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. 2026 May 14;110:204. doi: 10.1007/s00253-026-13852-8

Human anti-alpha-hemolysin scFvs mitigate toxin-mediated damage in a mouse model of MRSA pneumonia

Somayeh Piri-Gavgani 1, Hamid Reza Moradi 2, Farzaneh Nazari 1, Abolfazl Fateh 1, Seyed Davar Siadat 1, Masoumeh Azizi 3, Fatemeh Rahimi-Jamnani 1,✉
PMCID: PMC13341833  PMID: 42133074

Abstract

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia represents a serious and potentially fatal infectious complication, hallmarked by its considerable strain on healthcare infrastructure and well-documented association with elevated morbidity and mortality. The clinical burden is particularly pronounced among immunosuppressed individuals and hospitalized patients reliant on mechanical ventilation. Suboptimal therapeutic outcomes associated with conventional antibiotic treatment of MRSA infections have necessitated alternative therapeutic strategies targeting bacterial virulence factors. Among these, alpha-hemolysin has emerged as a critical determinant in the pathogenesis of MRSA-associated pneumonia, representing a promising therapeutic target. Our previous investigation revealed the remarkable neutralizing capabilities of two human single-chain variable fragments (scFvs), designated SP192 and SP220, which effectively target alpha-hemolysin in vitro. To delve deeper into their therapeutic potential in vivo, we administered SP192, SP220, or a combination of the two scFvs to immunocompromised mice with MRSA pneumonia, every 12 h for 72 h. The findings were significant, indicating a marked increase in survival rates among SP192- and SP220-treated mice, with outcomes similar to those observed in the vancomycin-treated control group. Furthermore, both SP192 and SP220, whether administered individually or in combination, significantly reduced bacterial load and mitigated renal and pulmonary damage compared with control groups that received normal saline or an unrelated scFv. These findings highlight the potential of targeted therapies to address a critical need in the management of MRSA pneumonia, which is relevant for clinicians seeking new treatment options.

Key points

• The efficacy of two anti-alpha-hemolysin scFvs was assessed in vivo.

• The SP192 and SP220 scFvs improve survival rates and reduce tissue damage.

• The combination of SP192 and SP220 has demonstrated the most effective treatment.

Keywords: Methicillin-resistant Staphylococcus aureus, Alpha-hemolysin, Single-chain variable fragment, Pneumonia

Introduction

Staphylococcus aureus is renowned as one of the most formidable opportunistic pathogens, persistently impacting human life with its broad spectrum of clinical complications. Its ability to adapt and thrive makes it a particularly challenging adversary in the realm of health (Tong et al. 2015). The annual prevalence of S. aureus infections fluctuates between 1 and 3%; these infections are diverse, yielding serious health consequences that notably elevate morbidity (such as dermonecrosis) and mortality (including bacteremia, endocarditis, meningitis, pneumonia, and sepsis) (Foletti et al. 2013; Thammavongsa et al. 2015). The high prevalence and elevated mortality rates of hospital- and community-acquired pneumonia caused by S. aureus underscore a critical unmet clinical need, imposing a considerable and growing burden on healthcare systems worldwide (Shekhar et al. 2025). Navigating the treatment of S. aureus infections has become more complex, particularly given the growing global challenge posed by multidrug-resistant (MDR) strains (François et al. 2018). The alarming rise of methicillin-resistant S. aureus (MRSA) has dramatically heightened the morbidity and mortality associated with S. aureus pneumonia over the past three decades (Pickens and Wunderink 2022). In light of this challenge, the Infectious Disease Society of America recommends vancomycin and linezolid as treatment options for MRSA pneumonia (Ke et al. 2024). Nonetheless, the lackluster clinical success rates, particularly in patients exposed to broad-spectrum antimicrobial agents, those requiring long-term mechanical ventilation, individuals on immunosuppressive therapies, and elderly patients, coupled with the toxicities reported with these medications, continue to impede their practical application (Hua et al. 2014).

The daunting nature of certain MRSA strains, driven by the production of extracellular cytotoxins, can lead to the life-threatening condition known as necrotizing pneumonia (Welte et al. 2019). Among these formidable cytotoxins, alpha-hemolysin stands out as a critical virulence factor in the pathogenesis of S. aureus pneumonia (Yu et al. 2017). This highly conserved pore-forming toxin with a molecular weight of 33 kDa binds to its receptor on host cell membranes, forming heptameric structures that compromise the integrity of the cell barrier (Chang et al. 2023). Moreover, alpha-hemolysin serves as a powerful chemoattractant, triggering the release of inflammatory molecules, including interleukin-1β, interleukin-6, and tumor necrosis factor-α. All of this orchestrates a profound cascade of events that leads to programmed cell death in leukocytes and endothelial cells, facilitates bacterial spread, induces immune dysregulation, and culminates in considerable tissue damage (Yu et al. 2017). Some studies have indicated that S. aureus strains deficient in alpha-hemolysin expression are markedly less virulent in animal models of pneumonia (Berube et al. 2014; Ragle and Wardenburg 2009; Wardenburg et al. 2007). Targeting alpha-hemolysin offers a compelling opportunity to mitigate the severity of S. aureus infections, and several studies have demonstrated that both active and passive immunization against alpha-hemolysin result in significant reductions in disease severity in mouse models of pneumonia (Diep et al. 2017; Foletti et al. 2013; Hua et al. 2014, 2015; Ragle and Wardenburg 2009; Surewaard et al. 2018).

Targeted therapy has ushered in a new era of innovative functional antibacterial agents, captivating the medical community with their growing prevalence (Rahimi-Jamnani et al. 2025). Anti-virulence therapeutic agents represent a strategic approach to addressing the challenges posed by antibiotic resistance, while simultaneously minimizing the side effects commonly associated with traditional antibiotics. Monoclonal antibodies (mAbs) are increasingly recognized as promising alternatives for clinicians due to their excellent safety profiles and precision in targeting hard-to-treat pathogens and their virulence factors (Tabor et al. 2018). Among the cutting-edge treatments, there are mAbs developed to neutralize alpha-hemolysin of S. aureus, including two fully human mAbs, known as tosatoxumab (AR-301) and suvratoxumab (MEDI4893; AR320), currently under rigorous evaluation in clinical trials (NCT03816956 and NCT05331885, respectively) for their potential in the prevention or treatment of S. aureus pneumonia (Diep et al. 2017; François et al. 2021). Mitigating the effects of these toxins by preventing their attachment to cellular receptors can be achieved effectively using antibody fragments (Piri-Gavgani et al. 2022; Rahimi-Jamnani et al. 2025; Soezi et al. 2022). Single-chain variable fragments (scFvs) are remarkable entities, crafted entirely from the variable regions of the heavy and light chains of mAbs (Ahamadi-Fesharaki et al. 2019). Their compact size (~25 kDa) enhances their ability to navigate and access targets with exceptional ease (Rahimi-Jamnani et al. 2025). This versatility is complemented by robust binding capabilities and the exciting potential to develop bispecific antibodies (Ahamadi-Fesharaki et al. 2019). Notably, scFvs achieve all of this while ensuring low immunogenicity and maintaining a cost-effective production process, making them a compelling choice in therapeutic applications (Ahamadi-Fesharaki et al. 2019; Basardeh et al. 2022; Soltanmohammadi et al. 2021). The remarkable ability of scFv antibodies to neutralize a range of bacterial toxins—including alpha-hemolysin (Piri-Gavgani et al. 2022), anthrax lethal toxin (Pelat et al. 2007), botulinum neurotoxin (Chahboun et al. 2011), diphtheria toxin (Wenzel et al. 2020), Escherichia coli Shiga toxin (Luz et al. 2015), and toxic shock syndrome toxin-1 (TSST-1) (Rahimi-Jamnani et al. 2025; Rukkawattanakul et al. 2017; Soezi et al. 2022)—highlights their immense potential as a groundbreaking strategy in the fight against these formidable pathogens.

In our prior investigation, we identified two fully human scFvs, SP192 and SP220, derived from a human scFv phage library (Piri-Gavgani et al. 2022). These scFvs exhibited remarkable specificity and binding affinity for alpha-hemolysin and effectively inhibited the alpha-hemolysin-mediated lysis of rabbit red blood cells (RBCs) (Piri-Gavgani et al. 2022). In the present study, we evaluated the safety of administering SP192 and SP220 in healthy mice. Subsequently, to more comprehensively explore their therapeutic potential, we administered SP192 and SP220, either individually or in combination, to immunocompromised mice with MRSA pneumonia. Our investigation meticulously focused on critical outcomes, including survival rates, bacterial burden, and the extent of tissue damage caused by the infection.

Materials and methods

Bacterial strains and growth media

The MRSA strains used in this study included S. aureus strain 48 (S.a. 48), obtained from cerebrospinal fluid; S. aureus strain 61 (S.a. 61), obtained from an infected catheter; and S. aureus strain 124 (S.a. 124), isolated from blood cultures (Soltanmohammadi et al. 2021). These strains were obtained from three distinct patients with S. aureus infections and were generously provided by the Department of Mycobacteriology and Pulmonary Research at the Pasteur Institute of Iran (Tehran, Iran) (Soltanmohammadi et al. 2021). In addition, we included two extensively drug-resistant (XDR) strains of Acinetobacter baumannii: A.b. 56, isolated from the endotracheal tube, and A.b. 58, isolated from the blood of patients with A. baumannii infections (Basardeh et al. 2022). The Microbiology Laboratory of the Pasteur Institute of Iran supplied these important strains. We also obtained S. aureus ATCC 6538, A. baumannii ATCC 19606, and Klebsiella pneumoniae ATCC 700603 from the American Type Culture Collection (ATCC) (Basardeh et al. 2022; Soltanmohammadi et al. 2021). For optimal growth, all strains were routinely cultured in Tryptic Soy Broth (TSB; Merck, Darmstadt, Germany) or Luria–Bertani (LB; Merck) agar at 37 °C, unless otherwise noted.

Determination of alpha-hemolysin secretion by S. aureus strains

First, a 96-well MaxiSorp plate (Nunc, Roskilde, Denmark) was coated with 100 μl of alpha-hemolysin protein (Merck, Calbiochem, Germany) at concentrations ranging from 0.5 to 10 μg/ml. Following a blocking step, the wells were incubated with 100 μl of mouse anti-staphylococcal alpha-hemolysin mAb (6C12; IBT Bioservices, Gaithersburg, MD, USA) at a 1:500 dilution for 1 h at room temperature (RT). After several washes, the wells were treated with goat anti-mouse IgG-horseradish peroxidase (HRP) antibody diluted 1:2000 (Santa Cruz Biotechnology Inc., Heidelberg, Germany) and incubated at RT for an additional hour. The color reaction was developed using a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Scientific, MA, USA), followed by the addition of 1 M H2SO4 (Merck) (Piri-Gavgani et al. 2022). The OD450 was measured using an Epoch enzyme-linked immunosorbent assay (ELISA) reader (BioTek Instruments, Inc., Winooski, VT, USA), and a standard curve was generated.

The production of alpha-hemolysin by various S. aureus strains (S.a.48, S.a.61, S.a.124, and ATCC 6538) was assessed according to the method described by Sharma-Kuinkel et al. (2015) with minor modifications. Briefly, S. aureus strains S.a.48, S.a.61, and S.a.124 were grown in TSB containing oxacillin (Sigma-Aldrich, Saint Louis, USA) at 37 °C overnight, while S. aureus ATCC 6538 was cultured in TSB (Soltanmohammadi et al. 2021). Additionally, A. baumannii ATCC 19606 and K. pneumoniae ATCC 700603 were cultured in TSB while A. baumannii strains A.b.56 and A.b.58 were cultured in TSB supplemented with imipenem (Sigma-Aldrich) (Basardeh et al. 2022). An aliquot of each overnight culture was diluted 1:50 in TSB (OD600~0.8) and incubated for an additional 4 h at 37 °C. The bacteria were then collected by centrifugation, and the supernatant was filter-sterilized and stored at −20 °C. The culture supernatants of S. aureus, A. baumannii, and K. pneumoniae strains were used to coat 96-well MaxiSorp microplates (Nunc) overnight at 4 °C, with alpha-hemolysin protein (2 μg/ml) included as a positive control. After blocking non-specific sites, the mouse anti-staphylococcal alpha-hemolysin mAb (IBT Bioservices) was added and incubated for 1 h, followed by incubation with goat anti-mouse IgG-HRP antibody (Santa Cruz Biotechnology Inc.). Extensive washes were performed after each step. The color reaction was developed using a TMB substrate solution, followed by the addition of 1 M H2SO4 solution. The OD450 was measured using an Epoch ELISA reader (BioTek Instruments, Inc.). Moreover, the wells containing culture supernatants from the mentioned bacterial strains and the wells containing uninfected medium were directly incubated with the mouse anti-staphylococcal alpha-hemolysin antibody (IBT Bioservices), which served as the controls.

Safety evaluation of the SP192 and SP220 scFvs in healthy mice

All in vivo assays were conducted using female C57BL/6 mice, aged seven to nine weeks and weighing 18 to 20 g, obtained from the Animal Laboratory at the Pasteur Institute of Iran (Tehran, Iran). The mice were randomly allocated to five groups (n = five per group), including three groups that received anti-alpha-hemolysin scFvs and two control groups for comparative analysis. In the scFv groups, the mice were administered 15 mg/kg of SP192, SP220, or a combination of the two scFvs (7.5 mg/kg of each) via intraperitoneal injection at 12-h intervals for 72 h. The control groups received 20 mg/kg of vancomycin or normal saline every 12 h for the same 72-h period.

At 72 h, all mice were euthanized with an intraperitoneal injection of an overdose, five times the anesthetic dose, of ketamine (100 mg/kg) and xylazine (20 mg/kg) (Alfasan, Woerden, Netherlands) (Basardeh et al. 2024). Their kidneys and livers were then harvested and visually examined for signs of toxicity. Subsequently, all samples underwent routine histological processing, including fixation in 10% formalin for 24 h, followed by paraffin embedding. Thin tissue sections were stained with hematoxylin and eosin. A veterinary pathologist, blinded to the experimental groups, analyzed the stained sections using a Dino-Lite digital lens, Dino Capture 2 software (AnMo Electronics Corp., New Taipei City, Taiwan), and a light microscope (Olympus CX21, Japan).

Assessing the therapeutic potential of the SP192 and SP220 scFvs in an immunocompromised mouse model of pneumonia induced by the MRSA strain S.a.124

The in vivo therapeutic efficacy of SP192 and SP220 in combating pneumonia caused by an MRSA strain was inspected in an immunocompromised mouse model, as previously delineated by Hua et al. (2015). Female C57BL/6 mice (n = eight per group) underwent immunosuppression through an intraperitoneal injection of 150 mg/kg of cyclophosphamide monohydrate (Sigma-Aldrich), dissolved in normal saline, administered 4 days before, and 100 mg/kg of cyclophosphamide one day before S. aureus inoculation (Hua et al. 2015; Pollitt et al. 2018). On the day of inoculation (day zero), the mice were subjected to anesthesia via intraperitoneal administration of ketamine (100 mg/kg) and xylazine (20 mg/kg) (Alfasan) in a 2:1 ratio (Chen et al. 2019; Schuetze et al. 2019). Once anesthetized, the mice were inoculated intranasally with a range of doses of the MRSA strain S.a.124 suspended in 40 µl of sterile phosphate-buffered saline (PBS) (ranging from 1 × 105 to 3.2 × 109 colony-forming units [CFU]/mouse) delivered into the left and right nares (Hua et al. 2014). The results revealed that immunocompromised mice inoculated with 1 × 107 and 3.2 × 109 CFU exhibited 50% and 100% lethality, respectively.

To explore the potential of passive immunotherapy for treating immunocompromised mice with pneumonia, SP192 (15 mg/kg), SP220 (15 mg/kg), or a combination of both scFvs (7.5 mg/kg of each) was administered intraperitoneally 2 h after the mice (n = eight per group) were inoculated intranasally with 1 × 107 CFU of the MRSA strain S.a.124. The administration continued every 12 h for 72 h. A series of carefully designed control groups was incorporated, including infected mice receiving a combination of the two scFvs (7.5 mg/kg of each, every 24 h), vancomycin (20 mg/kg, every 12 h) (Hua et al. 2014; Ohsawa et al. 2015), normal saline (every 12 h), and EB211, an unrelated scFv, targeting A. baumannii (15 mg/kg, every 12 h) (Basardeh et al. 2022, 2024).

For 7 days, all mice were closely monitored for clinical signs, including weight loss, hunched posture, ruffled fur, and lethargy (Chan et al. 2023). Survival rates were meticulously tracked over 7 days, and survival curves were analyzed using the Log-rank (Mantel-Cox) test (Hua et al. 2014; 2015). Mice were ethically euthanized with an overdose of ketamine and xylazine if they were unable to eat or drink, or if they became immobile, and these individuals were recorded as non-survivors in the survival censuses.

The MRSA burden in the vital organs of immunocompromised mice with pneumonia receiving the SP192 and SP220 scFvs

Immunocompromised mice (n = eight per group) were intranasally infected with the MRSA strain S.a.124 and subsequently administered treatment with SP192, SP220, a combination of both scFvs, vancomycin, normal saline, or EB211 2 h after infection. All mentioned treatments were scheduled every 12 h for a total of 72 h, and the group receiving a combination of both scFvs was scheduled every 24 h for the same 72 h. Mice that died or were euthanized during 24, 48, and 72 h of infection were assessed for further evaluation (Basardeh et al. 2024). The vital organs—heart, kidneys, liver, lungs, and spleen—were promptly extracted and weighed, allowing for a precise assessment of the MRSA burden. To quantify MRSA, organ homogenates were diluted in series and plated on LB agar (Merck) supplemented with oxacillin. After 18 h of incubation at 37 °C, the MRSA colonies were carefully enumerated, following methods previously outlined by Cohen et al. (2016) and Stulik et al. (2019). This thorough analysis not only illuminates the devastating impact of MRSA on critical organs but also underscores the potential for therapeutic interventions in addressing pneumonia-induced infections.

Pathological evaluation of the renal and pulmonary tissues in immunocompromised mice with pneumonia induced by MRSA

The kidneys and lungs of infected mice (n = eight per group) undergoing treatment, as well as those from healthy control mice (n = five per group), were carefully collected in an aseptic manner after the animals were sacrificed at 24, 48, and 72 h of infection. Thin sections of the paraffin-embedded tissues were meticulously stained with hematoxylin and eosin. Subsequent histopathological examinations were conducted using a Dino-Lite digital lens, the Dino Capture 2 software (AnMo Electronics Corp.), and a light microscope (Olympus CX21).

Statistical analyses

All results are presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was assessed using two-way analysis of variance (ANOVA) followed by Sidak’s or Dunnett’s multiple-comparison test. The survival curve was constructed using the Kaplan–Meier method, and significance was evaluated using the log-rank (Mantel-Cox) test. Differences were considered statistically significant at P < 0.05. All analyses were conducted using GraphPad Prism version 8 software (https://www.graphpad.com/).

Results

Antimicrobial susceptibility testing

The antibiotic susceptibility of S. aureus strains S.a.48, S.a.61, S.a.124, and ATCC 6538 was tested using the minimum inhibitory concentration (MIC) test strip assay. The results showed that the S. aureus ATCC 6538 was the only strain with susceptibility to oxacillin (MIC: 0.2 µg/ml), while S. aureus strains S.a.48, S.a.61, and S.a.124 were extremely resistant to oxacillin (MIC > 256 µg/ml). According to the broth microdilution assay, S. aureus strains S.a.48, S.a.61, S.a.124, and ATCC 6538 showed susceptibility to vancomycin (MICs ranged from 1 to 2 µg/ml). A. baumannii strains A.b.56 and A.b.58 were identified as imipenem-resistant strains (MIC: 32 µg/ml). A. baumannii ATCC 19606 and K. pneumoniae ATCC 700603 were susceptible to imipenem (MIC: 2 and 0.125 µg/ml, respectively). Furthermore, according to the MIC breakpoints of the Clinical and Laboratory Standards Institute (CLSI) for colistin sulfate, A. baumannii strains A.b.56, A.b.58, and ATCC 19606 were considered as colistin-susceptible strains (MIC: 1 µg/ml; susceptible if MIC ≤ 2 µg/ml) (Basardeh et al. 2022; Soltanmohammadi et al. 2021).

The analysis of S. aureus strains revealed varying levels of alpha-hemolysin secretion

Based on a standard curve drawn from various concentrations of alpha-hemolysin protein ranging from 0.5 to 10 μg/ml (Fig. 1A), the concentration of alpha-hemolysin in the supernatants from overnight cultures of various S. aureus strains was determined by ELISA (Fig. 1B). The results showed that the S. aureus strain S.a.124 was remarkably prolific, producing a high concentration of alpha-hemolysin at 2 µg/ml, which was significantly higher than the two other MRSA strains, S.a.48 and S.a.61 (Fig. 1B). In contrast, S. aureus ATCC 6538 exhibited the lowest level of alpha-hemolysin secretion, measuring only 0.7 µg/ml. These findings underscore the considerable variability in alpha-hemolysin production among the four strains examined. Furthermore, the control groups—including A. baumannii strains A.b.56, A.b.58, and ATCC 19606 and K. pneumoniae ATCC 700603—demonstrated no secretion of alpha-hemolysin, reinforcing the distinct profiles observed in the S. aureus strains (Fig. 1B).

Fig. 1.

Fig. 1

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Determination of alpha-hemolysin secretion by examined S. aureus strains. An enzyme-linked immunosorbent assay (ELISA) was conducted to establish A a standard curve using different concentrations of alpha-hemolysin. The wells were coated with 100 μl of alpha-hemolysin protein at concentrations ranging from 0.5 to 10 μg/ml. Subsequently, the wells were incubated with the mouse anti-staphylococcal alpha-hemolysin toxin monoclonal antibody (mAb), followed by incubation with the goat anti-mouse IgG-HRP antibody. The absorbance was measured at an optical density (OD) of 450 nm (OD450), facilitating the generation of a standard curve. B The release of alpha-hemolysin from the various S. aureus strains under investigation was quantified in bacterial culture supernatants through ELISA. Notably, the production of alpha-hemolysin was significantly higher in the S. aureus strain S. a. 124 compared to S. aureus strains S.a.48, S.a.61, and ATCC 6538. Statistical significance was determined by two-way ANOVA, followed by Sidak’s multiple-comparison test, with *P < 0.0001 indicating significant differences. A.b.: A. baumannii; K.p.: K. pneumonia; S.a.: S. aureus; Um: uninoculated media

The SP192 and SP220 scFvs demonstrated a favorable safety profile, as supported by histopathological evaluations indicating no toxic effects

To assess the impact of SP192 and SP220 on vital organs, we administered the scFvs (either individually or in combination) intraperitoneally at a dose of 15 mg/kg every 12 h for 72 h. At the 72-h mark, all mice were euthanized, and their kidneys and livers were then harvested and visually examined for signs of toxicity. Subsequently, all samples underwent routine histological processing, including fixation in 10% formalin for 24 h, followed by paraffin embedding. Thin tissue sections were stained with hematoxylin and eosin. Histopathological analysis revealed no toxicity in the kidneys (Fig. 2A) or livers (Fig. 2B) of the treated mice, consistent with the control group that received normal saline. In contrast, treatment with vancomycin at a dose of 20 mg/kg every 12 h over the same 72-h period led to partial liver toxicity (Fig. 2B).

Fig. 2.

Fig. 2

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Absence of toxic effects on the kidneys and liver of mice administered SP192 and SP220. Mice (n = five per group) were subjected to intraperitoneal injections of SP192 (15 mg/kg), SP220 (15 mg/kg), a combination of the two scFvs (7.5 mg/kg of each), or vancomycin (20 mg/kg), administered every 12 h for 72 h. All mice were euthanized with an intraperitoneal injection of an overdose of ketamine and xylazine 72 h after injection. Their kidneys and livers were then harvested and visually examined for signs of toxicity. The histopathological analysis showed no adverse or toxic effects on either A the kidneys or B the livers of mice after 72 h of receiving the scFv. These results were consistent with those observed in the group that received normal saline. In contrast, the administration of vancomycin resulted in partial liver toxicity. White arrow: infiltration of inflammatory cells; Yellow arrow: renal tubule

The SP192 and SP220 scFvs significantly improved survival rates in an immunocompromised mouse model of pneumonia induced by the MRSA strain S.a.124

To investigate the relationship between toxin binding affinity/in vitro neutralization potency and in vivo efficacy of anti-alpha-hemolysin scFvs, the functional activity of SP192 and SP220 (individually or in combination) was assessed in an immunocompromised mouse model of pneumonia. In this regard, mice were treated with cyclophosphamide before intranasal infection with the MRSA strain S.a.124 (the highly alpha-hemolysin-producing S. aureus strain). Next, immunocompromised mice were infected with varying bacterial inoculum sizes (1 × 105 to 3.2 × 109 CFU). The 50% lethal dose (LD50) inoculum was determined to be 1 × 107 CFU per mouse, resulting in 50% lethality within 7 days (data not shown). Then, SP192 (15 mg/kg), SP220 (15 mg/kg), the combination of both scFvs (7.5 mg/kg of each), and vancomycin (20 mg/kg) were administered every 12 h for 72 h to immunocompromised mice with pneumonia 2 h after intranasal infection with the MRSA strain S.a.124. Infected mice receiving normal saline (the vehicle) or EB211 (an unrelated scFv, against A. baumannii) every 12 h for 72 h lost significantly more weight and showed apparent signs of disease, including hunched posture, ruffled fur, and lethargy, by 8 to 10 h, demonstrating the quick advancement of the infection. Compared to a 50% survival rate observed in the vehicle and EB211 groups, immunocompromised mice with pneumonia receiving SP192, SP220, a combination of SP192 and SP220, and vancomycin afforded complete protection with 100% survival rates (log-rank test) (Fig. 3).

Fig. 3.

Fig. 3

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Therapeutic effects of SP192 and SP220 on survival rates in immunocompromised mice with MRSA pneumonia. Immunocompromised mice (n = eight per group) were administered treatments intraperitoneally every 12 h for 72 h. The treatments included SP192 (15 mg/kg), SP220 (15 mg/kg), a combination of both SP192 and SP220 (7.5 mg/kg of each), vancomycin (20 mg/kg), normal saline (the vehicle), or EB211 (an unrelated scFv, targeting A. baumannii; 15 mg/kg). These treatments were given 2 h after the intranasal challenge with 1 × 107 CFU of the MRSA strain S. a. 124. Additionally, a group of infected mice was given a combination of the two scFvs every 24 h (QD) for 72 h. The mortality rates of the mice were recorded daily for 7 days. Asterisks indicate statistical significance when compared to the vehicle group, as determined by the log-rank test (*P < 0.05)

Treatment of infected mice with a combination of SP192 and SP220 every 24 h for 72 h resulted in 75% survival, although this slight difference was not statistically significant (Fig. 3).

The SP192 and SP220 scFvs effectively reduced bacterial load in vital organs of immunocompromised mice with pneumonia induced by the MRSA strain S.a.124

The therapeutic potential of SP192 and SP220 in reducing bacterial burden in the lung and distal organs of immunocompromised mice infected intranasally with the MRSA strain S.a.124 was evaluated at 24, 48, and 72 h post-infection. Mice treated with SP192 (15 mg/kg), SP220 (15 mg/kg), or a combination of both scFvs (7.5 mg/kg of each) exhibited a remarkable reduction in bacterial CFUs in the lungs. These results closely resembled those observed in mice treated solely with vancomycin (Fig. 4D). Furthermore, within the treatment groups—including SP192, SP220, a combination of SP192 and SP220, and vancomycin—a significant decrease in bacterial dissemination to vital organs, including the heart, kidneys, liver, and spleen, was observed, surpassing the outcomes noted in the control groups (vehicle or EB211) (P < 0.05) (Fig. 4A–C and E). It is noteworthy that the highest reduction in bacterial burden in the examined organs was observed in mice administered a combination of SP192 and SP220 or vancomycin every 12 h for 72 h compared to other groups.

Fig. 4.

Fig. 4

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The effects of SP192 and SP220 on the bacterial burden in vital organs of immunocompromised mice with MRSA pneumonia. Immunocompromised mice (n = eight per group) were treated with SP192 (15 mg/kg), SP220 (15 mg/kg), a combination of both scFvs (7.5 mg/kg of each), vancomycin (20 mg/kg), normal saline (the vehicle), or EB211 (an unrelated scFv, targeting A. baumannii; 15 mg/kg) 2 h after intranasal challenge with 1 × 107 CFU of the MRSA strain S. a. 124. The treatment duration was every 12 h for 72 h. Moreover, a group of infected mice received the combination of the two scFvs every 24 h (QD) for 72 h. A The heart, B kidneys, C liver, D lung, and E spleen of mice that died or were ethically euthanized following 24, 48, or 72 h of infection were promptly extracted and weighed to allow for precise assessment of the MRSA burden. Statistical significance was determined by two-way ANOVA, followed by Dunnett’s multiple-comparison test against the vehicle group. *P < 0.05, **P < 0.01, and ***P < 0.001

The neutralizing activity of SP192 and SP220 against alpha-hemolysin significantly mitigated the pathogenesis of MRSA in target organs

The therapeutic efficacy of the SP192 and SP220 scFvs was evaluated through a histopathological analysis of the kidneys and lungs of infected mice that received SP192, SP220, a combination of both scFvs, or vancomycin after 24, 48, and 72 h of infection (Figs. 5 and 6).

Fig. 5.

Fig. 5

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The treatment effectiveness of SP192 and SP220 in alleviating renal damage in immunocompromised mice with MRSA pneumonia. Immunocompromised mice (n = eight per group) were intraperitoneally administered every 12 h for 72 h with either SP192 (15 mg/kg), SP220 (15 mg/kg), a combination of the two scFvs (7.5 mg/kg of each), vancomycin (20 mg/kg), normal saline (the vehicle), or EB211 (an unrelated scFv, targeting A. baumannii; 15 mg/kg) 2 h after intranasal challenge with 1 × 107 CFU of the MRSA strain S. a. 124. Furthermore, one group of infected mice received a combination of the two scFvs every 24 h (QD) for 72 h. Within 24, 48, and 72 h following infection, the histopathological evaluation of the kidneys in immunocompromised mice with pneumonia receiving normal saline (the vehicle) or EB211 was conducted to characterize the progression of renal pathology, indicating focal bacterial aggregates of S. aureus, significant disruption of standard renal architecture, renal tubular epithelial necrosis and degeneration, interstitial hemorrhage, as well as focal and patchy infiltration of mononuclear cells within the interstitium. In contrast, the groups receiving SP192 (15 mg/kg), SP220 (15 mg/kg), or a combination of both scFvs (7.5 mg/kg of each scFv) demonstrated a markedly different histopathological profile, underscoring the potential therapeutic impact of these treatments. Black arrow: focal bacterial aggregates; black arrowhead: disruption of normal renal architecture and interstitial hemorrhage; white arrow: focal or patchy cellular infiltrates (mononuclear cells) within the interstitium; white arrowhead: renal tubular epithelial necrosis and degeneration; yellow arrow: renal corpuscles; IC: immunocompromised mice with no infection; normal: healthy mice

Fig. 6.

Fig. 6

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The treatment effectiveness of SP192 and SP220 in reducing pulmonary injury in immunocompromised mice with MRSA pneumonia. Immunocompromised mice (n = eight per group) were intraperitoneally administered every 12 h for 72 h with either SP192 (15 mg/kg), SP220 (15 mg/kg), a combination of the two scFvs (7.5 mg/kg of each), vancomycin (20 mg/kg), normal saline (the vehicle), or EB211 (an unrelated scFv, targeting A. baumannii; 15 mg/kg) 2 h after intranasal challenge with 1 × 107 CFU of the MRSA strain S. a. 124. Furthermore, one group of infected mice received a combination of the two scFvs every 24 h (QD) for 72 h. Histopathological assessment of the lungs in immunocompromised mice with pneumonia treated with either normal saline (the vehicle) or EB211 revealed several significant findings. Within 24, 48, and 72 h following infection, the evaluation revealed focal accumulations of S. aureus, disruption of normal tissue architecture, bronchial epithelial necrosis and degeneration, peribronchial or alveolar mononuclear cell aggregates, and hemorrhage. In contrast, partial hemorrhage and limited infiltrations of inflammatory cells were observed in the alveolar spaces of the lungs from infected mice treated with SP192, SP220, or a combination of the two scFvs (QD). Furthermore, lung sections from mice administered the combination of the two scFvs every 12 h for 72 h showed no evidence of hemorrhage or infiltration of inflammatory cells. Black arrow: focal accumulations of S. aureus; black arrowhead: disruption of normal tissue architecture and hemorrhage; white arrow: peribronchial/alveolar mononuclear cell aggregates; white arrowhead: bronchial epithelial necrosis and degeneration; IC: immunocompromised mice with no infection; normal: healthy mice

By the 24-h mark, the evaluation of the kidney in mice treated with normal saline (the vehicle) or EB211 displayed focal bacterial aggregates of S. aureus, significant disruption of normal renal architecture, renal tubular epithelial necrosis and degeneration, interstitial hemorrhage, as well as focal and patchy infiltration of mononuclear cells within the interstitium (Fig. 5). Moreover, the kidney of mice administered the combination of scFvs every 24 h or vancomycin indicated tubular epithelial necrosis and degeneration, accompanied by limited interstitial infiltration of mononuclear cells. In contrast, the group receiving SP192 exhibited only low degenerated renal tubules, while no marked histopathological changes were evident in the groups treated with SP220 or the combination of both scFvs every 12 h (Fig. 5).

After 48 h, although the vehicle and EB211 groups persisted in displaying pathological findings—including focal bacterial aggregates (S. aureus), renal tubular epithelial necrosis and degeneration, as well as focal or patchy interstitial infiltration of mononuclear cells—no histopathological abnormalities were observed in the groups receiving the anti-alpha-hemolysin scFvs. The vancomycin-treated group maintained evidence of tubular epithelial necrosis and degeneration, albeit with limited interstitial cellular infiltrates (Fig. 5).

By 72 h, limited renal tubular degeneration was observed across all treatment groups, including those receiving scFvs or vancomycin. Nevertheless, mice in the vehicle and EB211 groups continued to exhibit focal bacterial aggregates, disruption of renal tissue architecture accompanied by interstitial hemorrhage, and renal tubular epithelial necrosis and degeneration (Fig. 5).

The gross pathological assessment of the lungs in infected mice from both the vehicle and EB211 groups demonstrated the neutralizing efficacy of the anti-alpha-hemolysin scFvs SP192, SP220, or their combination when administered to infected mice (Fig. 6).

At the 24-h mark, histopathological evaluations revealed that the lungs of infected mice treated with either normal saline (the vehicle) or EB211 exhibited disruption of normal tissue architecture, bronchial epithelial necrosis and degeneration, hemorrhage, and peribronchial and alveolar mononuclear cell aggregates. Additionally, focal accumulations of S. aureus were noted within the lungs of these groups. The presence of peribronchial and alveolar mononuclear cell aggregates was also observed in the groups receiving SP192, SP220, the combination of scFvs (administered every 24 h), and vancomycin. Notably, hemorrhage was seen in the groups treated with SP192 or the combination of scFvs administered every 24 h. The most favorable outcomes, characterized by the absence of histopathological changes, were observed in the group receiving the combined treatment of SP192 and SP220 every 12 h (Fig. 6).

All pathological findings documented in the vehicle and EB211 groups at the 24-h interval were similarly evident after 48 h. The groups administered SP192, SP220, the combination of scFvs (every 24 h), or vancomycin exhibited analogous results to those observed at 24 h as well. However, hemorrhage was exclusively detected in the mice receiving the combination of scFvs every 24 h. Again, no histopathological abnormalities were identified in the group treated with the combination of scFvs every 12 h (Fig. 6).

By the 72-h mark, the vehicle and EB211 groups displayed comparable pathological signs to those observed after 24 and 48 h. Peribronchial and alveolar mononuclear cell aggregates were recognized in selected microscopic fields of mice treated with SP192, SP220, the combination of scFvs administered every 12 h, the combination administered every 24 h, or vancomycin. Importantly, bronchial epithelial necrosis and degeneration were also noted in specific histological fields of the vancomycin-treated group (Fig. 6).

Discussion

The use of antibiotics has unquestionably enhanced the quality and length of life for countless people, and it was once thought that most bacterial infectious diseases had been conquered (Ferraz 2024). However, this earlier optimism is now challenged by the increasing emergence of antibiotic-resistant bacteria and the unavailability of effective antibiotics (Soltanmohammadi et al. 2021). In S. aureus infections, the incidence of MRSA-associated invasive infections, such as necrotizing pneumonia, with high morbidity and mortality, is continuing to increase, while the antibacterial agents available today against this pathogenic bacterium are extremely limited (Parimon et al. 2013; Soltanmohammadi et al. 2021). This formidable disease demands our attention and action, underscoring its prominence in public health. Therefore, new therapeutic strategies and antimicrobial agents for infections caused by S. aureus are greatly needed (Piri-Gavgani et al. 2022; Soltanmohammadi et al. 2021).

Interference with bacterial virulence has emerged as an effective and promising strategy for meeting the challenge. Although traditional strategies are aimed at cellular viability (bactericidal and bacteriostatic activity), which are highly effective, these modes of action can lead to the emergence of drug-resistant strains (Soltanmohammadi et al. 2021). Of note, anti-virulence strategies offer promising opportunities to abate pathogenicity and its consequences without directly killing the target bacteria, thereby presumably applying milder selective pressure on the development of antibiotic resistance (Piri-Gavgani et al. 2022; Rahimi-Jamnani et al. 2025; Soezi et al. 2022). The effectiveness of neutralizing recombinant antibodies against bacterial toxins such as anthrax toxin (Obiltoxaximab and Raxibacumab) and Clostridium difficile Toxin B (Bezlotoxumab) has been demonstrated (Soezi et al. 2022). S. aureus, similar to other Gram-positive bacteria, produces a variety of extracellular virulence factors that significantly enhance its potential to cause disease. Among these factors, cytolysins and superantigens play key roles in the bacterium’s ability to induce illness (Piri-Gavgani et al. 2022; Soezi et al. 2022). Alpha-hemolysin, a potent pore-forming toxin found in most S. aureus strains, stands out as a formidable cytolysin and a key player in the virulence of this bacterium. Its influence is particularly profound in a range of severe infections, including dermonecrosis, pneumonia, sepsis, endocarditis, and keratitis, showcasing its critical role in the pathogenic arsenal of S. aureus (Hua et al. 2014; Piri-Gavgani et al. 2022). Secreted alpha-hemolysin binds A-disintegrin and metalloprotease 10 (ADAM10), and generates heptameric pores in host membranes, resulting in cell lysis, tissue disruption, and evasion of the protective host immune response (Hilliard et al. 2015). Based on these findings, alpha-hemolysin may represent a promising anti-virulence target for the development of novel targeting agents for S. aureus (Hua et al. 2014; Piri-Gavgani et al. 2022).

In recent years, human-derived scFvs have been considered as remarkable alternatives for whole mAbs (Soltanmohammadi et al. 2021). The advantages of scFvs for immunotherapy derive from their smaller size compared to the mAbs, which helps them penetrate deeper and faster into tissues to target and easily clear from the blood (Piri-Gavgani et al. 2022; Soezi et al. 2022). Owing to the lack of constant regions, scFv retention by Fc receptors on tissues and organs decreases significantly, which in turn can reduce their side effects and make them less immunogenic (Rahimi-Jamnani et al. 2025). In this regard, in our previous study, we identified two human alpha-hemolysin neutralizing scFvs, designated SP192 and SP220, which showed stronger binding to alpha-hemolysin than the control proteins, including bovine serum albumin, human adiponectin, and TSST-1. Besides, both scFvs showed high-affinity binding to alpha-hemolysin in the nanomolar range, leading to marked inhibition of alpha-hemolysin-mediated lysis of rabbit red blood cells (Piri-Gavgani et al. 2022).

Methicillin-resistant S. aureus pneumonia has emerged as a critical global health concern, particularly affecting vulnerable populations such as infants, immunocompromised individuals, the elderly, and patients relying on ventilators (Lee et al. 2018). In response to this pressing issue, our objective was to initially provide additional evidence of the safety profile of SP192 and SP220 in healthy mice and then demonstrate their therapeutic efficacy in an immunocompromised mouse model of MRSA pneumonia.

Off-target toxicity and adverse effects, such as cardiotoxicity and nephrotoxicity, have been documented with mAbs; however, these occurrences are infrequently associated with scFvs (Ahamadi-Fesharaki et al. 2019; Basardeh et al. 2024; Rahimi-Jamnani et al. 2025; Soltanmohammadi et al. 2021). The safety profiles of SP192 and SP220 were systematically examined in healthy mice administered the scFvs—either individually at a dosage of 15 mg/kg or in combination at a dosage of 7.5 mg/kg of each—every 12 h for a duration of 72 h. Microscopic examination of tissue sections from the kidneys and liver demonstrated normal architecture, with no discernible differences in histological and cellular structures compared with those of mice receiving normal saline. Moreover, the results indicated superior outcomes relative to those observed in mice treated with vancomycin.

The therapeutic potential of anti-alpha-hemolysin mAbs for the treatment of pneumonia has been demonstrated in several studies (Diep et al. 2017; François et al. 2021; Hua et al. 2014, 2015). Initiating therapy promptly—ideally within 10 h—strongly enhances treatment effectiveness and significantly lowers mortality rates (Hilliard et al. 2015; Hua et al. 2015). This timely intervention may help achieve better outcomes for immunocompromised patients in acute care settings. To this end, in our study, immunocompromised mice with MRSA pneumonia received SP192, SP220, or a combination of the two scFvs 2 h post-infection. The treatment was continued every 12 h for 72 h. It has been demonstrated that S. aureus impairs lung function by secreting alpha-hemolysin, which directly lyses lung epithelial cells and, at sublytic concentrations, activates ADAM-10. Our results indicated that the initiation of therapy with anti-alpha-hemolysin scFvs after 2 h and every 12 h led to the lowest disruption of normal lung tissue architecture, bronchial epithelial necrosis and degeneration, hemorrhage, and focal accumulations of S. aureus in the lungs of the immunocompromised mice, compared to the group receiving the combination of scFvs every 24 h. It can be questionable how the SP192 and SP220 scFvs targeting alpha-hemolysin affect the viability of the bacteria or trigger opsonization, resulting in low focal accumulations of S. aureus. This finding would be consistent with the hypothesis that SP192 or SP220, by neutralizing alpha-hemolysin, could help preserve the effectiveness of the immune system to reduce the bacterial growth (François et al. 2018).

One of the most clinically advanced anti-alpha-hemolysin mAbs is MEDI4893 (suvratoxumab) (François et al. 2021). This human immunoglobulin G1(κ) (IgG1[κ]) specifically targets alpha-hemolysin and exhibits an extended half-life, achieved by incorporating YTE mutations into the Fc domain of the predecessor mAb, LC10 (also referred to as MEDI4893*) (Hua et al. 2014; Yu et al. 2017). In an initial study conducted by Hua et al. (2014), the therapeutic efficacy of LC10 (MEDI4893*) was evaluated in an immunocompetent mouse model of S. aureus pneumonia. The findings indicated that administering LC10 at 15 mg/kg 1 h post-infection resulted in a 30 to 40% survival rate (Hua et al. 2014). Furthermore, a histopathological examination of the lungs of mice treated with LC10 at 24 h post-infection revealed necrosis, edema, hemorrhage, multifocal loss of alveolar architecture, and severe inflammation with the presence of bacterial colonies (Hua et al. 2014). In a study similar to ours, Hua et al. (2015) evaluated the therapeutic effectiveness of MEDI4893* in immunocompromised mice with S. aureus pneumonia. They administered a single dose of MEDI4893* at 15 mg/kg 1 h post-infection. The results demonstrated that immunocompromised mice receiving MEDI4893* had a 50% survival rate (Hua et al. 2015). Furthermore, treatment with MEDI4893* not only reduced bacterial levels in the lungs but also preserved the structural integrity of lung tissues, thereby maintaining a robust epithelial barrier (Hua et al. 2015). Diep et al. evaluated the treatment efficacy of MEDI4893* in a rabbit model of necrotizing pneumonia (Diep et al. 2017). In their study, rabbits were given MEDI4893* at a dosage of 30 mg/kg, 90 min after infection. The results showed a 67% survival rate for rabbits treated with MEDI4893*, while the survival rates for those treated with vancomycin and c-IgG were 33% and 17%, respectively (Diep et al. 2017). Additionally, the bacterial burden in the lungs, spleen, and kidneys of the rabbits treated with MEDI4893* was not significant (Diep et al. 2017).

In a different study, Foletti et al. developed a human anti-alpha-hemolysin mAb, designated LTM14, and assessed its effectiveness in an intranasal mouse model of S. aureus pneumonia (Foletti et al. 2013). In this model, mice typically exhibited pronounced clinical signs of illness within 12 to 18 h, underscoring the swift and aggressive nature of the infection (Foletti et al. 2013). The experimental design was crafted to evaluate the impact of timely intervention. Mice were treated with either LTM14 (30 mg/kg) or linezolid (12 mg/kg) 12 or 18 h after being infected with 5 × 108 CFU of the S. aureus strain USA300 LAC. When treatment was administered within 12 h of infection, both LTM14 and linezolid resulted in a 100% survival rate. However, when the treatment was postponed until 18 h post-infection, survival rates plummeted to a stark drop to 40% to 50% for both treatments. These findings emphasized that delaying treatment can greatly diminish its effectiveness, underscoring the necessity of early intervention (Foletti et al. 2013).

Vancomycin stands out as a powerful antibiotic; however, it grapples with a considerable challenge in the treatment of pneumonia—its comparatively poor bioavailability in the lungs. This limitation often compels clinicians to resort to higher therapeutic doses to achieve the desired therapeutic effects. Unfortunately, these elevated doses may lead to adverse effects, including nephrotoxicity (Hua et al. 2015). In addition to this concern, vancomycin can increase alpha-hemolysin levels by disrupting cell wall biosynthesis, leading to cell lysis and the subsequent release of cytosolic alpha-hemolysin. Neutralizing this released alpha-hemolysin—whether originating from lysed bacteria or pre-existing levels—using SP192 and SP220 works by binding to alpha-hemolysin, thereby mitigating tissue damage and modulating inflammatory responses in conjunction with vancomycin administration, potentially reducing the need for high dosages (Hua et al. 2014).

Our study represents a pioneering investigation into the therapeutic efficacy of anti-alpha-hemolysin scFvs. We observed a significant improvement in survival rates, a reduction in bacterial burden in vital organs, and the preservation of healthy renal and pulmonary structures in an immunocompromised mouse model of MRSA-induced pneumonia. Notably, the combination of SP192 and SP220, administered every 12 h for a duration of 72 h, demonstrated the most pronounced protective efficacy among the treatment regimens evaluated in this study. Consequently, we anticipate that our forthcoming research, which will assess the therapeutic efficacy of vancomycin in conjunction with this combination of scFvs, may yield substantial findings relevant to the treatment of MRSA pneumonia in immunocompromised subjects.

Conclusions

We have developed two human alpha-hemolysin-neutralizing scFvs, designated SP192 and SP220, exhibiting high specific binding affinities in the nanomolar range (Kaff: 0.9 and 0.7 nM−1, respectively). These scFvs demonstrated marked inhibition of alpha-hemolysin-mediated lysis of rabbit RBCs. We further assessed the therapeutic effectiveness of these scFvs in an immunocompromised mouse model of MRSA pneumonia. The results revealed that both SP192 and SP220 provide substantial protection, resulting in increased survival rates and minimized renal and pulmonary damage. Although each scFv exhibited therapeutic activity on its own, the combination of SP192 and SP220, administered at 12-h intervals, was more effective than any individual treatment regimen investigated in our study. These findings suggest promising potential for the use of these scFvs in immunosuppressed patients with MRSA pneumonia, improving clinical outcomes. Therefore, the adjunctive therapy combining vancomycin with both SP192 and SP220 may represent a significant advancement in the treatment of immunocompromised patients facing this severe infection.

Acknowledgements

The results described in this paper were part of a Ph.D. thesis of the first author, conducted at the Human Antibody lab of the Mycobacteriology and Pulmonary Research Department. We thank the Mycobacteriology and Pulmonary Research Department of the Pasteur Institute of Iran for their cooperation.

Author contribution

FRJ supervised and directed the study. HRM performed histopathological analysis. SP performed experiments and contributed to manuscript preparation. FN was involved in the manuscript preparation. AF, SDS, and MA helped interpret the data. All authors read and approved the final manuscript.

Funding

This article was extracted from the Ph.D. thesis of Somayeh Piri-Gavgani, which the Pasteur Institute of Iran partly funded.

Data availability

All data generated or analyzed during this study are included in the manuscript.

Declarations

Ethics approval

All animal experiments were conducted in accordance with ARRIVE guidelines (https://arriveguidelines.org) and approved by the Animal Care and Use Committees of the Pasteur Institute of Iran (Ethics No.: IR.PII.REC.1398.031). All methods were performed in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Clinical trial number

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated or analyzed during this study are included in the manuscript.

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