Epitope Profiling of Diphtheria Toxoid Provides Enhanced Monitoring for Consistency Testing during Manufacturing Process Changes.

In the vaccine industry, multiple physicochemical, immunological, in vitro and in vivo analytical methods are applied throughout the manufacturing process to characterize and monitor the quality of vaccines. Presented here is the Single Epitope Antigenicity Test (SEAT), an innovative, quantitative epitope profiling method which provides an extended immunochemical analysis for diphtheria toxoid (DTxd) to be used for consistency testing during manufacturing process changes. The method uses BioLayer Interferometry (BLI) and a panel of monoclonal antibodies (mAbs) to independently assess nine individual antigenic sites of DTxd. The panel includes mAbs which are functional, bind distinct sites on DTxd and are able to distinguish intact DTxd from that which has been exposed to heat treatment. The SEAT method was qualified for precision, accuracy, and linearity, and was used to define a preliminary comparability range for DTxd made using the current manufacturing process. DTxd lots manufactured using alternate processes were assessed in the context of this range to determine the impact on DTxd antigenicity. Epitope profiling by SEAT provides quantitative information on the integrity of multiple important antigenic regions of DTxd, and therefore represents a valuable tool in a comprehensive analytical test package which can be used to support manufacturing process changes for vaccines.

Replacing the in vivo toxin challenge test with an in vitro assay for assessment of potency for diphtheria toxoid containing vaccines.

Replacement of the potency tests for diphtheria vaccines is a high priority for the international initiative to reduce, refine, and replace animal use in vaccine testing. Diphtheria toxoid containing vaccine products marketed in the US currently require potency testing by the United States Public Health Service (USPHS) test, which includes an in vivo passive protection test with a diphtheria toxin challenge. Here we describe an in vitro Diphtheria Vero Cell (DVC) assay which combines the immunization approach from the USPHS test and the use of a cell based neutralization assay for serological testing of vaccine potency. The DVC assay reduces the overall number of animals used compared to other serological potency tests and eliminates the in vivo toxin challenge used in the US test. The DVC assay can be used to test vaccine products with a low or high diphtheria toxoid dose. It has been optimized and validated for use in a quality control testing environment. Results demonstrate similar sera antibody unitage as well as agreement between the serum neutralization values determined using the USPHS test and the DVC assay and thus support the use of the DVC assay for routine and stability testing for diphtheria toxoid containing vaccine products.

Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

In vitro assessment of biological activity and stability of the ALVAC-HIV vaccine.

The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay.