S. aureus USA300 LAC strain was used for the in vivo model of skin infection and recurrence. Bacteria were grown in tryptic soy broth (TSB) at 37°C in agitation until the early exponential phase is reached and diluted 1:1 in a freezing solution, composed of phosphate-buffered saline (PBS) + 10% bovine serum albumin (BSA) + 10% L-glutamic acid. Aliquots were conserved at −80°C in cryovials until use.

For the inoculum preparation, 2-mL frozen stocks of bacteria were thawed and diluted in 48 mL of fresh TSB and incubated at 37°C at 250 rpm until bacteria reached the early exponential phase (OD₆₀₀ = 2.0), at approximately 1.5–2 × 10⁹ colony-forming units (CFUs)/mL. Bacteria were then washed once in sterile PBS and diluted with sterile PBS to obtain 4 × 10⁸ CFUs/mL or 8 × 10⁸ CFUs/mL for the skin infection or recurrence models, respectively.

S. aureus USA300 LAC strain expressing the green fluorescent protein (GFP) was used for the internalization assays. Bacteria were streaked on a tryptic soy agar (TSA) plate and incubated overnight (O/N) at 37°C + 5% CO₂. The following day, pre-inocula were prepared from single colonies and they were incubated O/N at 37°C at 250 rpm. The following day, the cultures were diluted in fresh TSB and incubated at 37°C and 250 rpm for approximately 3 h, until they reached the early exponential phase (OD₆₀₀ = 2). Bacteria were centrifuged at 2,400 rpm and 4°C for 10 min and washed twice with RPMI 1640 GlutaMAX + 25 mM HEPES + 0.05% BSA. After the final resuspension, the OD₆₀₀ of bacteria was measured and adjusted to an OD₆₀₀ of 1.0. Bacteria were aliquoted in 2-mL cryovials and stored at −20°C until use.

Animal husbandry and experimental procedures were ethically reviewed and carried out in accordance with European Directive 2010/63/EU, Italian Decree 26/2014, and GSK Vaccines’ Policy on the Care, Welfare and Treatment of Animals, and were approved by the Italian Ministry of Health (authorization 123/2015-PR). Animals were kept in an AAALAC-accredited facility. Upon arrival, animals were randomly distributed in different experimental groups in individually ventilated cages (IVC, Sealsafe Plus GM500 by Tecniplast). The acclimation lasted for a period of 5 days. At the end of the acclimation period, each animal was identified by an individual tattoo. All animals had ad libitum access to GMP-grade food (Mucedola 4RF25 TOP CERTIFICATE) and bottled, filtered, tap water. Certified, irradiated cellulose bags containing Mucedola SCOBIS UNO bedding and carboard tunnels (ANTRUM) or plexiglass mouse houses were provided within the cages. A few food pellets in the cage were also used as enrichment for foraging and additional gnawing. Cage and bedding changes were performed once every 2 weeks in agreement with the cage supplier’s indications. Air supplied in IVC was 100% fresh air filtered by an EPA filter via the IVC system, with 60–75 air changes per hour. The animal room conditions were as follows: temperature, 21°C (± 3°C); relative humidity, 50% (range 30%–70%); and 12-h light/12-h dark cycle. Pressure, temperature, and relative humidity were recorded continuously by room probes, while the IVC system recorded the individual motors’ performance. The light cycle setting was ensured by a validated alarm system.

The mouse model of skin infection and recurrence was performed as depicted in the Supplementary Materials (see Supplementary Material Figure S1 ). Five-week-old female specific pathogen-free (SPF) C57BL/6 mice were immunized twice intramuscularly (IM), 28 days apart. Animals were immunized with 10 µg of a SpA_mut adjuvanted with AS01, in a final volume of 50 µL, 25 µL/posterior leg. The control group (sham) was immunized with the formulation buffer alone (10 mM Na₂HPO₄ and 150 mM NaCl, pH 6.1).

For the in vivo vaccine efficacy studies, 4 weeks after the second immunization, the back of mice was shaved under anesthesia (3.5% isoflurane) using an electric razor and depilatory cream. For the primary skin infection, the day after shaving, mice were inoculated subcutaneously (SC) in the right flank with 2 × 10⁷ CFU/50 µL of S. aureus USA300 LAC strain, under anesthesia (3.5% isoflurane). Six weeks after, mice were shaved and infected again as previously described in the opposite flank with 4 × 10⁷ CFU/50 µL of S. aureus USA300 LAC strain to establish the model of skin infection recurrence.

Mice were observed daily for clinical signs of disease up to 14 days after infection. Mice were scored on the basis of pre-established parameters, and they were euthanized in case they exhibited pre-established humane endpoints in agreement with local Animal Welfare Policies (see Supplementary Materials Table 1 ). Dermonecrotic lesions were photographed and measured daily from day 4 to day 7 post-infection. At specific time points (which are shown in Supplementary Figure 1 ), blood was withdrawn from mice and serum was collected by centrifugation, for future serological analysis. At determined time points, mice were euthanized, and skin biopsies and kidneys were collected, homogenized, serially diluted and plated for bacterial burden, and expressed as CFU/mL for skin biopsies or as dissemination index for kidneys.

For all the Luminex-based assays, 20 µg of recombinant SpA_WT or SpA_mut was chemically coupled to 1.25 × 10⁶ MagPlex beads (Luminex Corporation), through an automated coupling method with a liquid handling workstation (Hamilton – Microlab STAR IVD). Briefly, antigens were coupled by a two-step carbodiimide procedure during which microsphere carboxyl groups are first activated with 1-ethyl-3-(3-dimethylaminopropryl) carbodiimide hydrochloride (EDC, Pierce), in the presence of sulfo-NHS (Pierce) to form a sulfo-NHS-ester intermediate. The reactive intermediate is then replaced by a reaction with the primary amine of the target molecule to form a covalent amide bond.

Total SpA_mut-specific IgG titers were evaluated with an automatized Luminex assay, incubating serially diluted mice sera with SpA_mut-coupled beads for 1 h with vigorous shaking. After washing, IgG titers were detected using an R-Phycoerythrin AffiniPure F(ab′)₂ Goat anti-mouse IgG (Jackson ImmunoResearch Labs, Cat #115-116-072, RRID: AB_2338627) through a FLEXMAP 3D reader (Luminex Corporation). Median fluorescence intensities (MFIs) of the samples, subtracted to the MFI of the blanks (MFI-Bkgd), were interpolated to a standard curve, expressing the IgG titer as relative light units per milliliter (RLU/mL). For each sample tested, the experiment was considered valid if a titer was observed in at least three points of the dilution and if these titers were interpolated in the linear portion of the standard curve; the final IgG titer of each value was expressed as the median value of the observed titers.

For the IgG subclass analysis, threefold serially diluted pools of sera collected after the second dose of immunization (D52, post II) or after skin infection (D72, 2 weeks post-infection) were incubated with SpA_mut-coupled beads for 1 h with vigorous shaking, repeating the assay in five different 96-well plates. After washing, the specific IgG subclass was detected using the appropriate R-Phycoerythrin AffiniPure F(ab′)₂ Goat anti-mouse IgG (Cat #115-116-072, RRID : AB_2338627), IgG1 (Cat #115-115-205, RRID : AB_2338620), IgG2a (Cat # 115-115-206, RRID : AB_2338621), IgG2b (Cat #115-115-207, RRID : AB_2338622), or IgG3 (Cat #115-115-209, RRID : AB_2338624) secondary antibody (Jackson ImmunoResearch Labs). Results were detected using a Luminex 200 instrument system (Luminex Corporation), and they were reported as the ratio between the RLU/mL of the different IgG subclasses.

For the characterization of the avidity, a mouse standard serum and mice sera obtained after immunization (D52, post II) and after skin infection (D72, 2 weeks post-infection) were threefold serially diluted and incubated in duplicate with SpA_mut-coupled beads for 1 h with vigorous shaking. After washing, one-half of the samples was incubated with vigorous shaking either with 2 M ammonium thiocyanate or with PBS for 30 min. After washing, the remaining IgGs were detected with an R-Phycoerythrin AffiniPure F(ab′)2 Goat anti-mouse IgG (Jackson ImmunoResearch Labs) using a Luminex 200 reader (Luminex corporation). Results were reported as avidity percentage, calculated on the basis of the ratio of the antibody titers recovered after incubation with ammonium thiocyanate with respect to titers observed after incubation with PBS.

The displacement assay was performed as described elsewhere (17). Briefly, SpA_WT-coupled beads (prepared as already described above) were saturated with 10 µg/mL of biotinylated-human IgGs for 30 min under vigorous shaking. After washing, saturated beads were incubated with threefold serially diluted total IgGs purified from mice sera collected after immunization (D52, post II), after skin infection (D72, 2 weeks post-infection) or with commercial total mouse IgGs as negative control (ChromPure Mouse IgG, ImmunoResearch Labs, Cat #015-000-003, RRID : AB_2337188), for 30 min with shaking. After washing, the amount of biotinylated-human IgGs bound to the SpA_WT-coupled beads was detected using 10 µg/mL of Streptavidin, R-Phycoerythrin Conjugate (SA-PE), through a FLEXMAP 3D reader. Results are shown as the percentage of antibody displaced, normalized on the signal obtained without adding mouse IgGs.

The staphylococcal protein microarray was generated by spotting relevant S. aureus antigens that were previously selected, expressed in competent E. coli cells, purified, and characterized as described elsewhere (18–20). Briefly, staphylococcal antigens were spotted randomly in quadruplicate on ultra-thin nitrocellulose-coated glass slides (Maine Manufacturing) using an ink-jet spotter (Arrayjet) in a cabinet at 18°C and 40% humidity (see Supplementary Table S2 for the complete list of spotted antigens). Slides were conserved in the dark at 4°C until use. After saturating the slides with a blocking buffer (BlockIt, Arraylt), diluted sera samples (n = 9) obtained 2 weeks after infection from immunized (SpA_mut/AS01 and infected) or non-immunized (sham and infected) mice were incubated on slides for 1 h in the dark in a humid chamber. After washing, signals were detected with an Alexa Flour 647 anti-mouse IgG secondary antibody (Jackson ImmunoResearch Labs, Cat #115-605-006, RRID : AB_2338903) and slides were rinsed and left to dry. Slides were scanned using an InnoScan 710 AL (INNOPSYS) and images were generated by the Mapix Microarray Image Analysis Software. Fluorescence intensities of spots were determined using ImaGene 9.0 software (Biodiscovery Inc.) and data analysis was performed using R scripts.

Each spotted antigen was considered as “positive” if recognized by the majority of tested sera (at least five out of nine).

THP-1 cells (ATCC TIB-202, RRID: CVCL_0006) were cultured in RPMI 1640 medium supplemented with GlutaMAX and HEPES (Gibco), 10% FBS, and 5 μg/mL of penicillin/streptomycin, according to manufacturer indications. Single sera collected from animal studies were heat-inactivated at 56°C for 30 min. The opsonophagocytosis assay was performed as described elsewhere (17). Briefly, 3–4 × 10⁶ CFUs/mL of S. aureus USA300 GFP strain were pre-opsonized with mouse sera obtained from normal mice (Pre-immune), immunized mice (SpA_mut/AS01 post II), 2 weeks after infection from non-immunized mice (sham and infected), and immunized mice (SpA_mut/AS01 and infected) for 30 min at 37°C under vigorous shaking. A total of 3–4 × 10⁴ THP-1 cells were added to allow opsonization of bacteria, and incubation continued for an additional 30 min (bacteria-to-cell ratio of 200:1). After adding lysostaphin (Sigma-Aldrich) to lyse non-internalized bacteria, bacteria and cells were fixed with 2% formaldehyde for 1 h on ice prior to acquisition at FACS CANTO II (Becton Dickinson) and data collected were analyzed using the FACSDiva 8.0.1 software. Gating strategy was as follows: (i) THP-1 cells; (ii) singlet cells; and (iii) FITC-A, representative of cells that have effectively internalized GFP-expressing bacteria. The median percentages of GFP⁺ THP-1 cells were normalized on the median signal obtained with pre-immune sera, and results were then represented as fold change with respect to the median of pre-immune sera.

Statistical analysis was performed using the GraphPad Prism Software version 8.0.0. The non-parametric Mann–Whitney U-test was used to assess differences between two groups, as for the evaluation of IgG titers against SpA_mut ( Figure 1 ). The one-way ANOVA Kruskal–Wallis and uncorrected Dunn’s post-test was used to assess differences between three or more groups, as in the in vivo results or in the internalization assay ( Figures 2A, B , 3 ). Finally, the Fisher’s exact t-test was used to assess differences between frequencies, as in the dissemination severity index or the protein microarray results ( Figures 2C , 4 ). A p (value) ≤ 0.05 was considered significant for all these analyses. Legend: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant, p > 0.05.

The area under the curve (AUC) was used to perform a cumulative data analysis on the dermonecrotic lesion areas ( Figure 2A ). The trapezoidal AUC of the dermonecrotic lesion areas, for a single day, was calculated using the following formula:

where B stands for major base, b denotes minor base, and t stands for time point.

The AUC was calculated for each time point after infection in the single animal and eventually summed together to have a final AUC value representing the whole time period after the infection.

Vaccination with SpA_mut was shown to protect guinea pigs against systemic disease and to enable the host to develop a broader antibody repertoire against several staphylococcal antigens otherwise masked by SpA activity, which could protect from a second encounter with S. aureus (8). Given these premises, we developed an animal model to evaluate the contribution of SpA-specific antibodies in mediating the protection to systemic sequelae of skin infection recurrences.

To achieve this, C57BL/6 mice were immunized with SpA_mut adjuvanted with AS01 (SpA_mut/AS01). Four weeks after immunization, mice were inoculated subcutaneously (SC) in the right flank with S. aureus USA300 LAC strain (primary infection). Six weeks after the primary infection, mice were infected again with the same bacterial strain in the opposite flank (secondary infection), mimicking a skin infection recurrence (see Supplementary Figure 1 for the experimental design of the model). Different readouts were used to monitor the effect of vaccination and of the infection, both locally and systemically. Results were compared with those obtained with mock immunized mice, infected either once or twice.

A previous exposure to the pathogen contributed to reduce dermonecrotic lesions developed by mice during a second infection by approximately 80% (the median value of naïve mice was 103.3 mm² and the median value of sham and infected mice was 18.25 mm²), but no additional effects due to a prior vaccination with SpA_mut/AS01 were observed ( Figure 2A ). Mouse vaccination contributed nevertheless to significantly reduce bacterial load in the skin compared to the sham and infected mice (p _val sham and infected vs. naïve was 0.07, p _val SpA_mut/AS01 and infected vs. naïve was 0.0057; Figure 2B ). Notably, a pre-existing immunity present in sham and infected mice did not have any impact on the ability of S. aureus to migrate from the skin to the kidneys, while immunization with SpA_mut/AS01 almost fully eradicated systemic complications during skin infection recurrence ( Figure 2C ).

We also evaluated a possible effect of immunization with the proposed vaccine in a model of skin infection but no reduction in dermonecrotic area size and CFU counts either in the skin or in the kidneys as observed (see Supplementary Material Figure 2 ).

Immunization with SpA_mut/AS01 expanded the immune response to the pathogen after infection of mice, which overall contributed to control local disease (bacterial load in skin lesions) and bacterial dissemination to kidneys in a skin infection recurrence model.

In an attempt to understand the mechanisms underlying this observation, we firstly evaluated the antibody response generated against the vaccine candidate after immunization and infection. As shown in Figure 1 , infection after vaccination did not boost the quantity of anti-SpA_mut antibodies generated by vaccination itself. Interestingly, no antibodies against SpA_mut were found in sera from sham and infected mice, underlying once more the ability of SpA to mask itself and, potentially, also other virulence factors to the host immune system ( Figure 1 ).

We then wondered whether a change in the quality of anti-SpA_mut IgG immune response could be detected after infection. IgG subclasses of anti-SpA_mut specific antibodies were therefore analyzed in mouse sera, showing that no significant changes in the antibodies repertoire occurred during infection ( Table 1 ). Eventually, affinity of anti-SpA_mut IgG was characterized in sera collected from mice before and after subcutaneous infection. Importantly, anti-SpA_mut antibodies strongly increased in affinity for their target during infection, with a relative percentage of IgG bound to the protein in the presence of 2 M ammonium thiocyanate that passed from 28% to 84%.

The observed increased affinity might lead to more functional antibodies. To verify this hypothesis, the functionality of SpA-specific antibodies was evaluated in an in vitro displacement assay that measured the ability of anti-SpA IgG to displace human immunoglobulins bound to the wild-type SpA protein mainly through the Fc region (SpA_WT). Figure 5 shows that immunoglobulins purified from vaccinated-only or infected-only mice sera displaced human IgG bound to the wild-type protein (40% at 500 µg/mL of purified IgGs) with a twofold increase as compared to the negative control group (almost 20% of background displacement). This effect was strongly enhanced when IgG purified from animals immunized and then infected were used with a 60% IgG displacement and an increase of threefold as compared to the negative control.

Moreover, immunization with SpA derivatives followed by S. aureus infection was previously shown to increase antibody response against several other S. aureus virulence factors (1, 8). Sera from sham and infected (only infected animals) and SpA_mut/AS01 and infected mice (animals immunized with SpA_mut/AS01 before subcutaneous S. aureus infection) were therefore compared for their ability to recognize S. aureus antigens on a protein chip where up to 96 antigens were spotted (see Supplementary Material Table 2 for the complete list of antigens). Among these 96 virulence factors, 23 reacted with higher frequencies when sera from SpA_mut/AS01 immunized and infected mice were used as compared to sera from sham and infected animals. Results with the 23 antigens are shown in Figure 4 and Table 2 .

Sera of SpA_mut/AS01 and infected mice, as compared to sera from only vaccinated or sham and infected mice, elicited antibodies that (i) showed an increased affinity for SpA,(ii) reduced SpA ability to bind immunoglobulins ( Figure 5 ), and (iii) recognized more S. aureus virulence factors both secreted and exposed on the bacterial surface ( Figure 4 ; Table 2 ). Antibodies present in sera of these animals before the second subcutaneous infection (recurrence) could therefore facilitate the clearance of bacteria, and this might explain why spreading of S. aureus from skin to kidneys was highly impaired in SpA_mut/AS01-immunized and infected animals.

To further evaluate the contribution of SpA-specific antibodies in the elimination of S. aureus from the blood circulation, a human monocytic cell line (THP-1) was used to evaluate bacterial internalization in the presence of sera from different groups of mice. As reported in Figure 3 , neither vaccination of mice with SpA_mut/AS01 (fold change equal to 0.7 with respect to the pre-immune sera, 10% percentile was 0.23, and 90% percentile was 1.18) nor bacterial infection (fold change equal to 3 with respect to the pre-immune sera, 10% percentile was 0.84, and 90% percentile was 5.8) significantly enhanced phagocytosis as compared to the negative control. Remarkably, the combination of vaccination and infection generated sera that significantly increased the phagocytic properties of THP-1 cells compared to the negative control (fold change equal to 5, 10% percentile was 2.8, and 90% percentile was 10, p _val = 0.0007) and, interestingly, also to vaccination alone (p _val< 0.0001).