Multimechanistic Monoclonal Antibody Combination Targeting Key Staphylococcus aureus Virulence Determinants in a Rabbit Model of Prosthetic Joint Infection.

In a rabbit model of methicillin-resistant Staphylococcus aureus prosthetic joint infection (PJI), prophylaxis with AZD6389*-a combination of three monoclonal antibodies targeting alpha-hemolysin, bicomponent cytotoxins (LukSF/LukED/HlgAB/HlgCB), and clumping factor A-resulted in significant reductions in joint swelling, erythema, intra-articular pus, and bacterial burden in synovial tissues and biofilm-associated prosthetic implants compared with isotype-matched control IgG. Targeting specific staphylococcal virulence factors may thus have potential clinical utility for prevention of PJI.

Pseudomonas aeruginosa Ventilator-Associated Pneumonia Rabbit Model for Preclinical Drug Development.

Development and validation of large animal models of Pseudomonas aeruginosa ventilator-associated pneumonia are needed for testing new drug candidates in a manner that mimics how they will be used clinically. We developed a new model in which rabbits were ventilated with low tidal volume and challenged with P. aeruginosa to recapitulate hallmark clinical features of acute respiratory distress syndrome (ARDS): acute lung injury and inflammation, progressive decrease in arterial oxygen partial pressure to fractional inspired oxygen PaO2:FiO2, leukopenia, neutropenia, thrombocytopenia, hyperlactatemia, severe hypotension, bacterial dissemination from lung to other organs, multiorgan dysfunction, and ultimately death. We evaluated the predictive power of this rabbit model for antibiotic efficacy testing by determining whether a humanized dosing regimen of meropenem, a potent antipseudomonal ß-lactam antibiotic, when administered with or without intensive care unit (ICU)-supportive care (fluid challenge and norepinephrine), could halt or reverse natural disease progression. Our humanized meropenem dosing regimen produced a plasma concentration-time profile in the rabbit model similar to those reported in patients with ventilator-associated bacterial pneumonia. In this rabbit model, treatment with humanized meropenem and ICU-supportive care achieved the highest level of survival, halted the worsening of ARDS biomarkers, and reversed lethal hypotension, although treatment with humanized meropenem alone also conferred some protection compared to treatment with placebo (saline) alone or placebo plus ICU-supportive care. In conclusion, this rabbit model could help predict whether an antibiotic will be efficacious for the treatment of human ventilator-associated pneumonia.

Protective Efficacy of Monoclonal Antibodies Neutralizing Alpha-Hemolysin and Bicomponent Leukocidins in a Rabbit Model of Staphylococcus aureus Necrotizing Pneumonia.

Staphylococcus aureus is a major human pathogen that causes a wide range of infections by producing an arsenal of cytotoxins. We found that passive immunization with either a monoclonal antibody (MAb) neutralizing alpha-hemolysin or a broadly cross-reactive MAb neutralizing Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysins HlgAB and HlgCB conferred only partial protection, whereas the combination of those two MAbs conferred significant protection in a rabbit model of necrotizing pneumonia caused by the USA300 methicillin-resistant S. aureus epidemic clone.

Monoclonal antibodies neutralizing alpha-hemolysin, bicomponent leukocidins, and clumping factor A protected against Staphylococcus aureus-induced acute circulatory failure in a mechanically ventilated rabbit model of hyperdynamic septic shock.

Background: Patients with septic shock caused by Staphylococcus aureus have mortality rates exceeding 50%, despite appropriate antibiotic therapy. Our objectives were to establish a rabbit model of S. aureus septic shock and to determine whether a novel immunotherapy can prevent or halt its natural disease progression. Methods: Anesthetized rabbits were ventilated with lung-protective low-tidal volume, instrumented for advanced hemodynamic monitoring, and characterized for longitudinal changes in acute myocardial dysfunction by echocardiography and sepsis-associated biomarkers after S. aureus intravenous challenge. To demonstrate the potential utility of this hyperdynamic septic shock model for preclinical drug development, rabbits were randomized for prophylaxis with anti-Hla/Luk/ClfA monoclonal antibody combination that neutralizes alpha-hemolysin (Hla), the bicomponent pore-forming leukocidins (Luk) including Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysin, and clumping factor A (ClfA), or an irrelevant isotype-matched control IgG (c-IgG), and then challenged with S. aureus. Results: Rabbits challenged with S. aureus, but not those with saline, developed a hyperdynamic state of septic shock characterized by elevated cardiac output (CO), increased stroke volume (SV) and reduced systemic vascular resistance (SVR), which was followed by a lethal hypodynamic state characterized by rapid decline in mean arterial pressure (MAP), increased central venous pressure, reduced CO, reduced SV, elevated SVR, and reduced left-ventricular ejection fraction, thereby reproducing the hallmark clinical features of human staphylococcal septic shock. In this model, rabbits pretreated with anti-Hla/Luk/ClfA mAb combination had 69% reduction in mortality when compared to those pretreated with c-IgG (P<0.001). USA300-induced acute circulatory failure-defined as >70% decreased in MAP from pre-infection baseline-occurred in only 20% (2/10) of rabbits pretreated with anti-Hla/Luk/ClfA mAb combination compared to 100% (9/9) of those pretreated with c-IgG. Prophylaxis with anti-Hla/Luk/ClfA mAb combination halted progression to lethal hypodynamic shock, as evidenced by significant protection against the development of hyperlactatemia, hypocapnia, hyperkalemia, leukopenia, neutropenia, monocytopenia, lymphopenia, as well as biomarkers associated with acute myocardial injury. Conclusion: These results demonstrate the potential utility of a mechanically ventilated rabbit model that reproduced hallmark clinical features of hyperdynamic septic shock and the translational potential of immunotherapy targeting S. aureus virulence factors for the prevention of staphylococcal septic shock.

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development.

Background: New drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections. Methods: We report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression. Results: In a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects. Conclusion: The rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia.

IBT-V02: A Multicomponent Toxoid Vaccine Protects Against Primary and Secondary Skin Infections Caused by Staphylococcus aureus.

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.

Proenergetic effects of resveratrol in the murine neuronal cell line Neuro2a.

SCOPE: Energy deficit is a common characteristic of neurodegenerative disorders, including Alzheimer's disease. Adenosine monophosphate activated protein kinase (AMPK) is a key enzyme maintaining energy balance by regulating the cellular uptake of glucose, ß-oxidation of fatty acids, and expression of glucose transporter 4. Since resveratrol has been shown to increase the activity of AMPK, we hypothesized that it might influence energy metabolism in a model neuron-like cell line, murine Neuro2a cells. METHODS AND RESULTS: Resveratrol caused an elevation of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) in a dose-dependent manner. The highest ATP and GTP levels achieved by treatment with resveratrol were 70.3 ± 8.2 nmol/mg protein (1.9-fold of control) and 27.2 ± 4.0 nmol/mg protein (1.7-fold of control), respectively, when cells were treated with 100 µM resveratrol for 6 h. Interestingly, increases in the total sum of all adenine nucleotides were found upon addition of resveratrol. Despite these increases in ATP, GTP, and the total adenine nucleotide pool, resveratrol treatment led to a decrease in glucose consumption and lactate release, suggesting that resveratrol does not increase energy production (e.g. via AMPK kinase activation) but rather inhibits energy-consuming processes. CONCLUSION: Resveratrol increases the levels of ATP and GTP, but without creating an additional glucose demand.