Nadine Frölich

Christian Dees

Christian Paetz

Xuan Ren

Martin J Lohse

Ph.D Viacheslav O Nikolaev
email: viacheslav.nikolaev@med.uni-goettingen.de

Meinhart H Zenk

Distinct pharmacological properties of morphine metabolites at G i -protein and -arrestin signaling pathways activated by the human µ-opioid receptor

Abstract

Morphine and several other opioids are important drugs for the treatment of acute and chronic pain. Opioid-induced analgesia is predominantly mediated by the µopioid receptor (MOR). When administered to humans, complex metabolic pathways lead to generation of many metabolites, nine of which may be considered major metabolites. While the properties of the two main compounds, morphine-6glucuronide and morphine-3-glucuronide, are well described, the activity of other morphine metabolites is largely unknown. Here we performed an extensive pharmacological characterization by comparing efficacies and potencies of morphine and its nine major metabolites for the two main signaling pathways engaged by the human MOR, which occur via G i -protein activation and β-arrestins, respectively. We used radioligand binding studies and FRET-based methods to monitor MORmediated G i -protein activation and β-arrestin recruitment in single intact 293T cells. This approach identified two major groups of morphine metabolites, which we classified into "strong" and "weak" receptor ligands. Strong partial agonists morphine, morphine-6-glucuronide, normorphine, morphine-6-sulfate, 6-acetylmorphine and 3acetylmorphine showed efficacies in the nanomolar range, while the weak metabolites morphine-N-oxide, morphine-3-sulfate, morphine-3-glucuronide and pseudomorphine activated MOR pathways only in the micromolar range.

Interestingly, three metabolites, normorphine, 6-acetylmorphine and morphine-6glucuronide, had lower potencies for Gi-protein activation but higher potencies and efficacies for -arrestin recruitment than morphine itself, suggesting that they are biased towards -arrestin pathways.

Key words: morphine, metabolite, FRET, G-protein, -arrestin A c c e p t e d M a n u s c r i p t

Introduction

Morphine is an important alkaloid from opium poppy and serves as the prototypical analgesic drug. Morphine and some other opioids, for example 3,6-di-acetylmorphine (heroin) are also relevant for drug abuse. Analgesic effects of opioids are achieved largely via activation of µ-opioid receptors (MOR) [START_REF] Corbett | 75 years of opioid research: the exciting but vain quest for the Holy Grail[END_REF][START_REF] Jordan | Molecular mechanisms of opioid receptor signal transduction[END_REF]. MOR is a G-protein-coupled receptor (GPCR), which can activate several cellular signaling pathways. The first pathway is mediated by the activation of inhibitory pertussis-toxin sensitive Gproteins (G i/o -proteins). The second pathway is induced by the recruitment of arrestins to the receptor, which leads to subsequent activation of other, so-called "non-classical" signaling cascades such as the mitogen activated protein kinase pathway [START_REF] Dang | Two distinct mechanisms mediate acute muopioid receptor desensitization in native neurons[END_REF][START_REF] Lefkowitz | Transduction of receptor signals by beta-arrestins[END_REF].

When administered to humans, morphine and other opiate alkaloids undergo extensive biotransformation by various metabolic pathways. The predominant metabolic pathway involves conjugation with glucuronic acid, which leads to the major metabolites morphine-3-glucuronide and morphine-6-glucuronide [START_REF] Hasselstrom | Morphine pharmacokinetics and metabolism in humans. Enterohepatic cycling and relative contribution of metabolites to active opioid concentrations[END_REF]. Other metabolic pathways include conjugation with sulfonic acid, leading to morphine-6sulfates and morphine-3-sulfates, acetylation to 3-and 6-acetylmorphines, oxidation to morphine-N-oxide and demethylation to normorphine [START_REF] Smith | Opioid metabolism[END_REF][START_REF] Skarke | Morphine metabolites: clinical implications[END_REF] (Fig. 1). Among all known morphine metabolites, the pharmacological properties have been best studied for morphine-6-glucuronide and morphine-3-glucuronide, which are generally considered as active and inactive metabolites, respectively [START_REF] Wittwer | Role of morphine's metabolites in analgesia: concepts and controversies[END_REF]. Much less is known about the activity of other metabolites, since no systematic pharmacological analysis has so far been performed, and only rare single reports on these compounds are A c c e p t e d M a n u s c r i p t 4 available. Lack of information about opiate metabolites does not allow definitive conclusions about their roles in analgesia and in adverse effects of morphine [START_REF] Smith | Opioid metabolism[END_REF][START_REF] Skarke | Morphine metabolites: clinical implications[END_REF].

To study the pharmacological properties of various ligands for G protein-coupled receptors, including MOR, we have developed a new real-time approach to monitor G i -protein activation in intact cells by using biosensors which are based on Förster resonance energy transfer (FRET) [START_REF] Bünemann | Gi protein activation in intact cells involves subunit rearrangement rather than dissociation[END_REF][START_REF] Nikolaev | Live cell monitoring of mu-opioid receptor-mediated G-protein activation reveals strong biological activity of close morphine biosynthetic precursors[END_REF][START_REF] Lohse | Optical techniques to analyze real-time activation and signaling of G-proteincoupled receptors[END_REF]. In addition, we have developed another technique to monitor -arrestin recruitment to various receptors by measuring FRET between a receptor and -arrestin [START_REF] Krasel | Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation[END_REF][START_REF] Klenk | Formation of a ternary complex among NHERF1, beta-arrestin, and parathyroid hormone receptor[END_REF]. These approaches allow us to study pharmacological properties of different receptors and their ligands in single intact cells with high temporal and spatial resolution [START_REF] Lohse | Kinetics of G-protein-coupled receptor signals in intact cells[END_REF][START_REF] Lohse | Kinetic analysis of G protein-coupled receptor signaling using fluorescence resonance energy transfer in living cells[END_REF].

Here we used these FRET assays together with radioligand binding studies to perform a systematic analysis of the pharmacological properties of all major morphine metabolites at the human MOR. We found that morphine metabolites exert either strong or weak signaling effects, often comparable to morphine itself, and that some metabolites show a previously unrecognized -arrestin-biased behavior and are more efficacious in recruiting -arrestin to the receptor than morphine itself.

A c c e p t e d M a n u s c r i p t 5

Material and methods

Substances

Morphine, morphine-6-glucuronide, morphine-3-glucuronide and DAMGO ([D-Ala 2 , N-MePhe 4 , Gly-ol]-enkephalin) were purchased from Sigma-Aldrich (Deisenhofen, Germany). Normorphine, 3-acetylmorphine and 6-acetylmorphine were from Lipomed GmbH (Weil am Rhein, Germany).

Morphine-N-oxide, morphine-3-sulfate, morphine-6-sulfate and pseudomorphine were synthesized as follows. Morphine-N-oxide and pseudomorphine were prepared as described by Garrido et al. [START_REF] Garrido | Voltammetric oxidation of drugs of abuse -I. Morphine and metabolites[END_REF]. Morphine-3-sulfate was prepared via 6acetylmorphine as previously described [START_REF] Wright | The enzymatic acetylation of heroin and related morphine derivatives by blood serum[END_REF] and further sulfonated and deprotected by a procedure described by Mori et al. [START_REF] Mori | Chemical synthesis and analgesic effect of morphine ethereal sulfates[END_REF]. Morphine-6-sulfate was synthesized from morphine hydrochloride according to Preechagoon et al. [START_REF] Preechagoon | Ester prodrugs of a potent analgesic, morphine-6-sulfate: syntheses, spectroscopic and physicochemical properties[END_REF]. Except where noted, all other chemicals were obtained from Sigma-Aldrich.

Plasmids

Human MOR-1A cDNA was obtained from imaGenes GmbH (Berlin, Germany) and subcloned into the pcDNA3 expression vector (Invitrogen) using HindIII and XbaI restriction sites. All fluorescent G i -protein subunits used to monitor G i activation by FRET have previously been described [START_REF] Bünemann | Gi protein activation in intact cells involves subunit rearrangement rather than dissociation[END_REF][START_REF] Nikolaev | Live cell monitoring of mu-opioid receptor-mediated G-protein activation reveals strong biological activity of close morphine biosynthetic precursors[END_REF]. Briefly, the rat PTX-insensitive G αi1protein (C351I mutant) was labeled with enhanced yellow fluorescent protein (YFP) between positions 91 and 92. Human G γ2 was C-terminally tagged with enhanced cyan fluorescent protein (CFP), and the untagged human G β1 cDNA was co-A c c e p t e d M a n u s c r i p t 6 expressed. For the FRET-based measurements of β-arrestin-2 recruitment, the plasmids encoding the human MOR-1A C-terminally tagged with CFP, bovine βarrestin-2 C-terminally tagged with YFP, and untagged human G protein-coupled receptor kinase (GRK) 2 were used [START_REF] Krasel | Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation[END_REF].

Cell culture and transfections

293T cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin (all from Pan Biotech GmbH, Aidenbach, Germany) at 37°C and 7% CO 2 . Cells were seeded onto poly-D-lysine (Sigma) coated glass cover slips for FRET experiments. Transient transfections were performed with Effectene Transfection reagent (Quiagen, Hilden, Germany) according to the manufacturer's protocol, and FRET measurements were done 48 h after transfection.

To monitor G i -protein activation, the following plasmids were transfected (µg DNA per 6-well plate): MOR 0.7, G αi1 -YFP 0.8, G β1 0.5, G γ2 -CFP 0.2. To measure β-arrestin recruitment, cells were transfected with (µg DNA per 6-well plate): MOR-CFP 0.3, GRK2 0.2, β-arrestin-2-YFP 0.2). For receptor binding studies, 1.9 µg MOR plasmid were transfected per 90 mm cell-culture dish.

Receptor binding assays

MOR expressing 293T cells were homogenized in 5 mM Tris, 2 mM EDTA, pH 7.4.

The cell homogenate was centrifuged for 10 min at 1700xg, and the remaining

FRET measurements

FRET experiments were performed on a Zeiss Axiovert 200 inverted microscope with a PLAN-Neofluar oil immersion 100x objective and a dual emission photometric system as previously described [START_REF] Nikolaev | Live cell monitoring of mu-opioid receptor-mediated G-protein activation reveals strong biological activity of close morphine biosynthetic precursors[END_REF]. Experiments were done at room temperature in a buffer containing 144 mM NaCl, 5.4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, pH 7.3. Cells were excited using the monochromatic light from a Polychrome IV (Till Photonics, Gräfelfing, Germany) at 436 nm ± 10 nm and a frequency of 5 Hz. Emitted light was separated by the DCLP 505 beam splitter into two emission channels recorded at 480 ± 20 nm (CFP) and 535 ± 10 nm (FRET channel). The F 535 /F 480 emission ratio was offline corrected for bleedthrough, direct YFP excitation and photobleaching using Origin software in order to obtain corrected F YFP/CFP ratios, which were used throughout the manuscript. Ratiometric traces were normalized to the initial ratio (set to 1).

A c c e p t e d

M a n u s c r i p t 8

Results

We analyzed the pharmacological properties of morphine and its nine major metabolites in three types of assays, namely radioligand binding studies, G-protein activation measurements and an assay to monitor -arrestin recruitment to the opiate receptor. These experiments were designed to compare the action of morphine metabolites and their efficacies and potencies for two distinct signaling pathways engaged by human MOR.

Radioligand binding studies

Initially, we determined the affinities of morphine and its major metabolites (plus DAMGO as a reference agonist) for MOR in radioligand binding studies with cell membranes prepared from 293T cells expressing the receptor, using the antagonist [ 3 H]naloxone as the radioligand. Competition displacement curves for morphine and DAMGO (Fig. 2) showed K i -values, which were comparable to published values for these ligands at the human MOR [START_REF] Befort | A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling[END_REF][START_REF] Spivak | Naloxone activation of mu-opioid receptors mutated at a histidine residue lining the opioid binding cavity[END_REF]. Next, we measured the K i -values of the major morphine metabolites (see Fig. 1). The radioligand displacement curves (Fig. 2) revealed two groups of metabolites, which we classified into "strong" and "weak" ligands. The "strong" metabolites, normorphine, 3-and 6-acetylmorphine, morphine-6-glucuronide and -6-sulfate showed affinities in the nanomolar range, while the "weak" ligands, pseudomorphine, morphine-N-oxide, morphine-3-glucuronide and -3sulfate, had micromolar K i -values (Fig. 2 and Table 1).

G i -protein activation

Based on these affinity measurements we then set out to investigate the metabolites' functional effects. To monitor ligand-induced activation of inhibitory G-proteins, cells A c c e p t e d M a n u s c r i p t 9 expressing human MOR and fluorescently labeled G i -protein subunits were treated with the different morphine metabolites (Fig. 3A). In this assay, receptor activation leads to a rapid and reversible decrease of the FRET ratio, which reflects structural rearrangements between the G-protein subunits, and [START_REF] Bünemann | Gi protein activation in intact cells involves subunit rearrangement rather than dissociation[END_REF]. In MOR expressing cells, we observed robust decreases of the FRET ratio upon morphine treatment, which were even further enhanced by the full receptor agonist DAMGO, suggesting that morphine acts only as a strong partial agonist at MOR (Fig. 3B). Addition of naloxone on top of these ligands led to a complete, relatively slow, deactivation of G-proteins, confirming that our system can be used to monitor specific pharmacological effects of receptor agonists, partial agonists and antagonists (Fig. 3B).

Next, we set out to compare FRET responses produced by morphine with those observed upon receptor activation with its various metabolites. Some of the metabolites, for example morphine-6-glucuronide, showed a strong G-protein activation signal, which had a similar amplitude as the response to morphine itself (Fig. 3C). Other metabolites, for example morphine-N-oxide, even at high concentrations, produced only partial effects, ranging from 70 to 90% of the morphine response (Fig. 3D).

To systematically analyze efficacies and potencies of all major morphine metabolites, we recorded concentration-response dependencies for the nine different compounds and compared them to morphine by normalizing their maximal FRET responses to the change of ratio evoked by the saturating morphine concentration of 10 µM. Again, these experiments identified the two groups of morphine metabolites that were seen in the radioligand binding experiments, i.e. the "strong" and "weak" ligands (Fig. 3E).

The strong metabolites all showed not only potencies in the nanomolar range, but also efficacies at the MOR, which were comparable to morphine itself. These strong A c c e p t e d M a n u s c r i p t 10 partial MOR agonists were morphine-6-glucuronide, normorphine, morphine-6-sulfate and both 3-and 6-acetylmorphine. The second group of "weak" metabolites had much lower potencies and slightly lower efficacies compared to morphine. These metabolites, morphine-N-oxide, morphine-3-sulfate and 3-glucuronide, as well as pseudomorphine, were able to activate G i -proteins only in the micromolar concentration range, and their maximal effects ranged from 75 to 100% of those of morphine (Fig. 3E, Table 1).

-Arrestin recruitment

To monitor the activity of the second important group of signaling pathways activated by MOR, we studied the effects of morphine metabolites on the ligand-induced arrestin translocation to this receptor. We chose the previously well established FRET-based approach which relies on the use of C-terminally CFP-tagged MOR and C-terminally YFP-tagged -arrestin-2 [START_REF] Krasel | Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation[END_REF], which were transfected into 293T cells together with the receptor kinase GRK2 to assure full agonist-triggered MOR phosphorylation. Ligand-dependent MOR activation leads to GRK-mediated receptor phosphorylation and subsequent -arrestin-YFP recruitment, which can be monitored as an increase of FRET (Fig. 4A). Again, morphine showed the typical response of a partial agonist, which was ~30% smaller than the change of FRET ratio evoked by the full MOR agonist DAMGO (Fig. 4B). Compatible with the need of prior GRKmediated receptor phosphorylation [START_REF] Krasel | Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation[END_REF], these responses were slower than those observed for G i (compare Figs. 3B and4B; note the different time scales). Next, we continued to compare the FRET responses produced by morphine metabolites with their parent compound. Strikingly, we found that some of the "strong" metabolites, such as normorphine (Fig. 4C), morphine-6-glucuronide and 6-acetylmorphine, A c c e p t e d M a n u s c r i p t 11 induced a significantly stronger -arrestin recruitment than morphine itself. Therefore, we normalized all metabolite responses to the maximal FRET response produced by 10 µM of DAMGO and measured concentration-response dependencies for all nine morphine metabolites (Figs. 4D,E). As in the case of G i -protein activation experiments, there were again the two groups of "strong" and "weak" partial agonists, which included the same members. Morphine-N-oxide and morphine-3-glucuronide showed lower potencies and significantly lower efficacies compared to DAMGO and morphine (Fig. 4D), while the other two weak metabolites were capable of translocating -arrestin to the same extent as morphine, but at much higher concentrations (Fig. 4E and Table 1). Overall, the spread of efficacies was much greater in this assay than in the G i -activation assay (compare Figs. 3E and4E).

Finally, we normalized the efficacies for all ligands measured in G i -and -arrestin assays to the maximal DAMGO response to graphically compare the extent of activation for each ligand and signaling pathway (Fig. 5)

Discussion

In this study, we performed a systematic analysis of the pharmacological properties of morphine metabolites at the human MOR. We analyzed the ability of these substances to bind to the receptor and to activate the two major independent downstream signaling pathways, which are activation of G i -proteins and -arrestin recruitment. The information obtained in these assays allows to predict, which of these metabolites might play a role in MOR signaling in vivo.

A c c e p t e d M a n u s c r i p t

To analyze MOR activation, we used novel FRET-based approaches for real-time monitoring of G i -protein activation and -arrestin recruitment. These approaches provide pharmacological information at the single-cell level and directly at the initiating biochemical event. While the results of FRET measurements of G i -protein activation generally match well with the data obtained in classical GTPS binding studies [START_REF] Nikolaev | Live cell monitoring of mu-opioid receptor-mediated G-protein activation reveals strong biological activity of close morphine biosynthetic precursors[END_REF], they provide fast kinetic readouts and can be done in intact cells, i.e. under much more physiological conditions. Likewise, measurements of -arrestin recruitment by FRET allow visualization of receptor/-arrestin interaction in real time.

These novel assays thus reflect the pharmacodynamic properties of receptor ligands at their target intact cells, but without the pharmacokinetic constraints typical for in vivo experiments. For example, morphine-6-glucuronide, when directly injected into mice, has been shown to induce its behavioral effects much slower than morphine [START_REF] Handal | Pharmacokinetic differences of morphine and morphine-glucuronides are reflected in locomotor activity[END_REF]. However, in our study, we observed similarly fast kinetics of G i -protein activation as well as comparable efficacies and potencies for the two substances. The discrepancy between the results of the single-cell experiments and in vivo studies can be explained by differences in pharmacokinetics, which largely depend on the chemical structure and parameters such as hydrophobicity of the various metabolites. Indeed, it has been demonstrated that both morphine glucuronides are much slower at crossing the blood-brain barrier than morphine [START_REF] Bickel | Poor permeability of morphine 3-glucuronide and morphine 6-glucuronide through the blood-brain barrier in the rat[END_REF]. The single-cell pharmacological analyses presented in this study are capable of directly reporting dynamic properties of signaling at the level of target cells and therefore reflect the real pharmacodynamics of these compounds at their receptor.

There are two sources of morphine and its metabolites which are relevant for the human body. One is exogenous morphine and other analgesic opiates, which are A c c e p t e d M a n u s c r i p t 13 administered for pain therapy or during opiate abuse. In this case, the circulating opiate concentrations can reach relatively high micromolar peak levels, which gradually decrease through metabolic pathways [START_REF] Handal | Pharmacokinetic differences of morphine and morphine-glucuronides are reflected in locomotor activity[END_REF][START_REF] Sawe | Oral morphine in cancer patients: in vivo kinetics and in vitro hepatic glucuronidation[END_REF]. The second source of opiates in the human body is the endogenous morphine biosynthesis, which is capable of producing high nanomolar concentrations of morphine from endogenous biosynthetic precursors [START_REF] Boettcher | How human neuroblastoma cells make morphine[END_REF][START_REF] Poeaknapo | Endogenous formation of morphine in human cells[END_REF][START_REF] Zhu | Human white blood cells synthesize morphine: CYP2D6 modulation[END_REF]. Much is known about the metabolic pathways which lead to morphine conjugation and biotransformation (see Fig. 1). 60-70% of exogenously administrated morphine is conjugated [START_REF] Hasselstrom | Morphine pharmacokinetics and metabolism in humans. Enterohepatic cycling and relative contribution of metabolites to active opioid concentrations[END_REF] to morphine-3-glucuronide (~55%) and morphine-6-glucuronide (~10%). After oral or parenteral morphine administration to patients, slightly higher plasma concentrations of morphine-6glucuronide and ~10-times higher concentration of morphine-3-glucuronide can be measured for up to 12 hours [START_REF] Sawe | Oral morphine in cancer patients: in vivo kinetics and in vitro hepatic glucuronidation[END_REF]. Morphine-3-glucuronide is a very weak metabolite, whereas morphine-6-glucuronide can activate MOR almost as strongly as morphine.

For morphine-3-and morphine-6-glucuronides we were able to confirm the previously observed differences [START_REF] Wittwer | Role of morphine's metabolites in analgesia: concepts and controversies[END_REF] in their pharmacological properties. The former metabolite has been classified as a weak MOR ligand, while the latter glucuronide is a strong activator of MOR-induced signaling with potencies comparable to morphine.

Much less has been known about the pharmacological properties of other metabolites of morphine, which include normorphine, morphine-3-and morphine-6sulfates, as well as 3-and 6-acetylmorphines. Normorphine is the N-demethylated metabolite of morphine which has been described as an active but slightly weaker opioid receptor ligand compared to morphine or morphine-6-glucuronide [START_REF] Sullivan | The spinal antinociceptive actions of morphine metabolites morphine-6-glucuronide and normorphine in the rat[END_REF][START_REF] Glare | Clinical pharmacokinetics of morphine[END_REF]. In our study, we confirmed this observation and showed that normorphine activates G iproteins at slightly higher concentrations than morphine. Morphine-6-sulfate, similarly A c c e p t e d M a n u s c r i p t 14 to morphine-6-glucuronide, is a potent ligand, which requires ~10-50-times lower parenteral dose to achieve an analgesic effect comparable to morphine [START_REF] Zuckerman | Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate[END_REF]. In contrast, morphine-3-sulfate is a weaker metabolite, which has been described to have much lower binding affinity and lower analgesic potency [START_REF] Brown | Analgesic potencies of morphine 3-and 6-sulfates after intracerebroventricular administration in mice: relationship to structural characteristics defined by mass spectrometry and nuclear magnetic resonance[END_REF][START_REF] Chen | Mu receptor binding of some commonly used opioids and their metabolites[END_REF]. In this study, we found that morphine-6-sulfate has binding and signaling properties similar to morphine, so that the analgesic potency of this metabolite cannot be explained by distinct pharmacodynamic properties but must be pharmacokinetic in origin.

Morphine-3-sulfate, however, was much less potent and fell into the group of weak metabolites, which is compatible with earlier observations. The 3-and 6acetylmorphines, which represent important heroin metabolites, have been reported to have similar or slightly less potent pharmacological effects than morphine [START_REF] Brown | Analgesic potencies of morphine 3-and 6-sulfates after intracerebroventricular administration in mice: relationship to structural characteristics defined by mass spectrometry and nuclear magnetic resonance[END_REF] which was also the case in our single-cell experiments. Likewise, the weak analgesic morphine-N-oxide, which has been found to be 10-90 times less potent than morphine [START_REF] Fennessy | The analgesic action of morphine-n-oxide[END_REF], showed almost 1000 times lower affinity and potencies in FRET assays.

Interestingly, we found that three metabolites, normorphine, 6-acetylmorphine and morphine-6-glucuronide, showed lower potencies for G i -protein activation but higher potencies and efficacies for -arrestin recruitment than morphine (compare Fig. 2E and Fig. 3E, see also Table 1 and Fig. 5). This finding suggests that such ligands might have a bias for -arrestin over G-protein signaling. Although they induce weaker responses at G-proteins, these metabolites can activate -arrestin signaling pathways much strongerpathways, which have been mostly associated with the pharmacological effects of opioid peptides. The most interesting aspect of this study is the observation that morphine metabolites show differential preference for the two major signalling pathways triggered by G i -proteins and -arrestins, respectively. A more detailed understanding of these two signalling pathways in eliciting the various pharmacological effects of morphine is certainly necessary before we might be able to exploit these differences for drug therapy. However, our data clearly show that there is such a potential for differential effects of morphine metabolites at the MOR. Data are presented as means ± SE (n=4-8). Individual data points were fitted to a single binding site model, and the obtained K i -values are presented in the Table 1.

A c c e p t e d M a n u s c r i p t 7 supernatant

 7 was centrifuged for 30 min at 50,000xg. The resulting pellet was resuspended in 50 mM of potassium phosphate buffer, pH 7.4, and aliquots were frozen in liquid nitrogen. 20 µg of membrane protein were mixed with 0.1-1 nM of 3[H]naloxone (American Radiolabeled Chemicals, Inc., St. Louis, USA), 100 µM GTP and various MOR ligand concentrations in 200 µl of 50 mM potassium phosphate buffer. Samples were incubated at room temperature for 3 h, then filtered through GF/F glass fiber filters (Millipore, Schwalbach, Germany) before liquid scintillation counting.

A

  c c e p t e d M a n u s c r i p t 15 Based on the results of this study, one can predict whether various opioid ligands, especially morphine metabolites, can have pharmacological effects in vivo. In physiological settings, weak metabolites would not activate MOR, because they do not reach high micromolar concentrations, unless high opioid amounts have been exogenously administrated. Several strong metabolites, such as normorphine and the main heroin metabolites 3-and 6-acetylmorphine, are much more relevant for receptor activation. Endogenously synthesized morphine and its strong metabolites might be pharmacologically relevant only if they are present at high nanomolar concentrations. In addition, normorphine and 6-acetylmoprhine, together with morphine-6-glucuronide, can trigger previously unrecognized stronger activation of the -arrestin signalling.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Competition radioligand displacement curves for [ 3 H]-naloxone measured

Figure 3 .

 3 Figure 3. Real-time monitoring of the morphine metabolite-induced effects on MOR-

Figure 4 .

 4 Figure 4. Measurements of -arrestin recruitment to the activated MOR by FRET. (A)

Figure 5 .

 5 Figure 5. Correlation of efficacies which various opioid ligands have at activating

Table 1 .

 1 Pharmacological properties of various morphine metabolites at the human µ-opioid receptor (MOR) measured by radioligand binding and FRET assays. EC 50 values for G i -protein activation and -arrestin recruitment in the FRET-based assays show concentrations at which a half-maximal response for each individual substance was observed. Data from competitive displacement experiments using 3 [H]-naloxone and MOR-expressing cell membranes are presented as K i values. All data are means ± SE calculated from three to seven independent experiments.

Figure 1

 1 Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image

Figure 5

 5 Figure 5 Click here to download high resolution image

Table 1

 1

			Assay
	Substance
		G i activation	β-arrestin recruitment	Binding
		EC 50 (µM),	EC 50 (µM),	K i (µM),
		means ± SE	means ± SE	means ± SE
	DAMGO	0.046 ± 0.004	0.060 ± 0.002	0.005 ± 0.002
	Morphine	0.015 ± 0.002	0.457 ± 0.157	0.022 ± 0.004
	Morphine-6-
	glucuronide	0.037 ± 0.008	0.338 ± 0.062	0.063 ± 0.014
	Normorphine	0.040 ± 0.007	0.267 ± 0.015	0.061 ± 0.013
	3-Acetylmorphine	0.057 ± 0.010	0.594 ± 0.019	0.098 ± 0.029
	Morphine-6-sulfate	0.053 ± 0.005	0.196 ± 0.036	0.047 ± 0.004
	6-Acetylmorphine	0.100 ± 0.021	0.339 ± 0.023	0.054 ± 0.022

Acknowledgements

The study was supported by the Deutsche Forschungsgemeinschaft (SFB487), the European Research Council (TOPAS) and the National Institutes of Health (NIH) (5R21DA024418-02).

Abbreviations: DAMGO, [D-Ala 2 , N-MePhe 4 , Gly-ol]-enkephalin; FRET, fluorescence (or: Förster) resonance energy transfer; GPCR, G-protein-coupled receptor; GRK, Gprotein-coupled-receptor kinase.


Dr Silva Holtfreter
email: s.holtfreter@auckland.co.nz

Joanna Jursa-Kulesza

Helena Masiuk

Nelianne J Verkaik

Corne De Vogel

Julia Kolata

Monika Nowosiad

Leif Steil

Willem Van Wamel

Alex Van Belkum

Uwe Völker

Stefania Giedrys-Kalemba

Barbara M Bröker

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

Keywords: autovaccination, Staphylococcus aureus, furunculosis, antibody, 2D-immunoblot, Luminex

Antibody responses in furunculosis patients vaccinated

with autologous formalin-killed

Introduction

Besides being a common colonizer of human skin and mucosa, Staphylococcus aureus also acts as a major human pathogen. The species can cause a broad range of infections, most frequently skin and soft tissue infections, such as wound infections, furuncles, carbuncles and abscesses, but also life-threatening systemic infections, such as pneumonia and sepsis [START_REF] Dryden | Complicated skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus: epidemiology, risk factors, and presentation[END_REF][START_REF] Lina | Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[END_REF][START_REF] Wertheim | Risk and outcome of nosocomial Staphylococcus aureus bacteraemia in nasal carriers versus non-carriers[END_REF]. Furunculosis is a common staphylococcal skin disease characterised by painful, deep infections of the hair follicle. Even mild lesions are painful and unsightly and often leave a scar after they heal [START_REF] Cu | A study of edema disease in pigs in Vietnam with particular reference to the use of autovaccine for the prevention of disease[END_REF]. Antibiotic treatment is frequently not effective, and many furunculosis patients suffer from recurrent episodes or develop chronic symptoms [START_REF] Cu | A study of edema disease in pigs in Vietnam with particular reference to the use of autovaccine for the prevention of disease[END_REF].

The alarming global spread of antibiotic resistant strains has spurred efforts to develop active and passive antistaphylococcal vaccines [START_REF] Boucher | Epidemiology of Methicillin-Resistant Staphylococcus aureus[END_REF][START_REF] Schaffer | Staphylococcal vaccines and immunotherapies[END_REF]. However, vaccine development is a challenging task, because both the species S. aureus and the host response that it induces are highly variable. Two S. aureus strains can differ drastically in their virulence gene content [START_REF] Lindsay | Staphylococcus aureus: superbug, super genome?[END_REF]. The variable genome consists of mobile genetic elements such as pathogenicity islands and phages that encode numerous virulence factors, including toxins, exoenzymes and immune modulators [START_REF] Lindsay | Staphylococcus aureus: superbug, super genome?[END_REF][START_REF] Holtfreter | Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates[END_REF][START_REF] Novick | The SaPIs: mobile pathogenicity islands of Staphylococcus[END_REF][START_REF] Goerke | Diversity of prophages in dominant Staphylococcus aureus clonal lineages[END_REF]. In concert with conserved virulence factors, these variable bacterial compounds could determine differential pathogenesis [START_REF] Holtfreter | Towards the immune proteome of Staphylococcus aureus -The anti-S. aureus antibody response[END_REF]. We observed a strong and strain-specific antibody response against these variable antigens in S. aureus carriers and during the natural course of S. aureus bacteremia [START_REF] Holtfreter | Staphylococcus aureus carriers neutralize superantigens by antibodies specific for their colonizing strain: a potential explanation for their improved prognosis in severe sepsis[END_REF][START_REF] Kolata | Distinctive patterns in the human antibody response to S. aureus bacteremia revealed by a prospective and personalized immune proteome approach submitted[END_REF].

Active vaccination can be based on mono-or multivalent subunit vaccines or on whole cell vaccines, which include autologous vaccines (short: autovaccines) [START_REF] Schaffer | Staphylococcal vaccines and immunotherapies[END_REF]. Autovaccines are individually prepared from the autologous infecting bacterial strain [START_REF] Halasa | Evaluation of certain immunological parameters in the course of autovaccine treatment in patients with chronic ostitis and carbunculosis[END_REF][START_REF] Wright | Notes on the treatment of furnculosis, sycosis, and acne by the inoculation of a staphylococcus vaccine and generally on the treatment of localised bacterial invasions by therapeutic inoculations of the corresponding bacterial vaccines[END_REF]. Following subculture, the bacteria and their secreted proteins are usually inactivated by fixation, heat or cell lysis, and then repeatedly applied orally or subcutaneously [START_REF] Halasa | Evaluation of certain immunological parameters in the course of autovaccine treatment in patients with chronic ostitis and carbunculosis[END_REF][START_REF] Ring | Chronic posttraumatic osteomyelitis. Attempt at oral autovaccine therapy][END_REF].

In contrast to subunit vaccines, autovaccines contain poorly characterized variegated cocktails of surface proteins and secreted virulence factors produced by the infecting strain.

Before the antibiotic era, chronic staphylococcal infections such as chronic furunculosis and osteomyelitis were frequently treated by therapeutic vaccination with autologous formalin-killed S. aureus cells [START_REF] Wright | Notes on the treatment of furnculosis, sycosis, and acne by the inoculation of a staphylococcus vaccine and generally on the treatment of localised bacterial invasions by therapeutic inoculations of the corresponding bacterial vaccines[END_REF][START_REF] Scott | A record of the treatment of bacterial infections by autogenous vaccines[END_REF][START_REF] Goodale | Clinical observations on use of autogenous vaccine made from cultures of coagulase positive hemolytic Staphylococcus aureus[END_REF][START_REF] Lewin | Therapy of disseminated furunculosis with autogenous and heterogenous vaccines[END_REF]. Today, autovaccination is still regularly performed in some Eastern European countries, including Poland and the Czech Republic [START_REF] Halasa | Evaluation of certain immunological parameters in the course of autovaccine treatment in patients with chronic ostitis and carbunculosis[END_REF][START_REF] Novotny | Treatment with autovaccines and the immunologic profile of patients]. [Czech][END_REF]. It is offered as a therapeutic alternative to patients with chronic S. aureus infections that are refractory to standard therapy. Moreover, bacterial whole cell vaccines are commonly used in veterinary medicine to treat chronic infectious diseases [START_REF] Cu | A study of edema disease in pigs in Vietnam with particular reference to the use of autovaccine for the prevention of disease[END_REF][START_REF] Nolte | Autovaccination of dairy cows to treat post partum metritis caused by Actinomyces pyogenes[END_REF][START_REF] Nolte | Homologe Autovakzination in der Behandlung von Infektionskrankheiten der Pferde -Ergebnisse einer retrospektiven Erhebung[END_REF]. The major argument against the use of autovaccines in human medicine is that safety and efficacy have not been determined in controlled clinical trials. Moreover, the mode of action is largely unknown.

In this prospective pilot study we analyzed if autovaccination influences the serum antibody response to a broad spectrum of secreted and surface-bound S. aureus antigens.

Materials and Methods

Autovaccination patients: The Department of Medical Microbiology and Immunology at the Pomeranian Medical University, Szczecin, Poland has long-lasting experience with autovaccination for therapy of chronic S. aureus furunculosis and osteomyelitis [START_REF] Halasa | Evaluation of certain immunological parameters in the course of autovaccine treatment in patients with chronic ostitis and carbunculosis[END_REF]. This prospective pilot study included four patients (3 female and one male) from the Szczecin area. They suffered from chronic or recurrent furunculosis and asked for autovaccination, one of the treatment options in Poland. In all four patients previous antibiotic treatment and surgical intervention had been unsuccessful. Patient data and anatomic location of the furuncles are shown in table 1. The patients showed no signs of immune suppression. All four patients provided their written informed consent and the study was approved by the Ethics Board of the University of Szczecin.

S. aureus strains and sera:

The S. aureus vaccine strains were isolated from infected lesions (pus) during episodes of furunculosis. Moreover, nasal swabs were taken repeatedly to determine the nasal colonization status. S. aureus was identified on the basis of generally accepted criteria: the presence of clumping factor (slide test) and extracellular coagulase (tube test), and biochemical identification (ID 32 Staph, BioMerieux). The infecting and colonizing S. aureus isolates were stored as glycerol stocks.

Antimicrobial susceptibility of isolates was estimated by the disc diffusion method according to current recommendations by the NCCLS/CLSI. Resistance to oxacillin/cephoxitin was confirmed by detection of PBP 2′ (Slidex MRSA detection, BioMerieux) and mecA PCR (see below).

Sera were obtained directly before and during autovaccination treatment and stored at -80 °C. We obtained four consecutive serum samples from patient 6466 (day 0, 86, 273 and 332), three samples from patients 7293 (day 0, 42, and 187) and 9105 (day 0, 85, and 166), and two samples from patient 7510 (day 0, and 63). Additionally, control sera from 11 healthy lab workers from the Department of Microbiology and Immunology, Pomeranian Medical University, Szczecin (age 37.6 ± 10.7 years; 36% male) were analyzed.

Preparation of autologous vaccines:

Autovaccines were individually prepared from the causative S. aureus strain, which was isolated from furunculosis lesions. Autologous strains were cultivated for a maximum of 24 h on oblique test tubes containing PPLO agar (Becton Dickinson) supplemented with 10% (w/v) glucose and 0.1% (v/v) Tween80. Bacteria were resuspended in 0.9% NaCl solution and fixed with 0.4% formalin solution for 48 h. The bacterial suspension was adjusted to 5x10 8 bacteria/ml (suspension I), 1x10 9 bacteria/ml (suspension II), and 2.5x10 9 bacteria/ml (suspension III) based on McFarland quantification. Afterwards, 0.1 mg/ml thiomersal was added to these suspensions as preservative. Finally, sterility of autovaccine preparation was tested by cultivation in thioglycollate medium (Becton Dickinson) for seven days.

Administration of autovaccines:

Autovaccines were prepared by the Department of Microbiology and Immunology, Pomeranian Medical University in Szczecin, and administered by the patients' general practitioners. Before the beginning of treatment, a skin test was performed to exclude hypersensitivity of the patient to any of the vaccine components. 0.5 ml of a 1:10 dilution of suspension I in Aqua ad injectionem was injected intradermally. Skin reaction was checked after 15 min and 24 hours. Autovaccines were then applied subcutaneously in 3-5 days intervals over 3 months. With each injection the dose was increased based on the following application scheme. Suspension I (5x10 8 bacteria/ml) was applied in volumes of 0.1, 0.2, 0.3, 0.4, 0.5 ml, followed by suspension II (1x10 9 bacteria/ml; 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml) and suspension III (2.5x10 9 bacteria/ml; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml). This immunization procedure was followed by several recall injections (suspension III, 1 ml) every 1-3 weeks for up to 5 -12 months. After each injection the patients were closely monitored for 30 minutes (swelling, pain or rash on injection side, blood pressure, breathing). After every vaccination, presumptive systemic symptoms (fever, headache, general malaise, sore throat etc.) were also analyzed by a physician to exclude any side effects associated with autovaccination. Moreover, laboratory tests were performed before autovaccination and after completion of the basic application scheme (blood count, CRP; urine: proteinuria).

Clinical evaluation:

The impact of autovaccination on the severity and frequency of furunculosis was retrospectively analyzed by reviewing the clinical records. Additionally, a telephone interview using a questionnaire was performed by a physician, which included questions about the chronicity, duration, location and severity of furuncles, as well as a patient's rating of the clinical improvement after autovaccination.

Detection of S. aureus virulence factors by PCR:

PCR was used to screen for a total of 26 virulence genes.

Single and multiplex-PCR were applied for the detection of gyrase (gyr), methicillin resistance (mecA), Panton-Valentine-leukocidin (pvl), staphylococcal enterotoxins (sea-seu), toxic shock syndrome toxin 1 (tst), exfoliative toxins (eta, etb, etd), and agr group 1-4 as previously reported [START_REF] Holtfreter | Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates[END_REF]. spa genotyping: PCR for amplification of the S. aureus protein A (spa) repeat region was performed according to the published protocol [START_REF] Harmsen | Typing of methicillinresistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management[END_REF][START_REF] Aires-De-Sousa | High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study[END_REF]. PCR products were sequenced by a commercial supplier using both amplification primers (Agowa, Berlin, Germany). The forward and reverse sequence chromatograms were analyzed with the Ridom StaphType software (Ridom GmbH, Würzburg, Germany). Spa types were clustered into different clonal clusters using the Based Upon Repeat Pattern (BURP) algorithm. Since spa typing and multilocus-sequence typing (MLST) are highly concordant, spa typing data could be mapped on MLST types by using the SpaServer database (www.spaserver.ridom.de).

2D-Immunoblots: Two-dimensional polyacrylamide gel electrophoresis (2-DE) with mini 2D gels and 2D

immunoblots (2D-IBs) were performed as described [START_REF] Holtfreter | Human immune proteome in experimental colonization with Staphylococcus aureus[END_REF]. Briefly, for antigen preparation, bacteria were inoculated in tryptic soy broth (TSB) to an optical density at 540 nm of 0.05 and cultivated in 100 ml cultures at 37°C and 180 rpm. Cultures were harvested 3.5 h after the bacterial culture entered the stationary phase, extracellular proteins were collected, and protein concentration was determined as previously described [START_REF] Holtfreter | Towards the immune proteome of Staphylococcus aureus -The anti-S. aureus antibody response[END_REF][START_REF] Holtfreter | Human immune proteome in experimental colonization with Staphylococcus aureus[END_REF].

Afterwards, isoelectric focusing was performed with 7 cm Immobiline Dry Strips of pI ranges 4-7 and 6-11 (GE Healthcare, Munich, Germany). Following separation according to molecular mass in the second dimension, the resolved staphylococcal proteins were blotted onto a PVDF membrane (Immobilon-P, Millipore) and incubated with the corresponding human sera at 1:10,000 dilution. IgG binding was detected by peroxidase-conjugated goat anti-human IgG and visualized with an ECL substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce). Consecutive serum samples from one patient were always analyzed simultaneously; at least three technical replicates were performed for each patient.

Analysis of the 2D-IB images was performed with the Delta-2D software package version 4.0 (Decodon GmbH, Greifswald, Germany) as described [START_REF] Holtfreter | Towards the immune proteome of Staphylococcus aureus -The anti-S. aureus antibody response[END_REF][START_REF] Holtfreter | Human immune proteome in experimental colonization with Staphylococcus aureus[END_REF]. A fused image of all IBs from one patient was obtained in a twostep procedure. First, all IB images from one time course experiment were matched with the most complex IB and a fusion image was obtained using the union fuse option. Second, the fusion images from the three technical replicates were matched and fused. Spots on the fusion image were automatically detected and manually validated by comparing the original blot images with the fusion image. Subsequently, the spot map and the corresponding labels from the fusion image were transferred to all blot images from one patient. For spot detection on IBs the signal intensity threshold was set to 0.2 arbitrary units [AU].

Flamingo® protein staining:

To correlate IB binding with protein abundance, representative IB images were matched with Flamingo®-stained 2D gels. Gels were stained with the Flamingo® Fluorescent Gel Stain (BioRad, Munich, Germany) according to the manufacturer's instructions [START_REF] Kolata | Distinctive patterns in the human antibody response to S. aureus bacteremia revealed by a prospective and personalized immune proteome approach submitted[END_REF]. Methods were as described previously [START_REF] Verkaik | Comparison of carboxylated and Penta-His microspheres for semi-quantitative measurement of antibody responses to His-tagged proteins[END_REF][START_REF] Verkaik | Anti-Staphylococcal Humoral Immune Response in Persistent Nasal Carriers and Noncarriers of Staphylococcus aureus[END_REF]. Tests were performed in independent duplicates and the Median Fluorescence Intensity (MFI) values, reflecting antibody concentrations, were averaged. In each experiment, control beads (no protein coupled) were included to determine non-specific antibody binding. In case of nonspecific binding, this background was subtracted from the MFI values. Human pooled serum was used as a standard.

Bead

Statistics: Differences in antibody binding (Luminex ® ) between furunculosis patients and healthy controls were assessed using the Mann-Whitney U test in conjunction with the Benjamini-Hochberg False Discovery Rate (FDR) multiple testing correction (FDR<0.05).

Results: Clinical outcome of autovaccination

To assess the impact of autovaccination on the severity and frequency of furunculosis, clinical records were screened and telephone interviews using a questionnaire were performed by a physician (Table 1). All four patients suffered from chronic (7293, 7510, and 9105) or recurrent (6466) furunculosis, which required repeated medical interventions.

Autovaccination reduced the frequency and severity of furunculosis or resulted in complete remission (Table 1).

Two patients (7293 and 9105) were free of symptoms after autovaccination treatment. The other two (6466, 7510) still developed single, self-healing furuncles, which did not require medical treatment. All patients reported subjective improvement (Table 1). Side effects occasionally observed by the patients were pain and inflammation at the side of injection and elevated temperature (< 38 °C) for two days after injection.

Molecular characterization of the furunculosis strains

The infecting S. aureus strains were repeatedly isolated from infected skin lesions. In parallel, nasal swabs were taken to determine the nasal colonization status. The infecting and colonizing S. aureus isolates were characterized by spa genotyping. Additionally, a panel of virulence genes, including pvl, mecA, exfoliative toxins and superantigens was detected by multiplex PCR (Table 2).

Three patients were nasal S. aureus carriers. Molecular characterization showed that all S. aureus furunculosis and nasal strains isolated from one patient over time belonged to the same S. aureus clone. Notably, two patients (7293 and 9105) were infected with a community-acquired methicillin-resistant S. aureus strain (CA-MRSA), which belonged to the clonal cluster CC59. Both strains harbored the characteristic mecA and pvl genes and displayed a macrolide-lincosamide-streptogramin B resistance (MLS B ) phenotype. Nasal and infecting strains from patient 6466 were pvl-positive and belonged to CC121, a lineage strongly associated with furunculosis [START_REF] Masiuk | Recurrent furunculosis -associations with Panton-Valentine leukocidin and the genetic background of Staphylococcus aureus[END_REF]. Patient 7510 carried a pvl-negative CC8 isolate (Table 2).

Anti-staphylococcal serum antibodies before autovaccination

Taking into account the pronounced variability of the species S. aureus we used a personalized approach to study the kinetics of the human antibody response in autovaccination. Two-dimensional immunoblots (2D-IB) served to analyze the binding of each patient's serum antibodies to the antigen spectra of the corresponding infecting S. aureus strain. Our analyses were focused on the more informative pH range of 6-11, where most extracellular proteins resolve, while protein A is excluded, so that unspecific IgG binding could be avoided [START_REF] Holtfreter | Human immune proteome in experimental colonization with Staphylococcus aureus[END_REF].

The furunculosis patients showed a strong antibody response against the extracellular proteins of their infecting S. aureus strain already before autovaccination treatment (Fig 1A). All four patients showed a relatively complex antibody binding pattern, especially in the basic pH range. However, there were also numerous protein spots which were not recognized by serum IgG. IB images from pH 4-7 were less complex and a number of spots were due to IgG binding to protein A. Overall, they corroborate the observations made in pH range 6-11 (data not shown).

Next, Luminex ® technology was used to compare the antibody response in furunculosis patients (before autovaccination) and healthy controls. Anti-staphylococcal IgG, IgA and IgM serum concentrations in the four investigated patients were comparable to those in healthy individuals (n=11) (Fig 2). Serum IgG and IgA from furunculosis patients reacted with a broad range of staphylococcal surface antigens, toxins (e.g. PVL, superantigens) and immunomodulatory proteins. There was little IgM binding to staphylococcal antigens in controls as well as two patients, but strong binding to many S. aureus antigens in the other two patients (Fig 2). Superantigens encoded by the enterotoxin gene cluster, egc, were on average bound less strongly than others. To conclude, furunculosis patients did not have a general lack of anti-staphylococcal antibodies.

Antibody response during autovaccination

To test whether autovaccination triggers an antibody response, we studied the kinetics of antibody binding using 2D-IBs and Luminex. First, we performed 2D-IBs with sera obtained over the course of autovaccination ( 2 Finally, we applied the Luminex assay to test whether autovaccines trigger an antibody response to surfacebound staphylococcal antigens. Indeed, three out of four patients showed a more than 2-fold increase in IgG binding to surface antigens, such as ClfA (7293, 7510, 9105), ClfB (9105), SdrD (7510) and SdrE (7293) (Fig 4). Similarly, we observed a boost in the IgA response against surface proteins (suppl. Fig. 1). Thus, vaccination induced a moderate boost of the antibody response against staphylococcal surface proteins. Novel antibody specificities against surface proteins were not generated.

Discussion

Autovaccines have been applied in chronic staphylococcal infections for decades, but their mechanism of action is still unknown. In this pilot study, we demonstrate that autovaccination only slightly boosts the antibody response to extracellular bacterial antigens and surface proteins. This pilot study was not designed to test the efficacy of autovaccination, but rather to characterize the kinetics of the antibody responses. Therefore, we analyzed only a small cohort and did not include placebo controls.

Moreover, some clinical outcome criteria (severity score, rating of clinical improvement) are subjective, which limits the evaluation of the clinical outcome. Nevertheless, it is interesting to note that based on the applied criteria all four patients, two of whom were chronically infected with CA-MRSA, clinically improved during treatment. In the seventies of the last century, Ring and co-workers reported that oral autovaccination treatment improved the clinical state in 11/18 patients with chronic posttraumatic osteomyelitis [START_REF] Ring | Chronic posttraumatic osteomyelitis. Attempt at oral autovaccine therapy][END_REF]. Similarly, a controlled study of 292 children with paranasal sinusitis demonstrated that antibiotic therapy plus autovaccine was superior to antibiotic therapy alone [START_REF] Okrasinska-Cholewa | Clinical evaluation of treating accessory nasal sinus diseases in children using autovaccine][END_REF]. Overall, little is published in English on the efficacy and safety of autovaccination. In particular, prospective, controlled, randomized, and, if possible, (double)-blind clinical studies are lacking.

Already before autovaccination, the study patients had serum antibodies that bound to a broad antigen spectrum of their infecting S. aureus strain. There was no obvious defect in their anti-staphylococcal antibody response.

Furunculosis patients suffer from chronic or recurrent infection for many months or even years and, therefore, the immune system is frequently and intensively exposed to the invasive strain and establishes a memory response. Moreover, most furunculosis patients are S. aureus carriers and the nasal and infecting strains are usually identical [START_REF] Masiuk | Recurrent furunculosis -associations with Panton-Valentine leukocidin and the genetic background of Staphylococcus aureus[END_REF]. In this study, three out of four patients were colonized with the infecting strain in the nose.

Thus, the nose appears to be a potential source for repeated endogenous infections. In agreement with our findings of a robust baseline anti-S. aureus antibody response in the furunculosis patients, we previously reported that healthy human carriers are pre-immunized with their colonizing strain [START_REF] Holtfreter | Staphylococcus aureus carriers neutralize superantigens by antibodies specific for their colonizing strain: a potential explanation for their improved prognosis in severe sepsis[END_REF][START_REF] Holtfreter | Human immune proteome in experimental colonization with Staphylococcus aureus[END_REF].

By applying 2D-IB and Luminex assays we found that autovaccination only slightly boosted the pre-existing antibody response to secreted bacterial antigens and surface proteins. Notably, autovaccination did not induce a profound de novo generation of antibody specificities. These findings suggest that repeated subcutaneous immunization does not strongly trigger the humoral immune response in these well pre-immunized patients. In line with this, Szkaradkiewicz et al. analyzed the IgG response in patients treated with autovaccines using 1Dimmunoblots and also observed no changes in IgG binding patterns to S. aureus polypeptides [START_REF] Szkaradkiewicz | Humoral response to Staphylococcus aureus polypeptides in patients treated with autovaccine[END_REF]. In contrast to autovaccination of furunculosis patients, systemic S. aureus infection strongly enhances the anti-staphylococcal antibody response in patients [START_REF] Kolata | Distinctive patterns in the human antibody response to S. aureus bacteremia revealed by a prospective and personalized immune proteome approach submitted[END_REF].

We previously reported a high degree of strain-specificity in the anti-S. aureus antibody response [START_REF] Holtfreter | Staphylococcus aureus carriers neutralize superantigens by antibodies specific for their colonizing strain: a potential explanation for their improved prognosis in severe sepsis[END_REF][START_REF] Kolata | Distinctive patterns in the human antibody response to S. aureus bacteremia revealed by a prospective and personalized immune proteome approach submitted[END_REF]. However, it is also possible that some antibody specificities are cross-reactive. For example, patient 6466 mounted an antibody response against SEC, but the strain used for autovaccination lacked the sec gene. SEC belongs to a family of 21 staphylococcal superantigens with up to 90% sequence homology, which could explain the observed cross-reactivity [START_REF] Bavari | Cross-reactive antibodies prevent the lethal effects of Staphylococcus aureus superantigens[END_REF][START_REF] Fraser | The bacterial superantigen and superantigen-like proteins[END_REF].

The pore-forming toxin PVL is epidemiologically strongly linked to S. aureus furunculosis [START_REF] Lina | Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[END_REF][START_REF] Masiuk | Recurrent furunculosis -associations with Panton-Valentine leukocidin and the genetic background of Staphylococcus aureus[END_REF]. In this study, three out of four patients were infected with pvl-positive strains. All patients had high levels of IgG antibodies against both subunits of the toxin, LukF and LukS, which did not increase during autovaccination.

Little is known about the mechanisms by which autovaccines might influence the disease course or impact on the immune response. Halasa et al. reported that autovaccination increases the concentration of S. aureus strainspecific agglutinating antibodies and enhances the phagocytosis of bacteria by peripheral blood granulocytes [START_REF] Halasa | Evaluation of certain immunological parameters in the course of autovaccine treatment in patients with chronic ostitis and carbunculosis[END_REF]. Similarly, clinical improvement of acne after autovaccination with killed Propionibacterium acnes was accompanied by the generation of specific antibodies against bacterial structures [START_REF] Zaluga | Skin reactions to antigens of propionibacterium acnes in patients with acne vulgaris treated with autovaccine[END_REF]. Still, it is difficult to imagine that the observed moderate changes of antibody titers should fully explain the clinical effects of the treatment. Other groups have reported that autovaccination down-regulates Th1 cell function and reduces delayed type hypersensitivity reactions [START_REF] Ring | Chronic posttraumatic osteomyelitis. Attempt at oral autovaccine therapy][END_REF][START_REF] Nolte | Autovaccination of dairy cows to treat post partum metritis caused by Actinomyces pyogenes[END_REF][START_REF] Rusch | Results of an open, non-placebo controlled pilot study investigating the immunomodulatory potential of autovaccine[END_REF]. In conjunction with the observation that T cells can influence the recruitment and anti-bacterial action of granulocytes [START_REF] Mcloughlin | IFN-gamma regulated chemokine production determines the outcome of Staphylococcus aureus infection[END_REF][START_REF] Mcloughlin | CD4+ T cells and CXC chemokines modulate the pathogenesis of Staphylococcus aureus wound infections[END_REF], this opens an interesting perspective for research. Moreover, the humoral immune response to non-protein staphylococcal antigens, such as lipoteichoic acid, wall teichoic acid and capsular polysaccharides, might be clinically important and will be addressed in future analyses [START_REF] Holtfreter | Towards the immune proteome of Staphylococcus aureus -The anti-S. aureus antibody response[END_REF][START_REF] Xia | The wall teichoic acid and lipoteichoic acid polymers of Staphylococcus aureus[END_REF]. Finally, we cannot rule out that formalin-fixation of the vaccine preparation influences the three-dimensional antigen structure, which might influence the antibody response, especially to surface antigens.

The rapid spread of multiresistant S. aureus strains enforces the development of new strategies to combat staphylococcal infections. In view of the pronounced variability of the bacterial species, personalized approaches could hold promise for patients with recurrent or chronic S. aureus infection. Autovaccination could serve as a model that merits further investigation.

  -based flow cytometry (Luminex ® ): Serum IgG, IgA and IgM directed against a panel of 40 recombinant staphylococcal antigens was quantified using the bead based flow cytometry technique (xMap ® , Luminex Corporation, Austin, Texas, USA). Antigens comprised surface proteins, such as clumping factor A and B (ClfA, ClfB), and serine-aspartate dipeptide repeat proteins (SdrD and SdrE), and secreted proteins, including toxins (superantigens, leukotoxins), and immune modulatory proteins (CHIPS, SCIN, SSL proteins).

  -4 samples). Superimposition of 2D-IB images from serum samples obtained before autovaccination (blue) and later (orange) showed almost identical antibody binding patterns as indicated by the dominance of black spots on the superimposed images (Fig 1B). Only a few new IgG-binding specificities, indicated by purely orange spots, were generated. Patient 7293 was exceptional, because we observed a boost in the IgG-binding as well as numerous novel IB-spots. Next, we quantified the individual IB-spot intensities and determined the changes in antibody binding during autovaccination (Fig 3). The patients' sera obtained before autovaccination reacted with 55-82 protein spots. Patient 7293 showed the weakest antibody binding before vaccination (Fig 3A). In this patient, vaccination induced a 2 to 5-fold intensity increase in most spots (59/67) (Fig 3B). A moderate boost was observed in patient 7510 with 22/83 spots increasing more than 2-fold. The antibody patterns of the other two patients (6466 and 9105) remained almost unchanged (Fig 3B). Notably, autovaccination induced only few novel antibody specificities. In conclusion, autovaccination boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected.

Fig. 1 Serum

 1 Fig. 1 Serum IgG response to extracellular S. aureus proteins in autovaccination.A) Overlay images of Flamingo® -stained 2D gels and corresponding 2D-immunoblots.The proteins released during post-exponential growth by the four invasive S. aureus strains were separated by 2D gel electrophoresis (pH 6-11) and stained with the fluorescence protein dye Flamingo® (red). The four bacterial isolates differed in their secretomes. In parallel, the extracellular bacterial antigens were blotted onto PVDF membranes and incubated with the corresponding patients' sera obtained before autovaccination. The overlay images (proteinred; IgG bindingblue) showed a strong antibody response to the infecting strain already before autovaccination. B) Kinetics of individual IgG responses to autovaccination. Extracellular antigens from the infecting S. aureus strains were separated by 2D gel electrophoresis and quantitative IBs were performed with the corresponding patients' sera obtained at different time points. Overlays of 2D-IBs with sera obtained before (blue) and during (orange) autovaccination. Autovaccination induced a moderate boost in the IgG response. One out of the three technical replicates is shown.

Fig. 2

 2 Fig. 2 Serum IgG, IgA and IgM response to S. aureus surface proteins and toxins in furunculosis patients and healthy controls. Serum IgG (A), IgA (B) and IgM (C) antibody binding to a panel of 40 S. aureus antigens was assessed with a Luminex assay and Ig binding to individual antigens is depicted as median fluorescence intensity (MFI). Each symbol represents a single individual. Median levels of anti-staphylococcal antibodies for patients (n=4; before autovaccination) and healthy controls (n=11) are indicated by horizontal lines. The antibody binding patterns of furunculosis patients and healthy controls were not significantly different (Mann-Whitney U test using the Benjamini-Hochberg False Discovery Rate multiple testing correction).ClfA, Clumping factor A; Efb, extracellular fibrinogen-binding protein; FnbpA, Fibronectin binding protein A; IsdA, iron-responsive surface determinant; Sas, S. aureus surface protein; Sdr, serine-aspartate dipeptide repeat proteins; CHIPS, chemotaxis inhibitory protein of S. aureus; Luk, leukocidin; SCIN, staphylococcal complement inhibitor; SSL, staphylococcal superantigen-like protein, SE, staphylococcal enterotoxin.

Fig. 3

 3 Fig. 3 Quantification of IgG binding to extracellular S. aureus antigens. Spot intensities on 2D-IB were determined from three or more replicate blots and median intensities (arbitrary units; AU) are depicted. The threshold was set at a volume of 0.2 AU.

Fig. 4 Suppl Fig 1 :

 41 Fig. 4 Kinetics of the serum IgG response to S. aureus surface proteins and toxins over the course of autovaccination. IgG binding to a panel of 40 S. aureus antigens was assessed with a Luminex assay. Each graph represents a single patient. Per patient, the level of antigen-specific antibodies in the time course of autovaccination is shown. Arrows indicate a more than 2-fold increase in antibody binding. See legend to figure 2 for more detailed information on the antigens.

1

 1

TABLE 1 : Clinical outcome of autovaccination.

 1

			Patient 6466	Patient 7293	Patient 7510	Patient 9105
	Patient data
	Age (years)		23	35	50	41
	Sex		female	female	male	female
	Risk factors 1		atopic dermatitis	-	-	atopic dermatitis
	Onset of furuncles		02/06	07/06	2002	07/06
	AV treatment		08/07 -12/08	10/06 -08/07	10/06 -10/07	12/06 -10/07
	Disease severity
	Location		face, neck, axilla	axilla, abdomen,	face, neck	axilla, buttocks
				back
	Mean No. of	Before AV	2	4	2	3.5
	episodes per month	During AV	0	1	2	0.2
		After AV	1	0	0.6	0
	Chronicity	Before AV	intermittently	permanently	permanently	permanently
		During AV	symptomfree	intermittently	intermittently	intermittently
		After AV	intermittently	symptom-free	intermittently	symptom-free
	Single/multiple sites Before AV	multiple	multiple	multiple	multiple
		During AV	-	multiple	single	single
				(1 episode)
		After AV	single	-	single	-
	Severity score 2	Before AV	2	2	2	3
		During AV	0	1	1	1
		After AV	1	0	1	0
	Clinical improvement 3		strong	strong	moderate	strong
	1 Risk factors: atopic dermatitis, chronic wounds, gout, diabetes mellitus, COPD, cancer, chronic liver disease,
	cardiovascular disease, rheumatoid diseases, HIV, chronic gut diseases (Morbus Crohn, Ulcerative Colitis),
	psoriasis, regular dialysis, immune suppressive drugs
	2 Disease severity was assessed based on a severity score.0 -No furuncles/abscesses; 1 -Few small furuncles,
	self-healing in less than two weeks, no medical intervention required; 2 -Furuncles of moderate severity,
	medical intervention required; 3 -Severe furuncles/carbuncles with strong pain and large fluid collections,
	medical intervention required.

3

Clinical improvement (classified as none, moderate or strong) based on the patients personal rating.

Acknowledgements:

This work was supported by the Deutsche Forschungsgemeinschaft (SFB-TR34 "Staphylococci in the postgenomic era", GRK840 "Host-Pathogen Interactions in Generalized Bacterial Infections").

2 MLST CC nomenclature was deduced from spa types using the Ridom SpaServer database.

6 3 The strain isolated from purulent furuncles was used to prepare the autovaccine.