MAbs were obtained as previously described [9]. Briefly, hybridoma cells were grown in Dulbecco’s modified eagle medium (DMEM, Life Technologies, Carlsbad, CA) with 10% of Ultra Low IgG Fetal Bovine Serum (Life Technologies, Carlsbad, CA) in 100-mL bottles at 37°C under 5% CO₂. Supernatants were harvested by centrifugation and passed through a 0.2-mm filter before liquid chromatography purification with MAbselect SuRe^™ protein A resin (GE Healthcare). Purified antibodies were dialyzed against phosphate-buffered solution (PBS) (10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.4), concentrated with Amicon ultrafiltration units, quantified at 280 nm using a Nanodrop spectrophotometer (Thermo Scientific), and stored at 4°C or in 40% glycerol at –20°C. A SDS-PAGE showing the purified MAb anti-PBP2a is represented in Supporting Information (S1 Fig).

Iberian Clone MRSA was obtained from the Unité des Agents Antibactériens—Institut Pasteur, and the BEC/BMB9393 (Brazilian Epidemic Clone) MRSA strain was obtained from the Instituto de Microbiologia Paulo de Goés—Universidade Federal do Rio de Janeiro. Both were epidemic MRSA strains of different genetic backgrounds and they were characterized by pulsed-field gel electrophoresis (PFGE) and multi-locus sequencing type (MLST) typing (BEC ST239, Iberian MRSA ST247) [15, 16].

All animal procedures were approved by the Ethics Committee on Animal Research (CEUA), FIOCRUZ (License number LW-30/12). Animals were provided by ICTB/Fiocruz and maintained in closed cages (dimensions: 37 cm in length, 24.2 cm in width, 24 cm in height) in a ventilated Microisolator Rack's system (ALESCO^®) with autoclaved pine flakes as a substrate. Cage changes were performed weekly. The mice were allocated in groups of four animals per cage and maintained under a 12 h/12 h (day/night) light cycle with unrestricted access to food and filtered water. Inoculations were performed simultaneously in all groups. Inoculation with the antibodies was performed during the first hours of the day cycle, and infection of the animals was performed during the last hours of the day cycle according to established protocols. For the therapeutic assays, infections were performed before the administration of antibodies or vancomycin, as described in the protocol. Using a clinical evaluation based on the ARRIVE guidelines, animals with severe clinical symptoms characterized by suffering or inability to access water or feed (humane endpoint) were euthanized with anesthesia at doses of 200 mg/kg ketamine hydrochloride and 20 mg/kg xilazine hydrochloride administered intramuscularly. After a few minutes of anesthetic administration, and in the absence of reflexes, sodium thiopental was administered intraperitoneally at a dose of 150 mg/kg.

The lethal dose and LD₅₀ were determined according to the Reed-Muench method with modifications for the MRSA strains employed in this study [17]. Four groups of 8-week-old female BALB/c mice (n = 4 per group, total of 16 animals) were inoculated intraperitoneally (IP) with bacteria (1x10⁸, 5x10⁸, 1x10⁹, and 5x10⁹ CFU [colony forming unit]) and observed over 7 days. Afterwards, all surviving animals were euthanized according to the approved CEUA protocol.

MRSA strains were grown until they reached exponential phase (OD₆₀₀ 0.6). They were then washed and resuspended in sterile PBS at OD₆₀₀ 0.5, which corresponded to approximately 2×10⁸ CFU (a sublethal dose). This concentration was calculated by a 10-fold serial dilution, and bacteria were cultured on Brain Heart Infusion (BHI) agar plates containing 10 μg/mL of oxacillin for 18 h at 37°C.

Three groups of 8-week-old female BALB/c mice (n = 7 per group, total of 21 animals) received an IP dose of either (a) 500 μg (25mg/kg) of purified anti-PBP2a MAb or (b) mock MAb (4G2 –a murine monoclonal antibody against Yellow fever) on the first day. To demonstrate in vivo antibody-neutralizing activity, another group (c) was treated with 350 μg (17.5 mg/kg) of anti-PBP2a F(ab’)₂. These fragments were obtained as previously described in Araujo et al. [14].

On the second day, mice were infected with a sublethal dose and observed until the fifth day. On the sixth day, animals were euthanized, and the kidneys were aseptically removed. These organs were then homogenized in 1 mL of sterile Luria Broth (LB), diluted by a 10-fold serial dilution, and plated as previously described. The resulting colonies were counted in order to calculate the concentration of bacteria in the kidneys. During the experiment, mice were monitored daily for clinical symptoms, such as apathy, rough hair coat, or fever. Anorexia and loss of mobility were considered humane endpoints.

MRSA strains were grown under the same conditions previously described, but an inoculum adjustment was performed for the previously established LD₅₀. Eight-week-old female BALB/c mice (n = 10 per group, total of 40 animals) received one IP dose of 500 μg (25 mg/kg) purified MAb on the first day. On the following day, these animals and a control group were inoculated IP with one dose of 3.3x10⁸ CFU (BEC—Brazilian Epidemic Clone MRSA strain), 4.2x10⁸ CFU (Iberian MRSA strain), or PBS (control group). The animals were observed for 10 days, and the survivors were subsequently euthanized.

Four groups of 8-week-old female BALB/c mice (n = 5 per group, total of 20 animals) received an infective dose (5–7.0×10⁶ CFU [BEC MRSA]). The animals received doses of purified MAb, vancomycin, MAb + vancomycin, or PBS (negative control) as described in the Table 1:

The first dose of MAb and vancomycin was administered 6 h after the administration of the infective dose. Animals were subjected to euthanasia on the fourth day. The animals’ kidneys were aseptically removed and subjected to bacterial quantification according to previously described methods.

Lethal and sublethal dose determinations were required for the in vivo protection assays, and these were evaluated using two models: 1.) bacterial load in the kidneys after challenge with a sublethal dose and 2.) a survival test after systemic infection with a higher bacterial inoculation that caused death in more than 50% of the animals. The IP route was chosen because of the easier method of administration and because it avoided bacterial loss. The COL MRSA strain was also tested; however, it proved to be less virulent, requiring a higher infective dose in relation to the epidemic BEC and Iberian MRSA strains (BEC ST239, Iberian ST247) evaluated. Therefore, it was not used in protection assays. For both MRSA clones, doses of 5.0×10⁸ CFU killed more than 50% of infected mice (n = 2/4), and doses of 1.0×10⁸ CFU generated a bacterial load in the kidneys without mortality.

Using a renal quantification model after infection, these assays demonstrated that a prophylactic treatment with anti-PBP2a MAbs reduced the number of bacteria in vital organs (kidneys) after a systemic infection. A reduction of more than 3 logs (1,000 times) was observed in independent assays using two epidemic MRSA strains (Fig 1), which showed similar levels of bacterial reduction (p<0.05 for assays with Iberian and BEC MRSA strains). No deaths, clinical signs, or morphological alterations in the kidneys were observed in the treated animals.

In order to demonstrate that in vivo protection is due to the antibody binding its target (PBP2a) and eliciting an antibody-neutralizing effect, an anti-PBP2a fragment F(ab’)₂ was tested. Since these MAb fragments lack the Fc region, they are unable to elicit an immune response to eliminate the pathogen. We used 350 μg of F(ab’)₂ instead of 500 μg for IgG MAb in order to maintain an equivalent molar concentration between both treated groups (F[ab’]₂ or IgG) since the molecular mass of IgGs (150 kDa) is greater than F(ab’)₂ (100 kDa).

There was a significant reduction in bacterial burden for the anti-PBP2a-fragment F(ab’)₂-treated group when compared to that of both control groups (p<0.05); however, a major response was noted in the group treated with IgG MAb (Fig 2).

To study the protection conferred by a previous treatment with anti-PBP2a MAb, the survival rate was determined after challenge with a bacterial load sufficient to kill more than 50% (5/10) of the animals. As shown in the Fig 3, for mice challenged with the BEC MRSA strain, 70% (7/10) of the animals survived the infection compared to only 10% (1/10) of the animals in the control group. Similarly, for mice challenged with the Iberian MRSA strain, 60% (6/10) of the treated animals survived, and 100% (10/10) died in the control group. Clinical symptoms were observed in treated and untreated animals. In addition, previous treatment with anti-PBP2a MAb prolonged survival. Differences observed in this assay could be due to a different bacteria inoculum and different levels of virulence among the MRSA strains used in this study. A second assay employing the Iberian MRSA strain was done and could be seen in Supporting information (S3 Fig).

Since vancomycin is considered the first antimicrobial choice for the treatment of severe MRSA infections, a study was conducted using a therapeutic assay in which mice were infected and treated 6 h after infection. The vancomycin dose was adjusted and administered in a similar manner to that of the human treatment (500 mg every 12 h). These results showed that the treatment with MAb conferred a similar level of protection to that for the group treated with vancomycin (96.2% versus 93.4% bacterial load reduction, respectively). Furthermore, a slightly greater level of protection was observed for the group treated with both the antibiotic and antibody (98.9% bacterial load reduction) (Fig 4). A second assay yielded similar results (S4 Fig). Thus, the protection conferred by an antibody dose corresponded to five vancomycin doses, and the simultaneous administration of MAbs and vancomycin was also efficient for reducing bacterial load in the kidneys of infected animals.

Since staphylococcal infections can affect different tissues in the body, it is important to investigate whether a therapeutic molecule can penetrate different organs. ELISA was used to estimate the MAb concentration in tissues at different times. The results demonstrated that the MAb reached different tissues (Fig 5). Twelve hours after MAb administration, MAb concentrations in the serum, kidneys, lungs, and spleen were 66.2, 3.20, 1.12, and 0.47 μg/mL, respectively, and at 96 hours, they were 42.36, 1.80, 0.86, and 0.28 μg/mL, respectively. MAb concentrations in tissues were lower than that present in the serum (about 4.5% in the kidneys, 1.9% in the lungs, and 0.7% in the spleen). No clinical symptoms were observed in the animals used in these assays.