Tryptic Shaving of Staphylococcus aureus Unveils Immunodominant Epitopes on the Bacterial Cell Surface.

The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics and by fast evolution into new lineages that rapidly spread within the healthy human population. This calls for development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available for clinical implementation, the present studies were aimed at identifying new leads for their development. For this purpose, we profiled the cell-surface-exposed staphylococcal proteome under infection-mimicking conditions by combining two approaches for "bacterial shaving" with immobilized or soluble trypsin and subsequent mass spectrometry analysis of liberated peptides. In parallel, non-covalently cell-wall-bound proteins extracted with potassium thiocyanate and the exoproteome fraction were analyzed by gel-free proteomics. All data are available through ProteomeXchange accession PXD000156. To pinpoint immunodominant bacterial-surface-exposed epitopes, we screened selected cell-wall-attached proteins of S. aureus for binding of immunoglobulin G from patients who have been challenged by different types of S. aureus due to chronic wound colonization. The combined results of these analyses highlight particular cell-surface-exposed S. aureus proteins with highly immunogenic exposed epitopes as potential targets for development of protective anti-staphylococcal immunization strategies.

A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface.

The human commensal bacterium Staphylococcus aureus is renowned as a causative agent of severe invasive diseases. Upon entering the bloodstream, S. aureus can infect almost every tissue and organ system in the human body. To withstand insults from the immune system upon invasion, several immune-evasive mechanisms have evolved in S. aureus, such as complement inhibition by secreted proteins and IgG-binding by surface-exposed protein A. While it is generally accepted that S. aureus cells bind a range of host factors for various purposes, no global analyses to profile staphylococcal host factor binding have so far been performed. Therefore, we explored the possibility to profile the binding of human serum proteins to S. aureus cells by "surface shaving" with trypsin and subsequent MS analysis of liberated peptides. This resulted in the identification of several components of the complement system, the platelet factor 4 and the isoform 1 of the inter-α-trypsin inhibitor heavy chain H4 on the staphylococcal cell surface. We conclude that surface shaving is a versatile tool to profile global interactions between human serum proteins and the S. aureus cell surface.

Strongly enhanced IL-10 production using stable galectin-1 homodimers.

Galectin-1 is the homodimeric form of a protein, which is present in a dynamic equilibrium with the beta-galactoside monomeric form and has potent anti-inflammatory and immunomodulating effects. These favorable effects are probably related to the induction of apoptosis in activated T cells and the induction of IL-10, which have been demonstrated to be characteristic for the dimeric form of the protein. Based on these findings it can be speculated that the in vivo effects of galectin-1 can be improved by the generation of stable galectin-1 homodimers (dGal). To test this hypothesis we produced leucine-zipper based stable galectin-1 homodimers and tested its apoptosis inducing effects on MOLT-4 cells and its immunomodulatory effects in vitro on PBMC of five independent donors. Phosphatidylserine exposure and a drop in mitochondrial membrane potential was strongly enhanced on MOLT-4 cells upon treatment with dGal as compared to wtGal. The minimal effective concentration was 20-fold reduced as compared to the minimal effective wtGal concentration. dGal showed enhanced induction of IL-10 on total PBMC as compared to treatment with wild-type protein (wtGal). The minimal effective dGal concentration was 100-fold lower than that of wtGal. Of the purified cell populations monocytes are the strongest IL-10 producers, whereas T cells induce IL-10 at a lower level and no induction is observed in B cells. Besides induction of IL-10, dGal caused an increase in IL-1beta production in all donors and a reduction of IL-2 production in 3 out of 5 donors, whereas no consistent changes were observed for other inflammatory cytokines. In summary, we demonstrated that dGal shows enhanced effects at strongly reduced concentrations. Application of dGal may therefore serve as an improved treatment of chronic inflammatory diseases.

A human monoclonal antibody that specifically binds and inhibits the staphylococcal complement inhibitor protein SCIN.

Staphylococcus aureus is a serious public health burden causing a wide variety of infections. Earlier detection of such infections could result in faster and more directed therapies that also prevent resistance development. Human monoclonal antibodies (humAbs) are promising tools for diagnosis and therapy owing to their relatively straightforward synthesis, long history of safe clinical use and high target specificity. Here we show that the humAb 6D4, which was obtained from a random screen of B-cells producing antibodies that bind to whole cells of S. aureus, targets the staphylococcal complement inhibitor (SCIN). The epitope recognized by 6D4 was localized to residues 26 to 36 in the N-terminus of SCIN, which overlap with the active site. Accordingly, 6D4 can inhibit SCIN activity as demonstrated through the analysis of C3b deposition on S. aureus cells and complement-induced lysis of rabbit erythrocytes. Importantly, while SCIN is generally regarded as a secreted virulence factor, 6D4 allowed detection of strongly increased SCIN binding to S. aureus cells upon exposure to human serum, relating to the known binding of SCIN to C3 convertases deposited on the staphylococcal cell surface. Lastly, we show that labeling of humAb 6D4 with a near-infrared fluorophore allows one-step detection of SCIN-producing S. aureus cells. Together, our findings show that the newly described humAb 6D4 specifically recognizes S. aureus SCIN, which can potentially be used for detection of human serum-incubated S. aureus strains expressing SCIN.

Short-term dietary adjustment with a hydrolyzed casein-based diet postpones diabetes development in the diabetes-prone BB rat.

From earlier studies it appears that weaning associated changes in the animal's physiology and that of the pancreas in particular, render diabetes-prone Bio-Breeding (DP-BB) rats susceptible to the induction and development of insulin-dependent diabetes mellitus (IDDM). In this study we tested whether a short-term dietary adjustment at weaning would influence the development of diabetes later in life. For this purpose a diet in which the protein source was replaced with hydrolyzed casein (HC) was given to the rats from weaning to 60 days of age and from weaning to 130 days of age. The control group received the cereal-based standard diet throughout the experiment. The short-term dietary adjustment resulted in a significant delay of diabetes development. The rats fed the HC diet from weaning to 130 days of age showed a lower incidence of diabetes at 130 days of age. No differences were seen in the histological insulitis scores between the rats of the different treatment groups. Interestingly, when testing (mucosal) immune functions of short-term HC-fed rats, their mesenteric lymph node cells (MLNC) showed increased interferon-gamma (IFN-gamma) and reduced interleukin-10 (IL-10) production after in vitro stimulation. These results demonstrate that short-term dietary adjustments at a young age can influence the course of diabetes later in life. The shift in cytokine profile of MLNC of the HC-fed rats suggests that mechanisms involved can be at the level of both the (mucosal) immune system and the beta cell.

Timing of pentoxifylline treatment determines its protective effect on diabetes development in the Bio Breeding rat.

Diabetes-prone Bio Breeding (DP-BB) rats spontaneously develop diabetes between 60 and 120 days of age. Diabetes-resistant (DR)-BB rats can be induced to develop diabetes by poly(I:C) and anti-RT6. Here, we studied the effect of pentoxifylline, a potent anti-inflammatory agent, on diabetes development in both BB rat models of insulin-dependent diabetes mellitus and investigated whether these effects were related to differential modulation of tumour necrosis factor (TNF)-alpha and interleukin-10. When DP-BB rats received pentoxifylline from day 60 onwards, diabetes development was delayed and reduced. The other treatment protocols had no effect. In DR-BB rats, pentoxifylline treatment resulted only in a delay of diabetes development. In both BB rat models, in vivo pentoxifylline treatment potently suppressed TNF-alpha, but only moderately affected interleukin-10 production in vitro. These results show that timing of pentoxifylline treatment determines its protective effect on diabetes development in DP-BB rats. The observed pentoxifylline-induced increase of the interleukin-10/TNF-alpha ratio might be a mechanism for protection or delay of the diabetes development.

Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax.

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-mug dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Human-derived, plant-produced monoclonal antibody for the treatment of anthrax.

The unpredictable nature of bio-terrorism compels us to develop medical countermeasures that will enable authorities to treat individuals exposed to agents such as anthrax. We report the feasibility of producing a protective, human-derived, monoclonal antibody directed against the protective antigen (PA) of Bacillus anthracis in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting antibody was able to neutralize toxin activity in vitro and in vivo at a comparable level to that seen for its hybridoma-produced counterpart.

Temporary, but essential requirement of CD8+ T cells early in the pathogenesis of diabetes in BB rats as revealed by thymectomy and CD8 depletion.

Autoimmunity-prone BB rats demonstrate a T lymphocytopenia and abnormal T cell subset distribution. To test whether the life span of all T cells or only of certain subsets is reduced in BB rats, we thymectomised 8-week-old BB and PVG rats and subsequently assessed size and composition of the T cell population over a 6-week-period. In both strains, thymectomy (Tx) was followed by a decrease in peripheral T cell numbers, which was proportionally larger in BB rats. The decline of the Thy-1+ recent thymic migrant (RTM) T cell phenotype was similar in both strains. BB rats showed a rapid preferential loss of CD8+ and CD45RC+ T cells, whereas the relative loss of RT6+ T cells was proportional to that of all T cells and not significantly different from that in PVG rats. Tx at 8-week did not prevent diabetes. Tx of 4-week-old BB rats revealed essentially the same changes in peripheral T cell subset distribution as in 8-week-old animals. However, Tx at week 4 did prevent diabetes. Since this raised the possibility of a temporary requirement of CD8+ T cells for the development of diabetes, we performed CD8 depletions during different pre-diabetic intervals. We found that CD8 depletion from 4 to 8 and 4 to 14 weeks, but not from 8 to 14 weeks of age prevented diabetes. We conclude that the protective effect of early adult Tx is, at least in part, due to the rapid loss of CD8+ T cells, and that these cells are only required between 4 and 8 weeks of age for diabetes to develop in BB rats.

The role of B cell-mediated T cell costimulation in the efficacy of the T cell retargeting bispecific antibody BIS20x3.

In this study, we investigated the role of the naturally occurring B cell-mediated T cell costimulation in the antitumor efficacy of the bispecific Ab BIS20x3. BIS20x3 has a dual specificity for both CD20 and CD3 and has previously been shown to effectively direct the lytic potential of cytolytic T cells toward malignant, CD20(+) B cells. BIS20x3 instigated T cell-B cell interaction caused a dose-dependent activation of T cells that was 30 times stronger when compared with T cell activation induced by monovalent anti-CD3 Abs. The activation of T cells by BIS20x3 and B cells appeared functional and resulted in the rapid induction of high lytic potential in freshly isolated peripheral T cells. BIS20x3-mediated T cell-B cell interaction resulted in a significant up-regulation of ICAM-1 on B cells and the activation of T cells was found to be dependent on the interaction of ICAM-1 with LFA-1 and trans-activation by the NF-kappaB pathway. Also, the lytic potential of freshly isolated T cells activated via BIS20x3 appeared to be dependent on NF-kappaB signaling in the target B cells. Interestingly, the costimulatory signaling effects described in this study appeared specifically related to the targeting against CD20 because targeting against CD19, by a CD3xCD19-directed bispecific Ab, was significantly less effective in inducing T cell activation and T cell-mediated B cell lysis. Together these results demonstrate that the malignant B cells actively contribute to their own demise upon CD20-directed bispecific Ab-mediated T cell targeting.