The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 Å resolution is determined for a complex of AAV-2 with the Fab′ fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

Influenza viruses affect millions of people worldwide on an annual basis. Although vaccines are available, influenza still causes significant human mortality and morbidity. Vaccines target the major influenza surface glycoprotein hemagglutinin (HA). However, circulating HA subtypes undergo continual variation in their dominant epitopes, requiring vaccines to be updated annually. A goal of next-generation influenza vaccine research is to produce broader protective immunity against the different types, subtypes, and strains of influenza viruses. One emerging strategy is to focus the immune response away from variable epitopes, and instead target the conserved stem region of HA. To increase the display and immunogenicity of the HA stem, nanoparticles are being developed to display epitopes in a controlled spatial arrangement to improve immunogenicity and elicit protective immune responses. Engineering of these nanoparticles requires structure-guided design to optimize the fidelity and valency of antigen presentation. Here, we review electron microscopy applied to study the 3D structures of influenza viruses and different vaccine antigens. Structure-guided information from electron microscopy should be integrated into pipelines for the development of both more efficacious seasonal and universal influenza vaccine antigens. The lessons learned from influenza vaccine electron microscopic research could aid in the development of novel vaccines for other pathogens.

Influenza virus afflicts millions of people worldwide on an annual basis. There is an ever-present risk that animal viruses will cross the species barrier to cause epidemics and pandemics resulting in great morbidity and mortality. Zoonosis outbreaks, such as the H7N9 outbreak, underscore the need to better understand the molecular organization of viral immunogens, such as recombinant influenza virus hemagglutinin (HA) proteins, used in influenza virus subunit vaccines in order to optimize vaccine efficacy. Here, using cryo-electron microscopy and image analysis, we show that recombinant H7 HA in vaccines formed macromolecular complexes consisting of variable numbers of HA subunits (range, 6 to 8). In addition, HA complexes were distributed across at least four distinct structural classes (polymorphisms). Three-dimensional (3D) reconstruction and molecular modeling indicated that HA was in the prefusion state and suggested that the oligomerization and the structural polymorphisms observed were due to hydrophobic interactions involving the transmembrane regions. These experiments suggest that characterization of the molecular structures of influenza virus HA complexes used in subunit vaccines will lead to better understanding of the differences in vaccine efficacy and to the optimization of subunit vaccines to prevent influenza virus infection.