Technical viability of the YF MAC-HD ELISA kit for use in yellow fever-endemic regions

Yellow fever (YF), an arboviral disease, affects an estimated 200,000 people and causes 30,000 deaths per year and recently has caused major epidemics in Africa and South America. Timely and accurate diagnosis of YF is critical for managing outbreaks and implementing vaccination campaigns. A YF immunoglobulin M (IgM) antibody-capture (MAC) enzyme-linked immunosorbent assay (ELISA) kit, the YF MAC-HD, was successfully introduced starting in 2018 to laboratories in Africa and South America. The YF MAC-HD kit can be performed in 3.5 hours, test up to 24 samples, and includes all reagents necessary to perform the test, except for water used to dilute wash buffer. In 2018 and 2019, a total of 56 laboratory personnel from 39 countries in Africa and South America were trained to use the kit during workshops, followed by take-home YF IgM proficiency testing (PT) exercises. Participants received either a 10- or 20-sample YF PT panel and performed testing using the YF MAC-HD kit. All countries obtained 90% or higher correct results. These results verified the technical viability and transferability of YF MAC-HD kit use for laboratories in YF-endemic countries.

CONSTRUCTION, EXPRESSION AND PURIFICATION OF BRUCELLA SPP. RECOMBINANT PROTEINS L7/L12 AND SODC IN E. COLI

Brucellosis is still an important public health problem as long as natural reservoirs of infection exist. Currently, live attenuated vaccines based on strains S19, RB51 and Rev1 are used for the prevention of brucellosis in animals, the main disadvantage of which is virulence for humans. However, animal immunization programs should be implemented to reduce the incidence of humans. The development of safe and effective new generation vaccines using “omix” technology is a promising direction of vaccinology. A number of immunogenic Brucella proteins that elicit both a humoral and cellular immune response has been identified. The aim of these research was to optimize the expression and purification conditions of the Brucella spp. recombinant proteins L7/L12 and SodC. As a result, expressing plasmids pET/Br-L7/L12 and pET/Br-SodC were obtained. The parameters of target genes expression in E. coli were established and the method for purification of recombinant proteins was optimized. Purification of the L7/L12 protein was performed under hybrid conditions on HisPur agarose using a binding buffer containing 6 M guanidine hydrochloride, a wash buffer with 20 mM imidazole and an elution buffer with 300 mM imidazole. Protein SodC was purified under denaturing conditions with the addition of 1 % Triton X-100 and 1 % sodium deoxycholate to the lysis buffer. Inclusions were solubilized with a buffer containing 8 M urea and 5 mM imidazole. The target protein was eluted from HisPur agarose with buffer containing 8 M urea and 100 mM imidazole. The use of modified purification protocols made it possible to obtain purified recombinant proteins with a yield of 13 mg/L for the L7/L12 protein and 10 mg/L for the protein SodC, respectively. The specificity of the proteins was confirmed by a Western blot. Immunization of mice with recombinant proteins led to the production of specific antibodies, the titer of which in ELISA was 1:20480 and 1:20480, respectively.

In Vitro Functionality of hiPSC Derived PLT+: The Utility of Microfluidic Assays in Determining Potency

Introduction: There is a growing shortage of platelets, the principal blood cells responsible for clot formation and blood vessel repair at sites of active bleeding. Platelet BioGenesis (PBG) is developing a commercial-scale, donor-free platelet (PLT+) production process using a cGMP-grade human induced pluripotent stem cell line (hiPSC) to address this issue. The PLT+ activity is being characterized extensively through multiple approaches that measure hemostatic function both in vitro and in vivo, with the ultimate goal of determining a clinical dose in humans. Alongside tests such as the current gold standard thrombin generation assay (TGA), we have developed an in-house microfluidic model to measure the ability of PLT+ to adhere to extracellular proteins under flow. The assay captures the surface receptor dynamics, as well as the ability of PLT+ to initiate coagulation. Materials and Methods: The thrombin generation assays are performed according to manufacturer's instructions. For the in-house microfluidic assay (MFA), fibrillar collagen, fibrinogen, or von Willebrand Factor is patterned in a commercial microfluidic device (Ibidi Slide VI 0.1). PLT+ (DNA-, calcein +, CD61+, CD42a+ cells from our bioreactor) are concentrated in normal pooled plasma, platelet additive solution, or platelet wash buffer to a normalized concentration of 1e8/mL, and are labeled with the mitochondrial dye DiOC6. Calcium chloride (7.5 mM) is added and the solution is perfused with a syringe pump at 50 1/s over the bioactive surface for 10 min. Images are captured every 10 seconds and quantified with ImageJ. After perfusion, the samples are fixed with 4% paraformaldehyde and can be stained for further characterization. Results and Discussion: PLTs+ are functionally non-inferior to human donor (blood) platelets and appear more active than Day 4/5 stored apheresis unit platelets. The TGA of PLT+ shows a more rapid generation of thrombin but similar total potential to donor platelets (Figure A). In the microfluidic assay, PLT+ (green DiOC6, red PAC1, figure B) adhere readily to the collagen surface under low shear flow, but do not adhere to non-functionalized surfaces, indicating GPVI functionality. As measured by surface coverage, the PLT+ resuspended in PAS adhere faster to the surface than either freshly washed donor platelets or apheresis platelets (p=0.01). When the PLT+ are preincubated in normal pooled plasma prior to perfusion, we observe fibrin formation and the aggregates show clot retraction, which supports the TGA results. Adhesion to surface bound fibrinogen and VWF is also comparable to donor platelets (GP IIb/IIa and GPIb function,). Combined, the TGA and MFA data reflect results of traditional more resource intensive assays, suggesting their viability as a rapid and flexible platform to study platelet function and function as release assays for in vitro generated PLTs+ Conclusion: Current work demonstrates that PLT+ are functional and a future alternative to donor derived platelet units. The MFA is a valuable tool for the characterization of this new therapeutic because of its specific control of surface protein-receptor interactions, shear forces, and flexibility to look at multiple markers. Alongside the TGA, the MFA will be a release assay for the PLT+. Figure Disclosures Lehmann: Platelet Biogenesis: Employment. Burton:Platelet Biogenesis: Employment. Szemethy:Platelet Biogenesis: Employment. Valdez:Platelet BioGenesis: Employment. Thon:Platelet BioGenesis: Employment.