INSTITUTO DE INVESTIGACIONES BIOTECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Structure-activity relationship and stability studies of the vaccine adjuvant U-Omp19.
RODOLFO RASIA; JULIANA CASSATARO; SEBASTIÁN KLINKE; LUCIA CHEMES; MARÍA LAURA DARRIBA; MARÍA LAURA CERUTTI; KARINA A. PASQUEVICH
Congreso; World Vaccine Congress; 2019
Oral delivery of vaccines represents the most attractive mode of administration over other routes of delivery. However, for inducing a proper immune response, subunit protein oral vaccines need to be adjuvanted and protected from the harsh environment of the gastrointestinal tract.In our laboratory we have been working with the Brucella protease inhibitor U-Omp19, a novel protein adjuvant that is in preclinical development. In the last years we have gained much knowledge about U-Omp19?s adjuvant properties. We have demonstrated that U-Omp19 can enhance the immune response against co-delivered antigens of diverse origins (bacterial, parasitic, viral). U-omp19 protects co-delivered antigens from degradation by partially inhibiting host gastrointestinal and endosomal proteases, promoting Ag arrival to inductive sites and activating cells of the immune system to finally enhance the elicited antigen-specific immune response. However, less is known about its physicochemical properties, its structure as well as its stability.In this work we have evaluated the structure of U-Omp19 by different techniques. SLS studies showed that it behaves as a globular monomer of 16,8 kDa and Far-UV CD spectra indicated a high predominance of β-strand secondary structure together with unstructured regions. Assignment of protein resonances in NMR studies using uniformly double labeled (15N, 13C) U-Omp19 confirmed that it bears a flexible N-terminal region (residues 1-64) and a C-terminal compact core of eight anti-parallel β-strands (residues 70-158). The structure of the C-terminal β-barrel was finally determined and refined by crystallization and X-ray diffraction analysis.To elucidate the function of each region on U-Omp19´s activities, the C-terminal and N-terminal domains were obtained. Far-UV CD spectra and NMR spectroscopy confirmed that the C-terminal domain retained the beta-barrel folding, however the inhibitor activity against α-chymotrypsin, trypsin and elastase was lost in both domains, indicating that U-Omp19 needs both regions for its inhibitory activity. Ongoing studies will shed light into the role of both domains in its adjuvanticity.Stability studies indicated that upon long term storage conditions, this adjuvant can be stored up to 3 years at ‒20 or ‒80˚C, 2 months at 4 ˚C and for 2 weeks at room temperature without significant loss of its physicochemical properties (inhibitory activity, protein integrity, folding and formation of aggregates). Moreover, U-Omp19 resists multiple cycles of freeze and thaw and upon lyophilization can be stored up to 6 months at room temperature recovering its full inhibitory activity when reconstituted. All together these results will contribute to a better understanding of U-Omp19 activity, the improvement of its activities will impact in the development of quality control tools in the journey of this novel adjuvant for oral vaccines from the lab bench to clinical development.