Structure of the staphylococcal enterotoxin B vaccine candidate S19 showing eliminated superantigen activity.

Four mutations (N23A, Y90A, R110A and F177A) were introduced into S19, a vaccine candidate for staphylococcal enterotoxin B (SEB), resulting in a lower binding affinity towards the T-cell receptor beta chain (TCB) and reducing its superantigen activity. The structure of S19 was solved and was superposed on the native or complex structure of SEB. In the superposition model, mutations that were introduced seemed to reduce the number of hydrogen bonds at the SEB-TCB interface. S19 also displayed an unexpected structural change around the flexible-loop region owing to the Y90A mutation. This local structural change provided evidence that the mutated form of S19 could have a lower affinity for major histocompatibility complex (MHC) class II than wild-type SEB.

Immunoinformatics analysis and in silico designing of a novel multi-epitope peptide vaccine against Staphylococcus aureus.

Staphylococcus aureus is a pathogen that causes a variety of infections in humans. Methicillin-resistant S. aureus, which is an antibiotic-resistant form, is responsible for nosocomial staphylococcal infections, whose frequency is increasing in healthy people. Thereby, the development of novel techniques is required to overcome this bacterial infection. In this context, the use of vaccines to control infections is an appropriate alternative. In this study, immunoinformatics analysis is used on three antigenic determinants as vaccine candidates, and a novel multi-epitope vaccine is designed to induce cellular, humoral, and innate immune responses against S. aureus. Alpha-enolase, clumping factor A, and iron surface determinant B were selected as the protective antigens; and phenol-soluble modulin alpha 4was applied as the adjuvant. Epitopes identification was done for each antigen using various immunoinformatics servers. Moreover, the tertiary structure of our protein vaccine was predicted and validated. Subsequently, the best-modeled protein structure was used for the refinement process. There fined model was then applied for docking studies with Toll-like receptor 2 (TLR2).In the next step, molecular dynamics (MD) simulation was used to evaluate the stability of vaccine molecule and TLR2-vaccine complex. The high ranked epitopes were selected from the mentioned antigens. The selected epitopes and the adjuvant were fused together by proper linkers. Then, the modeled protein structure was selected and validated. Validation results indicated that the initial model needs refinement. After a refinement process, the final model was generated. Finally, the best-docked model of vaccine and TLR2 complex was selected. In this research, we attempted to design an efficient subunit vaccine, which could stimulate humoral and cellular immune responses. Therefore, we expect that our designed vaccine could defeat antibiotic-resistant staphylococcal infections.

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

Immunization with heat-inactivated Staphylococcus aureus induced an antibody response mediated by IgG1 and IgG2 in patients with recurrent tonsillitis.

Currently Staphylococcus aureus is the predominant pathogen isolated from the respiratory tract of patients with recurrent tonsillitis. Because of an increase in multi-drug resistant strains of S. aureus, there is a pressing need for effective treatments and preventive approaches to reduce the risk of invasive and life-threatening infections. A preventive vaccine against S. aureus would have a tremendous clinical impact. However, multiple clinical trials have failed to identify an agent that can induce protective responses. Most trials have been based on subunit vaccines using one or a few purified antigens, which may not be enough to confer protection. Here, the impact of a whole-cell vaccine comprised of heat-inactivated S. aureus was investigated in patients with RT. The vaccine was well tolerated and had no significant local or systemic reactions. Immunization with heat-inactivated S. aureus elicited a significant antibody response characterized by production of IgG1 and IgG2 antibodies and, to a lesser extent, of IgA antibodies. Notably, this response was associated with an important decrease in the incidence of tonsillitis and bacterial colonization of the oropharyngeal mucosa. Our results show that whole-cell inactivated S. aureus is safe and capable of evoking specific antibody responses in patients with recurrent tonsillitis.

Bacterial protoplast-derived nanovesicles as vaccine delivery system against bacterial infection.

The notion that widespread infectious diseases could be best managed by developing potent, adjuvant-free vaccines has resulted in the use of various biological immune-stimulating components as new vaccine candidates. Recently, extracellular vesicles, also known as exosomes and microvesicles in mammalian cells and outer membrane vesicles in Gram-negative bacteria, have gained attention for the next generation vaccine. However, the more invasive and effective the vaccine is in delivery, the more risk it holds for severe immune toxicity. Here, in optimizing the current vaccine delivery system, we designed bacterial protoplast-derived nanovesicles (PDNVs), depleted of toxic outer membrane components to generate a universal adjuvant-free vaccine delivery system. These PDNVs exhibited significantly higher productivity and safety than the currently used vaccine delivery vehicles and induced strong antigen-specific humoral and cellular immune responses. Moreover, immunization with PDNVs loaded with bacterial antigens conferred effective protection against bacterial sepsis in mice. These nonliving nanovesicles derived from bacterial protoplast open up a new avenue for the creation of next generation, adjuvant-free, less toxic vaccines to be used to prevent infectious diseases.

Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.

[Influence of Staphylococcus vaccine on functional activity of antigen-presenting cells].

AIM: Study the influence of staphylococcus vaccine on functional activity of antigen-presenting cells. MATERIALS AND METHODS: Mice intraperitoneally received 500 µg of "Staphylovac" vaccine. Phagocytic activity of peritoneal macrophages against Staphylococcus aureus 1991 was determined in animals at various time intervals. Phagocytic index (PI) and phagocytic number (PN) in smears made at 30 and 60 minutes of incubation were calculated. Dendritic cells (DC) were obtained from bone marrow precursors during cultivation with 20 ng/ml GM-CSF and 20 ng/ml IL-4 (BioSource International Inc., Belgium). At day 6 of incubation staphylococcus vaccine (50 µg/ml) was added to immature cells for induction pf DC maturation. DC phenotype evaluation was carried out by flow cytometry using monoclonal antibodies against cell antigens (Beckman Culter, USA). RESULTS: PI at 30 and 60 minutes of incubation increased by 0.12 - 1.4 times and 1.11 - 1.52 times, respectively, compared with control. PN at 30 minutes of incubation of cells with microbial suspension increased from 8.6 to 11.4% against 5.9% in control, at 60 minutes of incubation--from 7.7 to 8.1% against 5.1% in control. In DC culture during their incubation with the vaccine, content of cells with expression of intercellular adhesion marker CD38, antigen presenting marker MHCII and DC terminal differentiation marker CD83 increased. Expression of CD34 and CD14 was also noted, that may give evidence on partial direction of cell differentiation to macrophages. CONCLUSION: "Staphylovac" vaccine during intraperitoneal administrationt to mice had activating influence on functional activity of antigen-presenting cells and peritoneal macrophages.

[Study of protective activivty of "Staphylovac-2" vaccine].

AIM: Study the protective properties of "Staphylovac-2" vaccinie. MATERIALS AND METHODS: Samples of the vaccine manufactured by SPA "Microgen" based on the developed technology were studied in balb/c mice during 3- and 6-fold immunization schemes. Protective activity of the preparation was determined in experiments with active and passive protection during intraperitoneal infection, seeding of the causative agent from spleen and kidneys during intravenous infection, of animals. RESULTS: In experiments with active protection of mice for both 3- and 6-fold immunization schemes, a significant protective activity of the studied series was determined, compared with the control group of mice. Sera obtained after animal immunization (rabbits, mice) by staphylococcus vaccine had protective properties. A reduction of spleen and kidneys seeding by Staphylococcus aureus in immunized mice compared with the control group was detected in the model of generalized staphylococci infection. CONCLUSION: The preclinical studies carried out with the "Staphylovac-2" vaccine, developed baed on the complex of protective staplylococci antigens, have confirmed the high protective activity of the preparation.

Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2.

Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. In the present paper we explore the structure and function of FhuD2 (ferric-hydroxamate uptake D2), a staphylococcal surface lipoprotein mediating iron uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2-ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully iron(III)-dependent. We determined the X-ray crystallographic structure of ligand-bound FhuD2 at 1.9 Å (1 Å=0.1 nm) resolution, revealing the bilobate fold of class III SBPs (solute-binding proteins). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2-siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other class III SBPs might also be exploited as vaccine candidates.

Anti-Staphylococcus aureus immunotherapy: current status and prospects.

Immunological prophylaxis and therapy for Staphylococcus aureus are attractive goals. However, there is nothing currently available in the clinic. Several approaches have failed and more are being undertaken. Here we assess current progress and the potential for success.

Revised structures for the capsular polysaccharides from Staphylococcus aureus Types 5 and 8, components of novel glycoconjugate vaccines.

Glycoconjugate vaccines based on the capsular polysaccharides (CPSs) from Staphylococcus aureus serotypes 5 and 8 conjugated to genetically detoxified recombinant exoprotein A (rEPA) from Pseudomonas aeruginosa have been shown, in Phase 3 clinical trials, to elicit a strong bactericidal immune response in end-stage renal disease patients. Such vaccines have the potential to reduce morbidity and mortality due to methicillin-resistant Staphylococcus aureus (MRSA), a major cause of hospital-acquired infection. The serotype 5 and 8 polysaccharides have been fully characterized by NMR spectroscopy and full structural analyses carried out. Published structures were found incorrect and the revised structures of the repeat units of the two polysaccharides are: [carbohydrate structure: see text]. Resonances indicative of the presence of peptidoglycan were observed in the spectra of both CPSs, consistent with reports that the CPS is covalently linked to peptidoglycan.