Development of a vaccine against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

A vaccine to prevent infections caused by Staphylococcus aureus would have a tremendously beneficial impact on public health. In contrast to typical encapsulated bacterial pathogens, such as Streptococcus pneumoniae, H. influenzae, and Neisseria meningitides, the capsule of S. aureus is not clearly linked to strain virulence in vivo. Furthermore, it is not clear that natural infection caused by S. aureus induces a protective humoral immune response, as does infection caused by typical encapsulated bacteria. Finally, pure B cell or antibody deficiency, in either animal models or in patients, does not predispose to more frequent or more severe S. aureus infections, as it does for infections caused by typical encapsulated bacteria. Rather, primary immune mechanisms necessary for protection against S. aureus infections include professional phagocytes and T lymphocytes (Th17 cells, in particular) which upregulate phagocytic activity. Thus, it is not clear whether an antibody-mediated neutralization of S. aureus virulence factors should be the goal of vaccination. Rather, the selection of antigenic targets which induce potent T cell immune responses that react to the broadest possible array of S. aureus strains should be the focus of antigen selection. Of particular promise is the potential to select antigens which induce both humoral and T cell-mediated immunity in order to generate immune synergy against S. aureus infections. A single-antigen vaccine may achieve this immune synergy. However, multivalent antigens may be more likely to induce both humoral and T cell immunity and to induce protection against a broader array of S. aureus isolates. A number of candidate vaccines are in development, raising the promise that effective vaccines against S. aureus will become available in the not-so-distant future. Possible development programs for such vaccines are discussed.     

Vaccine Protection of Leukopenic Mice against Staphylococcus aureus Bloodstream Infection

The risk for Staphylococcus aureus bloodstream infection (BSI) is increased in immunocompromised individuals, including patients with hematologic malignancy and/or chemotherapy. Due to the emergence of antibiotic-resistant strains, designated methicillin-resistant S. aureus (MRSA), staphylococcal BSI in cancer patients is associated with high mortality; however, neither a protective vaccine nor pathogen-specific immunotherapy is currently available. Here, we modeled staphylococcal BSI in leukopenic CD-1 mice that had been treated with cyclophosphamide, a drug for leukemia and lymphoma patients. Cyclophosphamide-treated mice were highly sensitive to S. aureus BSI and developed infectious lesions lacking immune cell infiltrates. Virulence factors of S. aureus that are key for disease establishment in immunocompetent hosts—α-hemolysin (Hla), iron-regulated surface determinants (IsdA and IsdB), coagulase (Coa), and von Willebrand factor binding protein (vWbp)—are dispensable for the pathogenesis of BSI in leukopenic mice. In contrast, sortase A mutants, which cannot assemble surface proteins, display delayed time to death and increased survival in this model. A vaccine with four surface antigens (ClfA, FnBPB, SdrD, and SpA_KKAA), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI.     

The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus

Vaccination with the recombinant N terminus of the candidal adhesin Als3p (rAls3p-N) protects mice from lethal candidemia. Candidal Als3p also is structurally similar to the microbial surface components recognizing adhesive matrix molecule adhesin, clumping factor, from Staphylococcus aureus. To determine the potential for cross-kingdom vaccination, we immunized mice with rAls3p-N or negative control proteins and challenged them via the tail vein with S. aureus or other gram-positive or gram-negative pathogens. The rAls3p-N vaccine, but neither tetanus toxoid nor a related Als protein (Als5p), improved the survival of vaccinated mice subsequently infected with multiple clinical isolates of S. aureus, including methicillin-resistant strains. The rAls3p-N vaccine was effective against S. aureus when combined with aluminum hydroxide adjuvant. However, the vaccine did not improve the survival of mice infected with other bacterial pathogens. Vaccinated, infected mice mounted moderated type 1 immune responses. T lymphocyte-deficient mice were more susceptible to S. aureus infection, but B lymphocyte-deficient mice were not. Furthermore, T but not B lymphocytes from vaccinated mice mediated protection in adoptive transfer studies. The passive transfer of immune serum was not protective. These data provide the foundation for cross-kingdom vaccine development against S. aureus and Candida, which collectively cause 200,000 bloodstream infections resulting in ≥40,000 to 50,000 deaths annually in the United States alone.     

Exploring <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> pathways to disease for vaccine development

Staphylococcus aureus is a commensal of the human skin or nares and a pathogen that frequently causes skin and soft tissue infections as well as bacteremia and sepsis. Recent efforts in understanding the molecular mechanisms of pathogenesis revealed key virulence strategies of S. aureus in host tissues: bacterial scavenging of iron, induction of coagulation pathways to promote staphylococcal agglutination in the vasculature, and suppression of innate and adaptive immune responses. Advances in all three areas have been explored for opportunities in vaccine design in an effort to identify the critical protective antigens of S. aureus. Human clinical trials with specific subunit vaccines have failed, yet provide important insights for the design of future trials that must address the current epidemic of S. aureus infections with drug-resistant isolates (MRSA, methicillin-resistant S. aureus).     

Recombinant ESAT-6-Like Proteins Provoke Protective Immune Responses against Invasive Staphylococcus aureus Disease in a Murine Model

Staphylococcus aureus is a common pathogen found in the community and in hospitals. Most notably, methicillin-resistant S. aureus is resistant to many antibiotics, which is a growing public health concern. The emergence of drug-resistant strains has prompted the search for alternative treatments, such as immunotherapeutic approaches. To date, most clinical trials of vaccines or of passive immunization against S. aureus have ended in failure. In this study, we investigated two ESAT-6-like proteins secreted by S. aureus, S. aureus EsxA (SaEsxA) and SaEsxB, as possible targets for a vaccine. Mice vaccinated with these purified proteins elicited high titers of anti-SaEsxA and anti-SaEsxB antibodies, but these antibodies could not prevent S. aureus infection. On the other hand, recombinant SaEsxA (rSaEsxA) and rSaEsxB could induce Th1- and Th17-biased immune responses in mice. Mice immunized with rSaEsxA and rSaEsxB had significantly improved survival rates when challenged with S. aureus compared with the controls. These findings indicate that SaEsxA and SaEsxB are two promising Th1 and Th17 candidate antigens which could be developed into multivalent and serotype-independent vaccines against S. aureus infection.     

Glioma Stem Cell-Targeted Dendritic Cells as a Tumor Vaccine Against Malignant Glioma

Purpose

Cancer stem cells have recently been thought to be closely related to tumor development and reoccurrence. It may be a promising way to cure malignant glioma by using glioma stem cell-targeted dendritic cells as a tumor vaccine. In this study, we explored whether pulsing dendritic cells with antigens of glioma stem cells was a potent way to induce specific cytotoxic T lymphocytes and anti-tumor immunity.

Materials and Methods

Cancer stem cells were cultured from glioma cell line U251. Lysate of glioma stem cells was obtained by the repeated freezing and thawing method. Dendritic cells (DCs) were induced and cultured from the murine bone marrow cells, the biological characteristics were detected by electron microscope and flow cytometry. The DC vaccine was obtained by mixing DCs with lysate of glioma stem cells. The DC vaccine was charactirizated through the mixed lymphocyte responses and cell killing experiment in vitro. Level of interferon-γ (IFN-γ) in the supernatant was checked by ELISA.

Results

After stimulation of lysate of glioma stem cell, expression of surface molecules of DC was up-regulated, including CD80, CD86, CD11C and MHC-II. DCs pulsed with lysate of glioma stem cells were more effective than the control group in stimulating original glioma cells-specific cytotoxic T lymphocytes responses, killing glioma cells and boosting the secretion of IFN-γ in vitro.

Conclusion

The results demonstrated DCs loaded with antigens derived from glioma stem cells can effectively stimulate naive T cells to form specific cytotoxic T cells, kill glioma cells cultured in vitro.     

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.Staphylococcus aureus is an opportunistic, nosocomial, community-acquired pathogen which causes several diseases ranging from minor skin infections to serious life-threatening infections like sepsis, endocarditis, pneumonia, and toxic shock syndrome (30). The rapid emergence of both hospital-associated methicillin (meticillin)-resistant S. aureus (MRSA) and community-acquired MRSA (CA-MRSA) is a major epidemiological problem worldwide (5, 25). A further threatening trend concerning S. aureus infections is the emergence of isolates with resistance to vancomycin, currently the antibiotic of choice against MRSA strains, and also to newly introduced drugs, such as daptomycin and linezolid (47). Hence, it is not surprising that interest in developing alternative approaches to prevent and treat staphylococcal infections has increased in recent years (34, 48).The major effector mechanism of the human immune system against S. aureus infection is comprised of professional phagocytes, such as neutrophils, that ingest and eliminate bacteria (16). However, phagocytosis of S. aureus relies on the opsonization of bacteria by antibodies and complement (7). Recognition of opsonizing antibodies bound to the surface of S. aureus via Fcγ receptors of neutrophils is a prerequisite for induction of the oxidative burst and therefore for killing of the phagocytosed bacteria (23) and induction of a long-term immune response (38). On the other hand, the presence of antistaphylococcal antibodies does not guarantee protection against reinfection. The reason for this apparent discrepancy is still not well understood. However, it was reported recently that antibodies against certain staphylococcal antigens present in healthy donors were missing or underrepresented in patient sera, indicating that antibodies reacting to these antigens are more efficient for induction of phagocytosis and for subsequent elimination of S. aureus than other antibodies (10, 12). Because of this, identification of protective antigens is a crucial step for vaccine development.Until now, most strategies for vaccination against S. aureus, active or passive, concentrated on single-component vaccines based on capsular polysaccharides or well-known virulence factors possessing the LPXTG motif, such as fibronectin binding protein (FnBP), collagen binding protein (CnBP), or clumping factor A (ClfA), as vaccination targets (14, 44). However, despite promising vaccination results obtained with animal models, so far most of the potential vaccines tested in clinical trials have failed to provide significant protection against S. aureus infection (48).It turns out that the efficacy of a monovalent vaccine may be hampered by the functional redundancy of adhesion proteins (17) or the appearance of escape mutants (56). Recently, it has been shown that a multivalent vaccine consisting of four antigenic determinants provides protection against lethal challenge with S. aureus in mice, whereas single-component immunization was much less effective (55). Therefore, identification of novel targets for an effective S. aureus vaccine has repeatedly been recognized as a high priority by experts in this field (20, 28, 34, 40, 43, 48). Indeed, numerous staphylococcal surface proteins predicted to be promising antigenic targets have been identified so far using recently adopted technologies, like proteomics (19, 36, 57) or protein selection methods based on expression libraries (10, 13, 58, 59). Unfortunately, most studies have not provided functional proof that identified proteins are vaccine candidates.Due to the fact that most of the previous experimental vaccine studies concentrated on candidates exhibiting the LPXTG sorting signal, we focused primarily on identification of noncovalently linked, cell wall-associated proteins, so-called anchorless cell wall (ACW) proteins. Proteins belonging to this class possess neither a conserved signal peptide nor an LPXTG motif and were recently recognized as novel virulence factors in gram-positive bacteria (9). Most of these ACW proteins are multifunctional; e.g., they are involved in different metabolic pathways and also in adhesion to extracellular matrix and invasion of host cells. Such proteins cannot be targeted by genome sequence screening due to the lack of conserved epitopes like LPXTG.For identification of new potential vaccine targets among ACW proteins we used intravenous immunoglobulin (IVIG) preparations to avoid limitation of the antibody source (i.e., individual sera). IVIG is a pool of immunoglobulins (Igs) from healthy persons that contains a broad spectrum of opsonizing antibodies against various pathogens, including S. aureus. By employing two-dimensional gel electrophoresis (2-DE), subtractive immunoblotting, and mass spectrometry, we identified several ACW proteins as novel vaccine targets. This method for identification of potential vaccines was called subtractive proteome analysis (SUPRA). The protective activity of antibodies raised against some of the identified proteins was evaluated in vitro and in a murine sepsis model.     

Staphylococcus aureus specific lung resident memory CD4+ Th1 cells attenuate the severity of influenza virus induced secondary bacterial pneumonia

Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4⁺ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4⁺ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4⁺ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4⁺ T cell responses. Interestingly, lack of CD4⁺ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4⁺ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4⁺ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.     

Inducing Humoral and Cellular Responses to Multiple Sporozoite and Liver-Stage Malaria Antigens Using Exogenous Plasmid DNA

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4⁺ and CD8⁺ T cell compartments. Furthermore, hepatic CD8⁺ lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8⁺ granzyme B⁺ T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.     

Role of Antibodies in Protection Elicited by Active Vaccination with Genetically Inactivated Alpha Hemolysin in a Mouse Model of Staphylococcus aureus Skin and Soft Tissue Infections

Due to the emergence of highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, S. aureus has become a major threat to public health. A majority of CA-MRSA skin and soft tissue infections in the United States are caused by S. aureus USA300 strains that are known to produce high levels of alpha hemolysin (Hla). Therefore, vaccines that contain inactivated forms of this toxin are currently being developed. In this study, we sought to determine the immune mechanisms of protection for this antigen using a vaccine composed of a genetically inactivated form of Hla (HlaH35L). Using a murine model of skin and soft tissue infections (SSTI), we found that BALB/c mice were protected by vaccination with HlaH35L; however, Jh mice, which are deficient in mature B lymphocytes and lack IgM and IgG in their serum, were not protected. Passive immunization with anti-HlaH35L antibodies conferred protection against bacterial colonization. Moreover, we found a positive correlation between the total antibody concentration induced by active vaccination and reduced bacterial levels. Animals that developed detectable neutralizing antibody titers after active vaccination were significantly protected from infection. These data demonstrate that antibodies to Hla represent the major mechanism of protection afforded by active vaccination with inactivated Hla in this murine model of SSTI, and in this disease model, antibody levels correlate with protection. These results provide important information for the future development and evaluation of S. aureus vaccines.     

Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.     

Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.     

Protective Immunity against Recurrent Staphylococcus aureus Skin Infection Requires Antibody and Interleukin-17A

Although many microbial infections elicit an adaptive immune response that can protect against reinfection, it is generally thought that Staphylococcus aureus infections fail to generate protective immunity despite detectable T and B cell responses. No vaccine is yet proven to prevent S. aureus infections in humans, and efforts to develop one have been hampered by a lack of animal models in which protective immunity occurs. Our results describe a novel mouse model of protective immunity against recurrent infection, in which S. aureus skin and soft tissue infection (SSTI) strongly protected against secondary SSTI in BALB/c mice but much less so in C57BL/6 mice. This protection was dependent on antibody, because adoptive transfer of immune BALB/c serum or purified antibody into either BALB/c or C57BL/6 mice resulted in smaller skin lesions. We also identified an antibody-independent mechanism, because B cell-deficient mice were partially protected against secondary S. aureus SSTI and adoptive transfer of T cells from immune BALB/c mice resulted in smaller lesions upon primary infection. Furthermore, neutralization of interleukin-17A (IL-17A) abolished T cell-mediated protection in BALB/c mice, whereas neutralization of gamma interferon (IFN-γ) enhanced protection in C57BL/6 mice. Therefore, protective immunity against recurrent S. aureus SSTI was advanced by antibody and the Th17/IL-17A pathway and prevented by the Th1/IFN-γ pathway, suggesting that targeting both cell-mediated and humoral immunity might optimally protect against secondary S. aureus SSTI. These findings also highlight the importance of the mouse genetic background in the development of protective immunity against S. aureus SSTI.     

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.     

Innate and adaptive immune responses against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> skin infections

Staphylococcus aureus is an important human pathogen that is responsible for the vast majority of bacterial skin and soft tissue infections in humans. S. aureus can also become more invasive and cause life-threatening infections such as bacteremia, pneumonia, abscesses of various organs, meningitis, osteomyelitis, endocarditis, and sepsis. These infections represent a major public health threat due to the enormous numbers of these infections and the widespread emergence of methicillin-resistant S. aureus (MRSA) strains. MSRA is endemic in hospitals worldwide and is rapidly spreading throughout the normal human population in the community. The increasing frequency of MRSA infections has complicated treatment as these strains are more virulent and are increasingly becoming resistant to multiple different classes of antibiotics. The important role of the immune response against S. aureus infections cannot be overemphasized as humans with certain genetic and acquired immunodeficiency disorders are at an increased risk for infection. Understanding the cutaneous immune responses against S. aureus is essential as most of these infections occur or originate from a site of infection or colonization of the skin and mucosa. This review will summarize the innate immune responses against S. aureus skin infections, including antimicrobial peptides that have direct antimicrobial activity against S. aureus as well as pattern recognition receptors and proinflammatory cytokines that promote neutrophil abscess formation in the skin, which is required for bacterial clearance. Finally, we will discuss the recent discoveries involving IL-17-mediated responses, which provide a key link between cutaneous innate and adaptive immune responses against S. aureus skin infections.     

Mucosal vaccination with a multivalent, live-attenuated vaccine induces multifactorial immunity against Pseudomonas aeruginosa acute lung infection

Many animal studies investigating adaptive immune effectors important for protection against Pseudomonas aeruginosa have implicated opsonic antibody to the antigenically variable lipopolysaccharide (LPS) O antigens as a primary effector. However, active and passive vaccination of humans against these antigens has not shown clinical efficacy. We hypothesized that optimal immunity would require inducing multiple immune effectors targeting multiple bacterial antigens. Therefore, we evaluated a multivalent live-attenuated mucosal vaccination strategy in a murine model of acute P. aeruginosa pneumonia to assess the contributions to protective efficacy of various bacterial antigens and host immune effectors. Vaccines combining 3 or 4 attenuated strains having different LPS serogroups were associated with the highest protective efficacy compared to vaccines with fewer components. Levels of opsonophagocytic antibodies, which were directed not only to the LPS O antigens but also to the LPS core and surface proteins, correlated with protective immunity. The multivalent live-attenuated vaccines overcame prior problems involving immunologic interference in the development of O-antigen-specific antibody responses when closely related O antigens were combined in multivalent vaccines. Antibodies to the LPS core were associated with in vitro killing and in vivo protection against strains with O antigens not expressed by the vaccine strains, whereas antibodies to the LPS core and surface proteins augmented the contribution of O-antigen-specific antibodies elicited by vaccine strains containing a homologous O antigen. Local CD4 T cells in the lung also contributed to vaccine-based protection when opsonophagocytic antibodies to the challenge strain were absent. Thus, multivalent live-attenuated vaccines elicit multifactorial protective immunity to P. aeruginosa lung infections.     

Activation of Bovine Lymphocyte Subpopulations by Staphylococcal Enterotoxin C

Staphylococcus aureus is a major mastitis-causing pathogen in cattle. The chronic nature of bovine staphylococcal mastitis suggests that some products or components of S. aureus may interfere with the development of protective immunity. One class of molecules that could be involved are superantigens (SAgs). Although a significant number of mastitis isolates produce SAgs, the effect of these molecules on the bovine immune system is unresolved. To determine if immunosuppression caused by SAgs could play a role in pathogenesis, we monitored bovine lymphocytes exposed to staphylococcal enterotoxin C1 (SEC1). Activation of bovine lymphocytes by either SEC1 or concanavalin A (ConA) was influenced by the γδ/αβ T-cell ratio in the culture. Compared to ConA-induced stimulation, cultures stimulated with SEC1 generated small numbers of CD4⁺ αβ T cells expressing high levels of interleukin-2 receptor α chain (IL-2Rα) and major histocompatibility complex class II (MHCII), suggesting that SAg exposure does not lead to full activation of these cells. This state of partial activation was most pronounced in cultures with a high γδ/αβ ratio. In contrast, significant numbers of CD8⁺ αβ T cells expressed high levels of IL-2Rα and MHCII, regardless of the γδ/αβ ratio and the stimulant used. CD8⁺ blasts in cultures stimulated with SEC1 also expressed another activation marker, ACT3, previously detected predominantly on thymocytes and CD4⁺ T cells. Although γδ CD2⁻ and CD2⁺ T cells expressed MHCII and IL-2Rα following stimulation with SEC1, only a few cells increased to blast size, suggesting that they were only partially activated. The results suggest ways in which SAgs might facilitate immunosuppression that promotes the persistence of bacteria in cattle and contributes to chronic intramammary infection.Staphylococcus aureus is a prominent pathogen in bovine mastitis (24). This organism is frequently isolated from milk (2, 16, 40) and from cows with intramammary infection (IMI) (17). IMI caused by S. aureus tends to become chronic and may resist antibiotic therapy (49). It has been postulated that persistent infection with S. aureus is associated with an impairment of the immune response, mediated by factors produced by S. aureus (34). Thus far, however, no single factor has been clearly implicated.Bovine isolates of S. aureus frequently produce one or more pyrogenic toxins (PTs), especially types C and D staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin (24). The staphylococcal PTs are prototype microbial superantigens (SAgs), characterized by the ability to bind to major histocompatibility complex class II (MHCII) molecules and to specific Vβ segments of αβ T-cell receptor (TCR) outside the binding groove associated with MHC-restricted immune system recognition of processed peptides. By bypassing antigenic specificity, SAgs stimulate abnormally large numbers of T cells and are able, at nanomolar concentrations, to induce T-cell proliferation (33). Few studies have been performed to investigate the effects of SAgs on the bovine immune system (53); most studies have involved other animals. In several species, SAgs exert wide-ranging and deleterious effects, including induction of shock (4), T-cell unresponsiveness and deletion (23), differential stimulation of CD4⁺ and CD8⁺ T-cell subsets (47), and B-cell differentiation (46). Thus, although largely unconfirmed, there is a clear potential for SEs, and other SAgs, to modulate immune responses and contribute to the virulence and persistence of S. aureus in cattle.The T-cell population consists of cells expressing either the αβ TCR (TCR2) or the γδ TCR (TCR1). While the roles of αβ T cells in immune responses of many species have been well characterized, the function of γδ T cells is less well understood (22). This is especially true in ruminants. Recent investigations have shown that the ruminant γδ T cells comprise two disparate subpopulations, characterized by constitutive expression of cell surface molecules. One subpopulation, similar in composition and tissue distribution to γδ T cells from other species, consists of cells that express CD2, CD5, and CD6 and are positive or negative for CD8 (10). These cells are present in low concentrations (3 to 5%) in peripheral blood and in high concentrations (35 to 40%) in spleen, gut epithelium, and mammary gland secretions (41). The second subpopulation, negative for CD2, CD6, and CD8, is unique and has been identified in only one other member of the Artiodactyla, swine (3, 28). This subpopulation is positive for CD5 and two lineage-restricted molecules, workshop cluster 1 (WC1) (32, 36, 50) and GD3.5 (21). The concentration of WC1⁺ GD3.5⁺ CD2⁻ CD6⁻ γδ T cells is high (30 to 50%) in the peripheral blood in young ruminants, decreasing with age, and is low (3 to 8%) in secondary lymphoid tissues and mammary gland secretions (41, 52). Definitive data on the function of either of these major subpopulations of γδ T cells have not been obtained. However, previous studies have suggested that they may be involved in regulating the proliferative response of CD4⁺ T cells to antigens (7).Park et al. (42) identified a subset of T cells, positive for CD2 and CD8, in mammary gland secretions of cows infected with S. aureus. These cells had the ability to inhibit the proliferative response of bovine CD4⁺ cells to staphylococcal antigens (42). Although the mechanisms by which these cells were induced and mediated their effect were not determined, they clearly have the potential to contribute to the pathogenesis of staphylococcal IMI. Although not all bovine staphylococcal isolates produce known SAgs, it is important to determine whether SAg production could induce these or other immunosuppressive subpopulations in cows and promote the development of some infections such as IMI. The objective of this study was to extend these initial observations. We examined the effect of a representative SAg (SEC1) on the major subpopulations of bovine αβ and γδ T cells.     

Antibody responses in furunculosis patients vaccinated with autologous formalin-killed Staphylococcus aureus

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex?). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens.     

Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus

Fifty methicillin-resistant Staphylococcus aureus (MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitive Staphylococcus aureus (MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen for S. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps. S. aureus strains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins of S. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negative S. aureus strains as methicillin resistant even prior to antimicrobial susceptibility testing.