Dissecting the Human Response to Staphylococcus aureus Systemic Infections.

Staphylococcus aureus is a common human commensal and the leading cause of diverse infections. To identify distinctive parameters associated with infection and colonization, we compared the immune and inflammatory responses of patients with a diagnosis of invasive S. aureus disease to healthy donors. We analyzed the inflammatory responses founding a pattern of distinctive cytokines significantly higher in the patients with invasive disease. The measure of antibody levels revealed a wide antibody responsiveness from all subjects to most of the antigens, with significantly higher response for some antigens in the invasive patients compared to control. Moreover, functional antibodies against toxins distinctively associated with the invasive disease. Finally, we examined the genomic variability of isolates, showing no major differences in genetic distribution compared to a panel of representative strains. Overall, our study shows specific signatures of cytokines and functional antibodies in patients with different primary invasive diseases caused by S. aureus. These data provide insight into human responses towards invasive staphylococcal infections and are important for guiding the identification of novel preventive and therapeutic interventions against S. aureus.

Staphylococcus aureus Vaccine Research and Development: The Past, Present and Future, Including Novel Therapeutic Strategies.

Staphylococcus aureus is one of the most important human pathogens worldwide. Its high antibiotic resistance profile reinforces the need for new interventions like vaccines in addition to new antibiotics. Vaccine development efforts against S. aureus have failed so far however, the findings from these human clinical and non-clinical studies provide potential insight for such failures. Currently, research is focusing on identifying novel vaccine formulations able to elicit potent humoral and cellular immune responses. Translational science studies are attempting to discover correlates of protection using animal models as well as in vitro and ex vivo models assessing efficacy of vaccine candidates. Several new vaccine candidates are being tested in human clinical trials in a variety of target populations. In addition to vaccines, bacteriophages, monoclonal antibodies, centyrins and new classes of antibiotics are being developed. Some of these have been tested in humans with encouraging results. The complexity of the diseases and the range of the target populations affected by this pathogen will require a multipronged approach using different interventions, which will be discussed in this review.

Staphylococcus aureus-Specific Tissue-Resident Memory CD4+ T Cells Are Abundant in Healthy Human Skin.

The skin is an immunocompetent tissue that harbors several kinds of immune cells and a plethora of commensal microbes constituting the skin microbiome. Staphylococcus aureus is a prominent skin pathogen that colonizes a large proportion of the human population. We currently have an incomplete understanding of the correlates of protection against S. aureus infection, however genetic and experimental evidence has shown that CD4+ T cells play a key role in orchestrating a protective anti-S. aureus immune response. A high S. aureus-specific memory CD4+ T cell response has been reported in the blood of healthy subjects. Since T cells are more abundant in the skin than in blood, we hypothesized that S. aureus-specific CD4+ T cells could be present in the skin of healthy individuals. Indeed, we observed proliferation of tissue-resident memory CD4+ T cells and production of IL-17A, IL-22, IFN-γ and TNF-ß by cells isolated from abdominal skin explants in response to heat-killed S. aureus. Remarkably, these cytokines were produced also during an ex vivo epicutaneous S. aureus infection of human skin explants. These findings highlight the importance of tissue-resident memory CD4+ T cells present at barrier sites such as the skin, a primary entry site for S. aureus. Further phenotypical and functional characterization of these cells will ultimately aid in the development of novel vaccine strategies against this elusive pathogen.

Human Organotypic Models for Anti-infective Research.

The use of human organotypic models for biomedical research is experiencing a significant increase due to their biological relevance, the possibility to perform high-throughput analyses, and their cost efficiency. In the field of anti-infective research, comprising the search for novel antipathogenic treatments including vaccines, efforts have been made to reduce the use of animal models. That is due to two main reasons: unreliability of data obtained with animal models and the increasing willingness to reduce the use of animals in research for ethical reasons. Human three-dimensional (3-D) models may substitute and/or complement in vivo studies, to increase the translational value of preclinical data. Here, we provide an overview of recent studies utilizing human organotypic models, resembling features of the cervix, intestine, lungs, brain, and skin in the context of anti-infective research. Furthermore, we focus on the future applications of human skin models and present methodological protocols to culture human skin equivalents and human skin explants.

Human Three-Dimensional Models for Studying Skin Pathogens.

Skin is the most exposed surface of the human body, separating the microbe-rich external environment, from the sterile inner part. When skin is breached or its homeostasis is perturbed, bacterial, fungal and viral pathogens can cause local infections or use the skin as an entry site to spread to other organs. In the last decades, it has become clear that skin provides niches for permanent microbial colonization, and it actively interacts with microorganisms. This crosstalk promotes skin homeostasis and immune maturation, preventing expansion of harmful organisms. Skin commensals, however, are often found to be skin most prevalent and dangerous pathogens. Despite the medical interest, mechanisms of colonization and invasion for most skin pathogens are poorly understood. This limitation is due to the lack of reliable skin models. Indeed, animal models do not adequately mimic neither the anatomy nor the immune response of human skin. Human 3D skin models overcome these limitations and can provide new insights into the molecular mechanisms of microbial pathogenesis. Herein, we address the strengths and weaknesses of different types of human skin models and we review the main findings obtained using these models to study skin pathogens.

Targeting Skin-Resident Memory T Cells via Vaccination to Combat Staphylococcus aureus Infections.

Tissue-resident memory T cells are important in adaptive immunity against many infections, rendering these cells attractive potential targets in vaccine development. Genetic and experimental evidence highlights the importance of cellular immunity in protection from Staphylococcus aureus skin infections, yet skin-resident memory T cells are, thus far, an untested component of immunity during such infections. Novel methods of generating and sampling vaccine-induced skin memory T cells are paralleled by discoveries of global, skin-wide immunosurveillance. We propose skin-resident memory CD4+ T cells as a potential missing link in the search for correlates of protection during S. aureus infections. A better appreciation of their phenotypes and functions could accelerate the development of preventive vaccines against this highly virulent and antibiotic-resistant pathogen.

One Dose of Staphylococcus aureus 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4+ T Cells, and IL-17A.

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.

MF59- and Al(OH)3-Adjuvanted Staphylococcus aureus (4C-Staph) Vaccines Induce Sustained Protective Humoral and Cellular Immune Responses, with a Critical Role for Effector CD4 T Cells at Low Antibody Titers.

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.

Oil-in-Water Emulsion MF59 Increases Germinal Center B Cell Differentiation and Persistence in Response to Vaccination.

Induction of persistent protective immune responses is a key attribute of a successful vaccine formulation. MF59 adjuvant, an oil-in-water emulsion used in human vaccines, is known to induce persistent high-affinity functional Ab titers and memory B cells, but how it really shapes the Ag-specific B cell compartment is poorly documented. In this study, we characterized the Ab- and Ag-specific B cell compartment in wild-type mice immunized with HlaH35L, a Staphylococcus aureus Ag known to induce measurable functional Ab responses, formulated with MF59 or aluminum salts, focusing on germinal centers (GC) in secondary lymphoid organs. Taking advantage of single-cell flow cytometry analyses, HlaH35L-specific B cells were characterized for the expression of CD38 and GL-7, markers of memory and GC, respectively, and for CD80 and CD73 activation markers. We demonstrated that immunization with MF59-, but not aluminum salt-adjuvanted HlaH35L, induced expanded Ag-specific CD73(+)CD80(-) GC B cells in proximal- and distal-draining lymph nodes, and promoted the persistence of GC B cells, detected up to 4 mo after immunization. In addition to increasing GC B cells, MF59-adjuvanted HlaH35L also increased the frequency of T follicular helper cells. This work extends previous knowledge regarding adaptive immune responses to MF59-adjuvanted vaccines, and, to our knowledge, for the first time an adjuvant used in human licensed products is shown to promote strong and persistent Ag-specific GC responses that might benefit the rational design of new vaccination strategies.

Phagocyte subsets and lymphocyte clonal deletion behind ineffective immune response to Staphylococcus aureus.

Lack of known mechanisms of protection against Staphylococcus aureus in humans is hindering development of efficacious vaccines. Preclinical as well as clinical data suggest that antibodies play an important role against S. aureus. For instance, certain hypogammaglobulinaemic patients are at increased risk of staphylococcal infections. However, development of effective humoral response may be dampened by converging immune-evasion mechanisms of S. aureus. We hypothesize that B-cell proliferation induced by staphylococcal protein A (SpA) and continuous antigen exposure, without the proper T-cell help and cytokine stimuli, leads to antigen-activated B-cell deletion and anergy. Recent findings suggest an important role of type I neutrophils (PMN-I) and conventionally activated macrophages (M1) against S. aureus, while alternatively activated macrophages (M2) favour biofilm persistence and sepsis. In addition, neutrophil-macrophage cooperation promotes extravasation and activation of neutrophils as well as clearance of bacteria ensnared in neutrophil extracellular traps. Activation of these processes is modulated by cytokines and T cells. Indeed, low CD4(+) T-cell counts represent an important risk factor for skin infections and bacteraemia in patients. Altogether, these observations could lead to the identification of predictive correlates of protection and ways for shifting the balance of the response to the benefit of the host through vaccination.

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

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

NAD-dependent ADP-ribosylation of the human antimicrobial and immune-modulatory peptide LL-37 by ADP-ribosyltransferase-1.

LL-37 is a cationic peptide belonging to the cathelicidin family that has antimicrobial and immune-modulatory properties. Here we show that the mammalian mono-ADP-ribosyltransferase-1 (ART1), which selectively transfers the ADP-ribose moiety from NAD to arginine residues, ADP-ribosylates LL-37 in vitro. The incorporation of ADP-ribose was first observed by Western blot analysis and then confirmed by MALDI-TOF. Mass-spectrometry showed that up to four of the five arginine residues present in LL-37 could be ADP-ribosylated on the same peptide when incubated at a high NAD concentration in the presence of ART1. The attachment of negatively charged ADP-ribose moieties considerably alters the positive charge of the arginine residues thus reducing the cationicity of LL-37. The cationic nature of LL-37 is key for its ability to interact with cell membranes or negatively charged biomolecules, such as DNA, RNA, F-actin and glycosaminoglycans. Thus, the ADP-ribosylation of LL-37 is expected to have the potential to modulate LL-37 biological activities in several physiological and pathological settings.

Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes.

Helicobacter pylori is a gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1ß, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.

Small molecule Toll-like receptor 7 agonists localize to the MHC class II loading compartment of human plasmacytoid dendritic cells.

TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, with 2 different fluorophores [5(6) carboxytetramethylrhodamine and Alexa Fluor 488] and monitored their intracellular localization in human plasmacytoid dendritic cells (pDCs). All fluorescent compounds induced the production of IFN-α, TNF-α, and IL-6 and the up-regulation of CD80 and CD86 by pDCs showing they retained TLR7-stimulating activity. Confocal imaging of pDCs showed that, similar to CpG-B, all compounds concentrated in the MHC class II loading compartment (MIIC), identified as lysosome-associated membrane protein 1(+), CD63, and HLA-DR(+) endosomes. Treatment of pDCs with bafilomycin A, an antagonist of the vacuolar-type proton ATPase controlling endosomal acidification, prevented the accumulation of small molecule TLR7 agonists, but not of CpG-B, in the MIIC. These results indicate that a pH-driven concentration of small molecule TLR7 agonists in the MIIC is required for pDC activation.

Coligation of the hepatitis C virus receptor CD81 with CD28 primes naive T lymphocytes to acquire type 2 effector function.

Costimuli provide supplementary signals required by naive T cells to become fully activated upon Ag encounter. Tetraspanins are a large family of transmembrane proteins that can costimulate T cells when engaged in vitro. In this study, we describe for the first time that coligation of the tetraspanins CD81, CD82, or CD9 with the costimulatory molecule CD28 in vitro leads to proliferation of naive T cells. When activated through this pathway, both CD4+ and CD8+ naive T cells differentiate into type 2 effector cells, which produce IL-4, IL-5, IL-13, and IL-10, together with IL-2 and TNF-alpha, but little to no IFN-gamma. These effector cells descend from precursors that display early and strong production of IL-4, STAT6 phosphorylation, and up-regulation of the transcription factor GATA-3, suggesting a direct skewing toward Th2 differentiation without a Th0 intermediate. The hepatitis C virus envelope protein E2 is the only ligand known for CD81. Therefore, we propose that this new type of Ag-independent T cell activation may occur in hepatitis C virus-infected individuals, contributing to liver inflammation, impaired type 1 immune responses, and recurrent flares of type 2 immunity associated with chronic infection.

Expression and selective up-regulation of toxin-related mono ADP-ribosyltransferases by pathogen-associated molecular patterns in alveolar epithelial cells.

Mono ADP-ribosyltransferases (ARTs) are a family of enzymes related to bacterial toxins that possess adenosine diphosphate ribosyltransferase activity. We have assessed that A549 constitutively expressed ART1 on the cell surface and shown that lipotheicoic acid (LTA) and flagellin, but not lipopolysaccharide (LPS), peptidoglycan (PG) and poly (I:C), up-regulate ART1 in a time and dose dependent manner. These agonists did not alter the expression of ART3 and ART5 genes. Indeed, LTA and flagellin stimulation increased the level of ART1 protein and transcript while ART4 gene was activated after stimulation of cells with LPS, LTA, PAM and PG via TLR2 and TLR4 receptors. These results show that human ARTs possess a differential capacity to respond to bacteria cell wall components and might play a crucial role in innate immune response in airways.

T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck.

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.