staphylococcus aureus▿ †

Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization—time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10- to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification

NOX2 Deficiency Permits Sustained Survival of S. aureus in Macrophages and Contributes to Severity of Infection

Although the crucial role of professional phagocytes for the clearance of S. aureus infections is well-established, several studies indicate an adverse role of leukocytes in the dissemination of S. aureus during infection. Since only little is known about macrophages in this context, we analyzed the role of macrophages, and in particular reactive oxygen species deficiency, for the seeding of S. aureus metastases. Infection of bone marrow-derived macrophages (BMDM) with S. aureus revealed that NADPH oxidase 2 (NOX2-) deficient, but not NOX1- or NOX4-deficient, BMDM failed to clear intracellular S. aureus. Despite of larger intracellular bacterial burden, NOX2-deficient BMDM showed significantly improved survival. Intravenous injection of mice with in vitro-infected BMDMs carrying intracellular viable S. aureus led to higher bacterial loads in kidney and liver of mice compared to injection with plain S. aureus. An even higher frequency of liver abscesses was observed in mice infected with S. aureus-loaded nox2(-/-) BMDM. Thus, the improved intracellular survival of S. aureus and improved viability of NOX2-deficient BMDM is associated with an aggravated metastatic dissemination of S. aureus infection. A combination of vancomycin and the intracellularly active antibiotic rifampicin led to complete elimination of S. aureus from liver within 48 h, which was not achieved with vancomycin treatment alone, underscoring the impact of intracellular S. aureus on the course of disease. The results of our study indicate that intracellular S. aureus carried by macrophages are sufficient to establish a systemic infection. This suggests the inclusion of intracellularly active antibiotics in the therapeutic regimen of invasive S. aureus infections, especially in patients with NADPH oxidase deficiencies such as chronic granulomatous disease

Murine Epidermal Ceramide Synthase 4 Is a Key Regulator of Skin Barrier Homeostasis

Epidermal barrier dysfunction is associated with a wide range of highly prevalent inflammatory skin diseases. However, the molecular processes that drive epidermal barrier maintenance are still largely unknown. Here, using quantitative proteomics, lipidomics, and mouse genetics, we characterize epidermal barrier maintenance versus a newly established barrier and functionally identify differential ceramide synthase 4 protein expression as one key difference. We show that epidermal loss of ceramide synthase 4 first disturbs epidermal lipid metabolism and adult epidermal barrier function, ultimately resulting in chronic skin barrier disease characterized by acanthosis, hyperkeratosis, and immune cell accumulation. Importantly, prolonged barrier dysfunction induced by loss of ceramide synthase 4 induced a barrier repair response that largely recapitulates molecular programs of barrier establishment. Collectively, this study provides an unbiased temporal proteomic characterization of barrier maintenance and disturbed homeostasis and shows that lipid homeostasis is essential to maintain adult skin barrier function to prevent disease

Epitope-specific immunity against Staphylococcus aureus coproporphyrinogen III oxidase

Staphylococcus aureus represents a serious infectious threat to global public health and a vaccine against S. aureus represents an unmet medical need. We here characterise two S. aureus vaccine candidates, coproporphyrinogen III oxidase (CgoX) and triose phosphate isomerase (TPI), which fulfil essential housekeeping functions in heme synthesis and glycolysis, respectively. Immunisation with rCgoX and rTPI elicited protective immunity against S. aureus bacteremia. Two monoclonal antibodies (mAb), CgoX-D3 and TPI-H8, raised against CgoX and TPI, efficiently provided protection against S. aureus infection. MAb-CgoX-D3 recognised a linear epitope spanning 12 amino acids (aa), whereas TPI-H8 recognised a larger discontinuous epitope. The CgoX-D3 epitope conjugated to BSA elicited a strong, protective immune response against S. aureus infection. The CgoX-D3 epitope is highly conserved in clinical S. aureus isolates, indicating its potential wide usability against S. aureus infection. These data suggest that immunofocusing through epitope-based immunisation constitutes a strategy for the development of a S. aureus vaccine with greater efficacy and better safety profile

A tissue-specific screen of ceramide expression in aged mice identifies ceramide synthase-1 and ceramide synthase-5 as potential regulators of fiber size and strength in skeletal muscle

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi-organ investigation of age-associated changes of ceramide species, a unique and distinctive change pattern of C-16:0 and C-18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C-16:0 and C-18:0 acyl chains, respectively, was down-regulated in skeletal muscle of aged mice. Similarly, an age-dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1(Delta/Delta) and CerS5(Delta/Delta) knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1- and CerS5-deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age-related muscle wasting disorders in humans