Through this highly focused and collaborative effort, we will create and validate new modeling techniques for developing antibodies and vaccine candidates against influenza as a model system for addressing diseases that cannot be addressed by classical exposure to naturally-occurring antigens (Ags). Successful `natural immunity' to viruses induces a protective immune response against disease during subsequent exposure (measles, mumps, rubella, poliovirus, and others) and represents the current state-of-the-art in vaccination technology. Having eradicated many of the scourges of our forebears, the human species now faces new and imminent threats from viruses (such as diverse avian influenza viruses) for which natural exposure does not induce protective immunity to different strains, clades and subtypes. While it has been possible to isolate monoclonal antibodies (mAbs) that inhibit or neutralize specific virus field strains of virtually all human viral pathoges, broad and sustained protection has been elusive. For influenza virus, the core obstacle to sustained immunity is antigenic variability. Many important human pathogens for which there is incomplete immunity and no licensed vaccine exhibit a high level of diversity in circulating field strains. Influenza virus causes yearly epidemics by a relatively slow process of antigenic drift mediated by point mutations in the surface proteins HA and NA. Occasional pandemics, where more rapid shifts are realized by reassortment of surface proteins with zoonotic viruses in birds and pigs, can have devastating consequences due to our immunological navety to novel Ags. Influenza is also a good model system for biodefense: Recent research described adaptations of influenza H5N1 that confer respiratory droplet transmissibility from ferret to ferret, which may mimic the future development of a highly pathogenic pandemic human H5 virus in nature. Current vaccine technologies may not abe dequate to induce the breadth of immunity needed to protect against all drifted or shifted influenza variants. We need to be able to design interfaces so we can control the cross- reactivity of the Abs (or immunogen) induced. To accomplish this goal, we propose to (i) generate an extremely large panel of Abs against diverse influenza HA molecules, (ii) delineate the molecular interactions that confer Ag-antibody (Ag-Ab) recognition, (iii) use these structural data to drive rational Ag design through advanced protein engineering, and (iv) validate designs in vivo, thus producing the next generation of vaccines. The feedback loop, with structural parameterization of Ags and in vitro/in vivo testing of designs, make the protein engineering efforts described here a tractable problem. Rational, structure-informed design also allows us to present native Ags to the immune system on novel scaffolds that are more stable and homogeneous than naturally occurring Ags, which may be flexible and degrade quickly. Our platform also will serve as a model for any biological system (viral, bacterial, and parasitic) or protein-protein design effort, in which it is desirable to control the affinity or specificity of an interaction.
The next generation of viral vaccines and biologics likely will be designed rationally, based on a structural understanding of the protective antigens. The multidisciplinary group in this application will develop and implement new structure based design methods, and then validate the power of the modeling approach with wet laboratory experiments focused and the structural basis of broad neutralization of influenza through recognition of the HA head domain. Project 1 - Identification of novel human antibodies and vaccines against influenza (Description as provided by applicant) The modeling projects in this application focus on development and implementation of new structure based design tools for influenza HA protein specific antibodies (RP2) or vaccine antigens (RP3). These projects will use knowledge about the structure and function of human neutralizing antibodies to the HA head that we derive in Aims 1 and 2 here in RP1 in order to design new antibodies or vaccines in silico. We have access to peripheral blood cells from a diverse panels of subjects with prior natural infection, or experimental infection with vaccines encoding HA molecules with both seasonal vaccines and unusual experimental influenza subtypes, including H3variant, H5, H6, H7, H9, and H10 viruses. The immune B memory cell populations from these individuals are the ideal materials with which to search for unusual heterosubtypic antibodies. Thus, in Aim 1, we will isolate broadly heterosubtypic human mAbs to the receptor binding domain of the HA head. In Aim 2, we will determine the immunome of the responding heterosubtypic clones using high-throughput next generation sequencing of antibody gene repertoires that comprise the clonal lineages of the most heterosubtypic antibodies isolated in Aim 1. These sequences will provide an order of magnitude or more increased information on the sequences encoding heterosubtypic antibody clones. Once antibodies with unusual breadth or activity are isolated, the structure of these antibodies will be determined in complex with purified HA molecules in the Structural Core at Scripps using crystallography and single particle EM. Such structures will provide the coordinates for the modeling experiments in RP2 and RP3. RP2 will computationally design in silico maturation of antibodies to increase affinity for the HA antigen of specific virus types and use multi-state design to create antibodies that recognize HAs of multiple different clades, subtypes, groups, or even types. In RP1 Aim 3 we then will synthesize and express these novel designed antibodies and determine neutralization activity, binding affinity, and competition binding groups of designed antibodies, using a diverse HA panel and pseudotyped viruses with all type A HAs in nature. RP3 similarly will use computational methods to design structurally stable epitope-focused immunogens and experimentally test those methods by evaluating immune responses to designed model antigens. In work in RP1 Aim 3 we will validate these designs by testing the interaction of human mAbs we isolate in detailed interaction studies with the novel immunogens, to validate their likely immunogenicity. Then, we will use the novel immunogens to isolate new antibodies from subjects naturally exposed to influenza, to show that the immunogens present antigens recognized by natural immune responses
Showing the most recent 10 out of 24 publications
