David E. Tabor

Cloning and assessment of tumorigenicity and oncogenicity of a Madin-Darby canine kidney (MDCK) cell line for influenza vaccine production.

An Madin-Darby canine kidney (MDCK) cell line, 9B9-1E4, was cloned by limit dilution from a heterologous cell population and chosen as a potential production cell substrate for cell culture-based influenza vaccine manufacture. Since MDCK cells are transformed cells of canine origin, extensive characterization, including evaluation of tumorigenicity and oncogenicity, was performed to ensure the safety of this cell line for vaccine production. Injection of intact MDCK cells into adult and newborn athymic nude mice did not lead to progressive tumor formation in two separate tumorigenicity studies. In addition, neither MDCK cell lysate nor cellular DNA induced tumors in newborn rodents (athymic nude mice, hamsters and rats) in six oncogenicity studies. Observations from these studies demonstrate the low tumorigenic and oncogenic potential of the MDCK cell clone 9B9-1E4. These observations coupled with other characterization study results strongly suggest a high safety assurance level can be achieved through cell cloning and selection of low tumorigenic and oncogenic cells for influenza vaccine production.

Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

ABSTRACT Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

Staphylococcus aureus Alpha-Toxin Is Conserved among Diverse Hospital Respiratory Isolates Collected from a Global Surveillance Study and Is Neutralized by Monoclonal Antibody MEDI4893

ABSTRACT Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development.

Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.

Characterisation of anti-alpha toxin antibody levels and colonisation status after administration of an investigational human monoclonal antibody, MEDI4893, against Staphylococcus aureus alpha toxin

MEDI4893 is a novel, long‐acting human monoclonal antibody targeting Staphylococcus aureus (SA) alpha toxin (AT). This report presents the results of the exploratory analyses from a randomised phase 1 dose‐escalation study in healthy human subjects receiving single intravenous MEDI4893 doses or placebo.

Application of Flow Cytometry for Rapid Bioburden Screening in Vaccine Virus Production

Sensitive and timely detection of bioburden in presterile filtration product in aseptic processing of biologics is a critical parameter for microbial control and assurance of final product sterility. An application of automated flow cytometry system was developed for rapid microbial assessment and in-process control in vaccine virus production. In order to minimize the background signal caused by the components of the chicken egg substrate sample matrix, a sample processing method to clear somatic cell debris was included. The sample processing and the automated analysis take approximately 5 to 7 min per test sample and the method provides objective results in real time, enabling uninterrupted processing. The flow cytometry method was compared with the standard aerobic plate count method using tryptic soy agar in a parallel study of 1566 independent production-scale samples. The method was further characterized by spike recovery of five model bacterial organisms in representative sample matrix. In comparison to the culture method, the flow cytometry method was shown to be 96.2% sensitive and 98.2% specific for the detection of bioburden at a level of sensitivity suitable for the process stage requirement with the advantage of a nearly instantaneous time to result. LAY ABSTRACT: In-process bioburden control in the manufacturing of biopharmaceuticals is essential for final product sterility and integrity. In manufacturing contexts where an in-process hold time is infeasible or in cases where uninterrupted processing is desired, conventional culture-based bioburden detection methods cannot be used, as they require significant time to results that may not fit within the time constraints. In this case study we demonstrate the use of flow cytometry as an alternative rapid method that provides real-time results to enable uninterrupted processing.