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Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and 1H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and (1)H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.

Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.

St. Leger et al. identify and examine innate-like αβ T cells that circumvent canonical STAT-3 phosphorylation to produce protective IL-17. These cells can exist in the ocular mucosa and protect the ocular surface from pathogenic Staphylococcus aureus infection., Appropriate regulation of IL-17 production in the host can mean the difference between effective control of pathogens and uncontrolled inflammation that causes tissue damage. Investigation of conventional CD4+ T cells (Th17 cells) has yielded invaluable insights into IL-17 function and its regulation. More recently, we and others reported production of IL-17 from innate αβ+ T cell populations, which was shown to occur primarily via IL-23R signaling through the transcription factor STAT-3. In our current study, we identify promyelocytic leukemia zinc finger (PLZF)–expressing iNKT, CD4−/CD8+, and CD4−/CD8− (DN) αβ+T cells, which produce IL-17 in response to TCR and IL-1 receptor ligation independently of STAT-3 signaling. Notably, this noncanonical pathway of IL-17 production may be important in mucosal defense and is by itself sufficient to control pathogenic Staphylococcus aureus infection at the ocular surface., Graphical Abstract

Multiple candidate vaccines against Staphylococcus aureus infections have failed in clinical trials. Analysis of a recent prematurely halted vaccine trial revealed increased mortality rates among vaccine recipients in whom postsurgical S. aureus infection developed, emphasizing the potential for induction of detrimental immune responses and the need to better understand the requirements for protective immunity against S. aureus. These failures of single-antigen vaccines have prompted ongoing development of multicomponent vaccines to target the multitude of S. aureus virulence factors. In the current study, we used lethally irradiated S. aureus as a model multicomponent vaccine and showed that vaccination of mice decreased survival in a bacteremia challenge model. These deleterious effects were due to a CD4 T-cell-dependent interferon γ response and could be prevented by inhibiting development of this response during vaccination. Our results identify the potential for vaccination to induce pathological immune responses, and they have implications for recent vaccine failures and the design of future staphylococcal vaccines.

Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recently discovered leukotoxin LukAB kills human phagocytes and monocytes and it is present in all known S. aureus clinical isolates. While using a structure-guided approach, we generated a library of mutations that targeted functional domains within the LukAB heterodimer to identify attenuated toxoids as potential vaccine candidates. The mutants were evaluated based on expression, solubility, yield, biophysical properties, cytotoxicity, and immunogenicity, and several fully attenuated LukAB toxoids that were capable of eliciting high neutralizing antibody titers were identified. Rabbit polyclonal antibodies against the lead toxoid candidate provided potent neutralization of LukAB. While the neutralization of LukAB alone was not sufficient to fully suppress leukotoxicity in supernatants of S. aureus USA300 isolates, a combination of antibodies against LukAB, &alpha, toxin, and Panton-Valentine leukocidin completely neutralized the cytotoxicity of these strains. These data strongly support the inclusion of LukAB toxoids in a multivalent toxoid vaccine for the prevention of S. aureus disease.

Staphylococcal enterotoxin B (SEB) is a protein exotoxin found on the cell surface of Staphylococcus aureus that is the source for multiple pathologies in humans. When purified and concentrated in aerosol form, SEB can cause an acute and often fatal intoxication and thus is considered a biological threat agent., Staphylococcal enterotoxin B (SEB) is a protein exotoxin found on the cell surface of Staphylococcus aureus that is the source for multiple pathologies in humans. When purified and concentrated in aerosol form, SEB can cause an acute and often fatal intoxication and thus is considered a biological threat agent. There are currently no vaccines or treatments approved for human use. Studies with rodent models of SEB intoxication show that antibody therapy may be a promising treatment strategy; however, many have used antibodies only prophylactically or well before any clinical signs of intoxication are apparent. We assessed and compared the protective efficacies of two monoclonal antibodies, Ig121 and c19F1, when administered after aerosol exposure in a uniformly lethal nonhuman primate model of SEB intoxication. Rhesus macaques were challenged using small-particle aerosols of SEB and then were infused intravenously with a single dose of either Ig121 or c19F1 (10 mg/kg of body weight) at either 0.5, 2, or 4 h postexposure. Onset of clinical signs and hematological and cytokine response in untreated controls confirmed the acute onset and potency of the toxin used in the challenge. All animals administered either Ig121 or c19F1 survived SEB challenge, whereas the untreated controls succumbed to SEB intoxication 30 to 48 h postexposure. These results represent the successful therapeutic in vivo protection by two investigational drugs against SEB in a severe nonhuman primate disease model and punctuate the therapeutic value of monoclonal antibodies when faced with treatment options for SEB-induced toxicity in a postexposure setting.

To develop an antibody (Ab) therapeutic against Staphylococcal Enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “Class T” anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “Class M” Abs. A systematic engineering approach was applied to affinity mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) revealed that the Class T epitope on SEB overlapped with the T-cell receptor (TCR) binding site, whereas other evidence suggested that the Class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ~50-fold more potent toxin-neutralizing efficacy than the best Class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two Class M binding sites and two Class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that Class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bispecific Ab engineering.

Staphylococcal enterotoxin B (SEB) is a protein exotoxin found on the cell surface ofStaphylococcus aureusthat is the source for multiple pathologies in man. When purified and concentrated in aerosol form, SEB can cause an acute and often fatal intoxication, and thus is considered a biological threat agent. There are currently no vaccines or treatments approved for human use. Studies in rodent models of SEB intoxication show that antibody therapy may be a promising treatment strategy, however many have used antibodies only prophylactically or well before any clinical signs of intoxication are apparent. We assessed and compared the protective efficacy of two monoclonal antibodies, Ig121 and c19F1, when administered after aerosol exposure in a uniformly lethal nonhuman primate model of SEB intoxication. Rhesus macaques were challenged using small particle aerosols of SEB, and then were infused intravenously with a single dose of either Ig121 or c19F1 (10 mg/kg) at either 0.5, 2 or 4 hours postexposure. Onset of clinical signs, hematological, and cytokine response in untreated controls confirmed the acute onset and potency of the toxin used in the challenge. All animals administered either Ig121 or c19F1 survived SEB challenge, whereas the untreated controls succumbed to SEB intoxication 30-48 hours postexposure. These results represent the successful therapeuticin vivoprotection by two investigational drugs against SEB in a severe nonhuman primate disease model and punctuate the therapeutic value of monoclonal antibodies hold when faced with treatment options for SEB-induced toxicity in a postexposure setting.One Sentence Summary: Two high-affinity monoclonal antibodies were tested for therapeutic efficacy using a rhesus macaque challenge model of aerosolized SEB

Spores of Bacillus subtilis are encased in a protein coat composed of ∼80 different proteins. Recently, we reconstituted the basement layer of the coat, composed of two structural proteins (SpoVM and SpoIVA) around spore-sized silica beads encased in a lipid bilayer, to create synthetic spore-like particles termed ‘SSHELs’. We demonstrated that SSHELs could display thousands of copies of proteins and small molecules of interest covalently linked to SpoIVA. In this study, we investigated the efficacy of SSHELs in delivering vaccines. We show that intramuscular vaccination of mice with undecorated one micron-diameter SSHELs elicited an antibody response against SpoIVA. We further demonstrate that SSHELs covalently modified with a catalytically inactivated staphylococcal alpha toxin variant (Hla(H35L)), without an adjuvant, resulted in improved protection against Staphylococcus aureus infection in a bacteremia model as compared to vaccination with the antigen alone. Although vaccination with either Hla(H35L) or Hla(H35L) conjugated to SSHELs similarly elicited the production of neutralizing antibodies to Hla, we found that a subset of memory T cells was differentially activated when the antigen was delivered on SSHELs. We propose that the particulate nature of SSHELs elicits a more robust immune response to the vaccine that results in superior protection against subsequent S. aureus infection.

A complex interplay between host and bacterial factors allows Staphylococcus aureus to occupy its niche as a human commensal and a major human pathogen. The role of neutrophils as a critical component of the innate immune response against S. aureus, particularly for control of systemic infection, has been established in both animal models and in humans with acquired and congenital neutrophil dysfunction. The role of the adaptive immune system is less clear. Although deficiencies in adaptive immunity do not result in the marked susceptibility to S. aureus infection that neutrophil dysfunction imparts, emerging evidence suggests both T cell- and B cell-mediated adaptive immunity can influence host susceptibility and control of S. aureus. The contribution of adaptive immunity depends on the context and site of infection, and can be either beneficial or detrimental to the host. Furthermore, S. aureus has evolved mechanisms to manipulate adaptive immune responses to its advantage. In this chapter, we will review the evidence for the role of adaptive immunity during S. aureus infections. Further elucidation of this role will be important to understand how it influences susceptibility to infection and to appropriately design vaccines that elicit adaptive immune responses to protect against subsequent infections.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 -/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 -/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans., Author Summary CARD9 is a molecule expressed by mammalian immune cells and is centrally positioned downstream of several C-type lectin receptors, which sense fungi. The critical role of CARD9 in the activation of antifungal host defense has been highlighted by the demonstration that human mutations that disrupt the function of CARD9 are associated with the development of spontaneous life-threatening fungal infections, many of which target the central nervous system (CNS). However, why CARD9-deficient patients develop fungal disease in the CNS remained unclear. Here, we show that CARD9 is required for the recruitment of neutrophils, an immune cell critical for antifungal host defense, to the fungal-infected CNS in both mice and humans. Furthermore, we show that CARD9-deficient mice and humans are unable to produce the molecules (chemoattractants) needed to recruit neutrophils to the site of infection, thus identifying the mechanism for the striking absence of these cells in the infected CNS. Our work helps explain why CARD9 deficiency associates with CNS fungal disease, and furthers our understanding of how antifungal immunity operates in this complex tissue.

The virulence of SCC mec type IV hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates belonging to the major sequence type 8 (ST8 [Lyon clone]) and to a minor upcoming clone, ST5, was compared with that of methicillin-susceptible S. aureus (MSSA) isolates of matching sequence types. In vitro adhesion to human airway epithelial cells (HAECs) as an indicator of dissemination and mortality in a murine sepsis model as an indicator of virulence were evaluated. Ten MRSA isolates and 8 MSSA isolates of ST8 and 8 MRSA isolates and 8 MSSA isolates of ST5 were characterized with respect to multilocus sequence type; agr, spa , and capsule typing; in vitro doubling time; toxin and adhesin gene profiles; and adherence to HAECs. Adherence was significantly lower in the MRSA ST5 group than in the ST8 groups. Infections with MRSA and MSSA isolates ST8 and ST5 were compared. No change in virulence related to the presence of SCC mec was observed, since ST8 but not ST5 caused a significantly lower mortality in its presence. Despite their similar genetic backgrounds, individual clonal MRSA and MSSA isolates were heterogeneous in adherence and virulence. No one of these specific virulence factors determined in vitro was related to mouse mortality. In conclusion, in a bacteremic model, mortality was dependent on the ST and was differentially modulated by SCC mec ; within an ST, clonality was not associated with a homogenous outcome.

Glycopeptide resistance, in a set of in vitro step-selected teicoplanin-resistant mutants derived from susceptible Staphylococcus aureus SA113, was associated with slower growth, thickening of the bacterial cell wall, increased N -acetylglucosamine incorporation, and decreased hemolysis. Differential transcriptome analysis showed that as resistance increased, some virulence-associated genes became downregulated. In a mouse tissue cage infection model, an inoculum of 10 4 CFU of strain SA113 rapidly produced a high-bacterial-load infection, which triggered MIP-2 release, leukocyte infiltration, and reduced leukocyte viability. In contrast, with the same inoculum of the isogenic glycopeptide-resistant derivative NM67, CFU initially decreased, resulting in the elimination of the mutant in three out of seven cages. In the four cages in which NM67 survived, it partially regained wild-type characteristics, including thinning of the cell wall, reduced N -acetylglucosamine uptake, and increased hemolysis; however, the survivors also became teicoplanin hypersusceptible. The elimination of the teicoplanin-resistant mutants and selection of teicoplanin-hypersusceptible survivors in the tissue cages indicated that glycopeptide resistance imposes a fitness burden on S. aureus and is selected against in vivo, with restoration of fitness incurring the price of resistance loss.

Background Modification of bacterial spore surfaces with proteins has previously been proposed as vaccine display platforms, however, relied on viable spores. Objective In the present study we describe how synthetic spore husk-encased lipid bilayers (SSHELs) can be utilized as a display platform to present protein antigens like ovalbumin and mutant staphylococcal alpha toxin (HlaH35L) as vaccines. This platform is based on 2 self-assembling proteins SpoVM and SpoIVA from Bacillus subtilis. Methods C57BL/6 mice were immunized with SSHEL (with or without adjuvant), antigen or antigen covalently bound to SSHEL (SSHEL_antigen) thrice intramuscularly at two-week intervals and serum antibody titers against SpoIVA or antigens were determined 7 days after each immunization. In select experiments mice were challenged intravenously with a lethal dose of S. aureus USA300 2 weeks after last vaccination. Untreated mice served as controls. Results Immunization of mice with SSHEL induced a similar antibody response towards SpoIVA as SSHEL that was adjuvanted in CpG or MPL-A suggesting adjuvant properties of this synthetic lipid-bilayer construct. Post vaccination serum of mice immunized with ovalbumin covalently bound to SSHEL (SSHEL_OVA) or HlaH35L covalently bound to SSHEL (SSHEL_HlaH35L) contained SpoIVA specific as well as antigen-specific (OVA or HlaH35L) antibodies. Infection of mice with a lethal dose of USA300 resulted in 100% survival of mice immunized with SSHEL_HlaH35L compared to 20% survival of non-immunized mice (P=0.01), and 40% survival of mice immunized with HlaH35L (P=0.049). In progress In ongoing studies we are evaluating the mechanism of action of SSHEL_HlaH35L that lead to protection during infection.

Staphylococcus aureus causes a wide array of diseases, ranging from superficial skin infections to life threatening diseases like sepsis. Treatment is difficult due to emerging multidrug-resistant strains and importantly, several approaches to develop an efficacious vaccine have either failed or augmented disease in clinical trials. In the present study, gamma-irradiated S. aureus USA300 (IRS) was used as a whole-cell vaccine to evaluate the immune response towards vaccination and subsequent infection. C57BL/6 mice were vaccinated thrice intramuscularly, adjuvanted with or without Al(OH)3, at two-week intervals and challenged with USA300 intravenously 2 weeks after the last vaccination. Vaccination enhanced mortality and failed to control bacterial growth in blood and organs when compared to PBS controls and was worse without Al(OH)3. USA300-specific antibodies were induced by vaccination but did not cause enhanced mortality when tested in serum transfer studies. Furthermore, B cell deficient and WT mice were similarly susceptible to infection after vaccination. Infection increased frequency of CD44hiTbet+ and CD44hiIFNg+ (Th1 effector) cells in spleens of both vaccinated groups but was higher in mice vaccinated without Al(OH)3. Frequency of CD44hiRORgt+ and CD44hiIL-17+ (Th17 effector) cells increased only in groups vaccinated with Al(OH)3. Depletion of CD4 T cells during infection reversed the lethal phenotype. Similarly, neutralization of IFNg but not IL-17 during infection improved survival and reduced organ pathology. Taken together, our findings show a detrimental role for IFNg+CD4 Teff cells generated by vaccination with IRS, and might shed some light on recent vaccine failures.

Background. Staphylococcus aureus has numerous virulence factors, including exotoxins that may increase the severity of infection. This study was aimed at assessing whether preexisting antibodies to S. aureus toxins are associated with a lower risk of sepsis in adults with S. aureus infection complicated by bacteremia. Methods. We prospectively identified adults with S. aureus infection from 4 hospitals in Baltimore, MD, in 2009–2011. We obtained serum samples from prior to or at presentation of S. aureus bacteremia to measure total immunoglobulin G (IgG) and IgG antibody levels to 11 S. aureus exotoxins. Bacterial isolates were tested for the genes encoding S. aureus exotoxins using polymerase chain reaction (PCR). Results. One hundred eligible subjects were included and 27 of them developed sepsis. When adjusted for total IgG levels and stratified for the presence of toxin in the infecting isolate as appropriate, the risk of sepsis was significantly lower in those patients with higher levels of IgG against α-hemolysin (Hla), δ-hemolysin (Hld), Panton Valentine leukocidin (PVL), staphylococcal enterotoxin C-1 (SEC-1), and phenol-soluble modulin α3 (PSM-α3). Conclusions. Our results suggest that higher antibody levels against Hla, Hld, PVL, SEC-1, and PSM-α 3m ay protect against sepsis in patients with invasive S. aureus infections.

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.

Panton-Valentine leukocidin (PVL) is a bipartite toxin that plays an important role in the pathogenesis of methicillin-resistant Staphylococcus aureus. Recent clinical data suggest a correlation between PVL and severe cases of S. aureus pneumonia. A clear understanding of the structure and function of PVL is critical to the development of novel, effective treatments. Here, we report an all-atom model of the macromolecular structure of Panton-Valentine leukocidin in its octameric, pre-pore conformation that confirms and extends our understanding of the toxin's mechanism of action.

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Staphylococcal superantigens are exotoxins that play a major role in disease pathogenesis. Using a T cell epitope prediction tool from the Immune Epitope Database Analysis Resource (IEDB) site, sequences within staphylococcal enterotoxin A and B (SEA, SEB) and Toxic Shock Syndrome Toxin with high affinity towards H2-Iab were identified and oligopeptides SEA_aa116-128, SEB_aa16-28 and TSST-1_aa212-224 were generated. C57BL/6 mice were immunized with a cocktail of oligopeptides or whole toxins in Al(OH)3. Serum samples from peptide-immunized mice tested positive towards individual peptides and whole toxins, however, samples from mice immunized with whole toxins did not bind to any of the peptides. To determine peptide-specific T cell response primed CD4 T cells isolated from spleens and total LNs of immunized mice were co-cultured with peptide-pulsed BMDCs and cytokine response was evaluated. Only cells of peptide-immunized mice resulted in elevated levels of IL-4, IL-5, IL-10, IL-13, GM-CSF, IFNg and TNFa, while cells obtained from whole toxin-immunized mice did not differ from baseline levels. To evaluate protective efficacy of peptides against toxin expressing strains, immunized mice were infected intravenously with MNHOCH (SEB+) or Newman (SEA+) and monitored up to 13 days. Mice that were infected with MNHOCH showed significantly extended survival compared to non-immunized mice (P=0.03). No significant difference in survival was observed when infected with strain Newman.

The ocular surface represents a mucosal interface between the environment and the host. Similarly to the lymphoid tissue of the gut, cells of the conjunctiva-associated lymphoid tissue (CALT) are organized into follicles. Humans exhibit CALT in healthy eyes, and the number and size of follicles increase with inflammation caused by infections, allergies, dry eye, and conjunctivitis, suggesting a functional role for CALT in immunity at the ocular surface. Due to the limited amount of data from animal models, functional studies of CALT are lacking. We aimed to develop a mouse model to study the dynamics and functional parameters of CALT in normal and immunologically perturbed hosts. Towards that end, we used mice deficient in IL-10 and/or IL-22. Upon ocular infection with Staphylococcus aureus (Strain USA300 LAC), we observed an expansion of immune cells in the CALT. This included elements of the adaptive and innate immune systems, with prominent expansion of group 3 innate-like lymphocytes (ILC3), which have hitherto only been shown to localize to the gut and lungs. The increase in cellular infiltrate correlated with an increase in the numbers of TNFα and IL-17 producing cells. Our studies lay the basis for a working mouse model to analyze the functional characteristics of CALT, an understudied component of the mucosal immune system. Also, this model may permit identification of factors that induce ocular inflammation leading to various ocular pathologies.

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.