Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens.

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Current advances in biopharmaceutical informatics: guidelines, impact and challenges in the computational developability assessment of antibody therapeutics.

Therapeutic monoclonal antibodies and their derivatives are key components of clinical pipelines in the global biopharmaceutical industry. The availability of large datasets of antibody sequences, structures, and biophysical properties is increasingly enabling the development of predictive models and computational tools for the "developability assessment" of antibody drug candidates. Here, we provide an overview of the antibody informatics tools applicable to the prediction of developability issues such as stability, aggregation, immunogenicity, and chemical degradation. We further evaluate the opportunities and challenges of using biopharmaceutical informatics for drug discovery and optimization. Finally, we discuss the potential of developability guidelines based on in silico metrics that can be used for the assessment of antibody stability and manufacturability.

Isolation of an in Vitro Affinity-Matured, Thermostable "Headless" HA Stem Fragment That Binds Broadly Neutralizing Antibodies with High Affinity.

The surface glycoprotein hemagglutinin (HA) of influenza virus is the primary target for the design of an effective universal influenza vaccine as it is capable of eliciting broadly cross-reactive antibodies against different HA subtypes. Several monoclonal antibodies targeting the stem region of HA that are able to neutralize various subtypes of influenza virus have been isolated in the recent past. Designing a stable, HA stem immunogen that attains a native-like conformation and can elicit such antibodies has been a challenge. We describe the affinity maturation of a previously designed stem immunogen (H1HA6) by random mutagenesis, followed by selection using yeast surface display. The affinity-matured mutant protein (H1HA6P2), upon bacterial expression, attained a stable, native-like, trimeric conformation without any heterologous trimerization motif and showed a significant improvement in thermal stability and binding to several stem specific, conformation-sensitive, broadly neutralizing antibodies (bnAbs) relative to H1HA6. These results point to an effective strategy for the design of stabilized HA stem immunogens that can be tested for their protective ability.

Unusual effects of crowders on heme retention in myoglobin.

Myoglobin (Mb) undergoes pronounced heme loss under denaturing conditions wherein the proximal histidine gets protonated. Our data show that macromolecular crowding agents (both synthetic and protein based) can appreciably influence the extent of heme retention in Mb. Interestingly, glucose and sucrose, the monomeric constituents of dextran and ficoll-based crowders were much more effective in preventing heme dissociation of Mb, albeit, at much higher concentrations. The protein crowders BSA and lysozyme show very interesting results with BSA bringing about the maximum heme retention amongst all the crowding agents used while lysozyme induced heme dissociation even in the native state of Mb. The stark difference that these protein crowders exhibit when interacting with the heme protein is a testament to the varied interaction potentials that a test protein might be exposed to in the physiological (crowded) milieu.