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

The Department of Medical Microbiology and Immunology at the Pomeranian Medical University, Szczecin, Poland has long-standing experience with autovaccination for therapy of chronic S. aureus furunculosis and osteomyelitis [13]. This prospective pilot study included four patients (3 female and 1 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.

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.

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.

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) Tween 80. Bacteria were resuspended in 0.9% NaCl solution and fixed with 0.4% formalin solution for 48 h. The bacterial suspension was adjusted to 5 × 10⁸ bacteria/ml (suspension I), 1 × 10⁹ bacteria/ml (suspension II), and 2.5 × 10⁹ 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 7 days.

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. Intradermally, 0.5 ml of a 1:10 dilution of suspension I in Aqua ad injectionem was injected. Skin reaction was checked after 15 min and 24 hours. Autovaccines were then applied subcutaneously in 3–5 day intervals over 3 months. With each injection the dose was increased based on the following application scheme. Suspension I (5 × 10⁸ bacteria/ml) was applied in volumes of 0.1, 0.2, 0.3, 0.4 and 0.5 ml, followed by suspension II (1 × 10⁹ bacteria/ml; 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml) and suspension III (2.5 × 10⁹ 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).

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.

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 groups 1–4 as previously reported [8].

PCR for amplification of the S. aureus protein A (spa) repeat region was performed according to the published protocol [23, 24]. 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).

Two-dimensional polyacrylamide gel electrophoresis (2-DE) with mini 2D gels and 2D immunoblots (2D-IBs) were performed as described [25]. 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 [11, 25].

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 [11, 25]. A fused image of all IBs from one patient was obtained in a two-step 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).

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.

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).

Methods were as described previously [26, 27]. 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 non-specific binding, this background was subtracted from the MFI values. Human pooled serum was used as a standard.

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).