SBIR-STTR Award

Shigella, a major cause of diarrhea for military personnel and civilians, is often associated with Shigella flexneri serotypes 2a, 3a, and 6, and Shigella sonnei. These shigella strains express serologically distinct O-antigen polysaccharides at the distal end of LPS molecules. To control shigella diseases, various shigella antigens are being investigated as vaccine candidates. In the wake of the highly successful pneumococcal conjugate vaccines, multivalent shigella conjugate vaccines (SCVs) are actively being developed. SCVs contain a mixture of several epidemiologically important serotype-specific lipopolysaccharides (LPS) that are conjugated with a carrier protein. SCVs are designed to elicit antibodies to O-antigens that can kill shigella expressing the targeted O-antigen (but not others) in the presence of complement. In developing SCVs, it is necessary to measure the functional capacity of antibodies such as the bactericidal activity induced by the vaccine. Since a serum bactericidal assay (SBA) measures antibody-mediated killing activity in vitro, that killing activity can be used as a surrogate for vaccine efficacy in vaccine studies. However, as explained below, SBAs are difficult assays that must be run once for each serotype, and SCVs have multiple serotypes. Consequently, SCV development requires a sample to be analyzed multiple times with a difficult SBA. Thus, it is highly desirable to have a simple, high-throughput, multiplexed SBA. In this proposal, we recommend two approaches for a two-fold multiplexed SBA: one using two antibiotic-resistant bacterial strains and the other using two fluorescent strains. Specifically, we propose to develop the necessary target strains, assess the two approaches, develop a data acquisition pipeline generating a “killing index” as an output parameter, and begin to evaluate assay performance with a panel of sera and other types of samples.

In response to DHA202-002, SunFire Biotechnologies (SFB) proposed to develop a 2-fold multiplexed serum bactericidal assay (mSBA) for Shigella antibodies and to demonstrate its operational feasibility. With that Phase I funding, SFB developed a 4-fold mSBA, based on antibiotic resistance, which allows the simultaneous testing of functional antibodies against S. flexneri serotypes 2a, 3a, and 6, as well as S. sonnei. The mSBA was found to be reliable, with good reproducibility and repeatability. SFB is now proposing to transition the mSBA to commercial use using two approaches. The first is to refine the mSBA created in Phase I to create an assay that can be utilized in Shigella vaccine development. To increase throughput, reduce testing turn-around times, and decrease cost, the assay will be optimized. With analytical qualification of the assay and application of our current quality assurance systems to ensure regulatory compliance, SFB will be able to offer mSBA testing for pre-clinical as well as early-stage clinical trials of Shigella vaccines on a fee-for-service basis. Additionally, SFB can improve the performance of mSBA further by developing an “in-house kit.” The mSBA is amenable to a kit since all critical mSBA reagents can be prepared in large amounts, stored in aliquots until needed, and characterized ahead of time. SFB will explore other options, such as automation, to further improve assay performance. The second approach to commercial transition is to produce and market mSBA kits, which were originally developed for an in-house use. The key to this approach is to demonstrate that the “in-house kit” allows an external laboratory to reliably perform the mSBA. For this purpose, we have identified an external laboratory to test the SFB in-house kits. If the kits perform well for the outside laboratory, SFB will be able to produce and market Shigella mSBA kits on a larger scale. The work will be performed in SFB under the direction of Mr. Burton, the Director of SFB and PI for the Phase I project. SFB has identified a subcontractor with experience in Shigella SBAs to test the kits, and several outstanding consultants that will help with this project. Furthermore, SFB is currently functioning as a contract research organization (CRO) for pneumococcal vaccine industry and can do the same for Shigella. Therefore, SFB can easily transition the assay developed in Phase I to these commercial applications.