Hatice Karauzum

Lower Antibody Levels to Staphylococcus aureus Exotoxins Are Associated With Sepsis in Hospitalized Adults With Invasive S. aureus Infections

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-α3 may protect against sepsis in patients with invasive S. aureus infections.

Novel Structurally Designed Vaccine for S. aureus α-Hemolysin: Protection against Bacteremia and Pneumonia

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.

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

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.

Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome

Background: SEB is a potent superantigen that can cause toxic shock in humans and can be incapacitating and lethal when used as a bioweapon. Results: Therapeutic administration of highly affinity-matured synthetic anti-SEB human monoclonal IgGs protects mice from lethal SEB challenge. Conclusion: Affinity maturation can enhance neutralizing efficacy and lead to successful postexposure therapeutics. Significance: No vaccines or therapeutics are currently available against SEB. 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.

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.

Adaptive Immunity Against Staphylococcus aureus

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.

Lethal CD4 T Cell Responses Induced by Vaccination Against Staphylococcus aureus Bacteremia

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.

STAT-3-independent production of IL-17 by mouse innate-like αβ T cells controls ocular 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 &agr;&bgr;+ 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) &agr;&bgr;+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.

Therapeutic Treatment of Aerosolized Staphylococcal Enterotoxin B in Nonhuman Primates with two Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a protein exotoxin found on the cell surface of Staphylococcus aureus that 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 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 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