Synthesis and Evaluation of a Conjugate Vaccine Composed of Staphylococcus aureus Poly-N-Acetyl-Glucosamine and Clumping Factor A

The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA) and deacetylated poly-N-β-(1-6)-acetyl-glucosamine (dPNAG). Mice immunized with 1, 5 or 10 μg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA) goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to augment the OPK and protective activities of antibodies to the PNAG cell surface polysaccharide.     

Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens of increasing importance due to the dissemination of antibiotic-resistant strains. Evidence suggests that the ability to form matrix-encased biofilms contributes to the pathogenesis of S. aureus and S. epidermidis. In this study, we investigated the functions of two staphylococcal biofilm matrix polymers: poly-N-acetylglucosamine surface polysaccharide (PNAG) and extracellular DNA (ecDNA). We measured the ability of a PNAG-degrading enzyme (dispersin B) and DNase I to inhibit biofilm formation, detach preformed biofilms, and sensitize biofilms to killing by the cationic detergent cetylpyridinium chloride (CPC) in a 96-well microtiter plate assay. When added to growth medium, both dispersin B and DNase I inhibited biofilm formation by both S. aureus and S. epidermidis. Dispersin B detached preformed S. epidermidis biofilms but not S. aureus biofilms, whereas DNase I detached S. aureus biofilms but not S. epidermidis biofilms. Similarly, dispersin B sensitized S. epidermidis biofilms to CPC killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. We concluded that PNAG and ecDNA play fundamentally different structural roles in S. aureus and S. epidermidis biofilms.     

Hydrogen Peroxide-Mediated Interference Competition by Streptococcus pneumoniae Has No Significant Effect on Staphylococcus aureus Nasal Colonization of Neonatal Rats

It has been proposed that the relative scarcity of Staphylococcus aureus and Streptococcus pneumoniae cocolonization in the nasopharynxes of humans can be attributed to hydrogen peroxide-mediated interference competition. Previously it has been shown in vitro that H₂O₂ produced by S. pneumoniae is bactericidal to S. aureus. To ascertain whether H₂O₂ has this inhibitory effect in the nasal passages of neonatal rats, colonization experiments were performed with S. aureus and S. pneumoniae. The results of these experiments with neonatal rats are inconsistent with the hypothesis that hydrogen peroxide-mediated killing of S. aureus by S. pneumoniae is responsible for the relative scarcity of cocolonization by these bacteria. In mixed-inoculum colonization experiments and experiments where S. aureus invaded the nasopharynxes of rats with established S. pneumoniae populations, the density of S. aureus did not differ whether the S. pneumoniae strain was H₂O₂ secreting or non-H₂O₂ secreting (SpxB). Moreover, the advantage of catalase production by S. aureus in competition with a non-catalase-producing strain (KatA) during nasal colonization was no greater in the presence of H₂O₂-producing S. pneumoniae than in the presence of non-H₂O₂-producing S. pneumoniae.     

Synthesis of Staphylococcus aureus type 5 capsular polysaccharide repeating unit using novel l-FucNAc and d-FucNAc synthons and immunochemical evaluation

Staphylococcus aureus is a major cause of nosocomial infections. Glycoconjugates of type 5 and 8 capsular polysaccharides have been investigated for vaccine application. The proposed structure of type 5 polysaccharide is: →4-β-d-ManNAcA-(1→4)-α-l-FucNAc(3OAc)-(1→3)-β-d-FucNAc-(1→. The stereocontrolled insertion of these three glycosydic bonds is a real synthetic challenge. In the present paper we report the preparation of two novel versatile l- and d-fucosamine synthons from commercially available starting materials. In addition we applied the two building blocks to the synthesis of type 5 trisaccharide repeating unit. The immunochemical properties of the synthesized trisaccharide were assessed by competitive ELISA and by immunodot blot analysis using sera of mice immunized with type 5 polysaccharide conjugated to CRM₁₉₇. The results suggest that although the type 5 S. aureus trisaccharide is recognized by specific anti polysaccharide antibodies in dot blot, structures longer than the trisaccharide may be needed in order to significantly compete with the native type 5 polymer in the binding with sera from mice immunized with S. aureus type 5 polysaccharide–CRM₁₉₇ conjugate.     

Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

A different path: Revealing the function of staphylococcal proteins in biofilm formation

Staphylococcus aureus and Staphylococcus epidermidis cause dangerous and difficult to treat medical device-related infections through their ability to form biofilms. Extracellular poly-N-acetylglucosamine (PNAG) facilitates biofilm formation and is a vaccination target, yet details of its biosynthesis by the icaADBC gene products is limited. IcaC is the proposed transporter for PNAG export, however a comparison of the Ica proteins to homologous exo-polysaccharide synthases suggests that the common IcaAD protein components both synthesise and transport the PNAG. The limited distribution of icaC to the Staphylococcaceae and its membership of a family of membrane-bound acyltransferases, leads us to suggest that IcaC is responsible for the known O-succinylation of PNAG that occurs in staphylococci, identifying a potentially new therapeutic target specific for these bacteria.     

Staphylococcus aureus Colonization of the Mouse Gastrointestinal Tract Is Modulated by Wall Teichoic Acid,Capsule, and Surface Proteins

Staphylococcus aureus colonizes the nose, throat, skin, and gastrointestinal (GI) tract of humans. GI carriage of S. aureus is difficult to eradicate and has been shown to facilitate the transmission of the bacterium among individuals. Although staphylococcal colonization of the GI tract is asymptomatic, it increases the likelihood of infection, particularly skin and soft tissue infections caused by USA300 isolates. We established a mouse model of persistent S. aureus GI colonization and characterized the impact of selected surface antigens on colonization. In competition experiments, an acapsular mutant colonized better than the parental strain Newman, whereas mutants defective in sortase A and clumping factor A showed impaired ability to colonize the GI tract. Mutants lacking protein A, clumping factor B, poly-N-acetyl glucosamine, or SdrCDE showed no defect in colonization. An S. aureus wall teichoic acid (WTA) mutant (ΔtagO) failed to colonize the mouse nose or GI tract, and the tagO and clfA mutants showed reduced adherence in vitro to intestinal epithelial cells. The tagO mutant was recovered in lower numbers than the wild type strain in the murine stomach and duodenum 1 h after inoculation. This reduced fitness correlated with the in vitro susceptibility of the tagO mutant to bile salts, proteases, and a gut-associated defensin. Newman ΔtagO showed enhanced susceptibility to autolysis, and an autolysin (atl) tagO double mutant abrogated this phenotype. However, the atl tagO mutant did not survive better in the mouse GI tract than the tagO mutant. Our results indicate that the failure of the tagO mutant to colonize the GI tract correlates with its poor adherence and susceptibility to bactericidal factors within the mouse gut, but not to enhanced activity of its major autolysin.     

Phase Variation of Poly-N-Acetylglucosamine Expression in Staphylococcus aureus

Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1–6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenic icaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production.     

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG₁ and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.     

Evaluation of Genetically Inactivated Alpha Toxin for Protection in Multiple Mouse Models of Staphylococcus aureus Infection

Staphylococcus aureus is a major human pathogen and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. While S. aureus protective antigens have been identified in the literature, the majority have only been tested in a single animal model of disease. We wished to evaluate the ability of one S. aureus vaccine antigen to protect in multiple mouse models, thus assessing whether protection in one model translates to protection in other models encompassing the full breadth of infections the pathogen can cause. We chose to focus on genetically inactivated alpha toxin mutant HlaH35L. We evaluated the protection afforded by this antigen in three models of infection using the same vaccine dose, regimen, route of immunization, adjuvant, and challenge strain. When mice were immunized with HlaH35L and challenged via a skin and soft tissue infection model, HlaH35L immunization led to a less severe infection and decreased S. aureus levels at the challenge site when compared to controls. Challenge of HlaH35L-immunized mice using a systemic infection model resulted in a limited, but statistically significant decrease in bacterial colonization as compared to that observed with control mice. In contrast, in a prosthetic implant model of chronic biofilm infection, there was no significant difference in bacterial levels when compared to controls. These results demonstrate that vaccines may confer protection against one form of S. aureus disease without conferring protection against other disease presentations and thus underscore a significant challenge in S. aureus vaccine development.     

Synthetic Teichoic Acid Conjugate Vaccine against Nosocomial Gram-Positive Bacteria

Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria.     

Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from β-hCG subunit

To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the β-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ∼1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the β5, β9 and β8 BCEs of β-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional β5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of β-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.     

Functional basis for complement evasion by staphylococcal superantigen‐like 7

The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen‐like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA‐FcαRI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b‐9‐mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal‐induced C5a‐mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA‐binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a‐induced phagocytosis of S. aureus and oxidative burst in an in vitro whole‐blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a‐driven influx of neutrophils in mouse peritoneum.     

Identification of Poly-N-acetylglucosamine as a Major Polysaccharide Component of the Bacillus subtilis Biofilm Matrix

Bacillus subtilis is intensively studied as a model organism for the development of bacterial biofilms or pellicles. A key component is currently undefined exopolysaccharides produced from proteins encoded by genes within the eps locus. Within this locus are four genes, epsHIJK, known to be essential for pellicle formation. We show they encode proteins synthesizing the broadly expressed microbial carbohydrate poly-N-acetylglucosamine (PNAG). PNAG was present in both pellicle and planktonic wild-type B. subtilis cells and in strains with deletions in the epsA–G and -L–O genes but not in strains deleted for epsH–K. Cloning of the B. subtilis epsH–K genes into Escherichia coli with in-frame deletions in the PNAG biosynthetic genes pgaA–D, respectively, restored PNAG production in E. coli. Cloning the entire B. subtilis epsHIJK locus into pga-deleted E. coli, Klebsiella pneumoniae, or alginate-negative Pseudomonas aeruginosa restored or conferred PNAG production. Bioinformatic and structural predictions of the EpsHIJK proteins suggest EpsH and EpsJ are glycosyltransferases (GT) with a GT-A fold; EpsI is a GT with a GT-B fold, and EpsK is an α-helical membrane transporter. B. subtilis, E. coli, and pga-deleted E. coli carrying the epsHIJK genes on a plasmid were all susceptible to opsonic killing by antibodies to PNAG. The immunochemical and genetic data identify the genes and proteins used by B. subtilis to produce PNAG as a significant carbohydrate factor essential for pellicle formation.     

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM₁₉₇ carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.     

Structurally Designed Attenuated Subunit Vaccines for S. aureus LukS-PV and LukF-PV Confer Protection in a Mouse Bacteremia Model

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.