Designed nanoparticles elicit cross-reactive antibody responses to conserved influenza virus hemagglutinin stem epitopes.

Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes.

Structural analysis of influenza vaccine virus-like particles reveals a multicomponent organization.

Influenza virus continues to be a major health problem due to the continually changing immunodominant head regions of the major surface glycoprotein, hemagglutinin (HA). However, some emerging vaccine platforms designed by biotechnology efforts, such as recombinant influenza virus-like particles (VLPs) have been shown to elicit protective antibodies to antigenically different influenza viruses. Here, using biochemical analyses and cryo-electron microscopy methods coupled to image analysis, we report the composition and 3D structural organization of influenza VLPs of the 1918 pandemic influenza virus. HA molecules were uniformly distributed on the VLP surfaces and the conformation of HA was in a prefusion state. Moreover, HA could be bound by antibody targeting conserved epitopes in the stem region of HA. Taken together, our analysis suggests structural parameters that may be important for VLP biotechnology such as a multi-component organization with (i) an outer component consisting of prefusion HA spikes on the surfaces, (ii) a VLP membrane with HA distribution permitting stem epitope display, and (iii) internal structural components.

Characterization of Hemagglutinin Antigens on Influenza Virus and within Vaccines Using Electron Microscopy.

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.

Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform.

While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.

Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures.

Ribonucleoprotein (RNP) complexes of influenza viruses are composed of multiple copies of the viral nucleoprotein (NP) that can form filamentous supra-structures. RNPs package distinct viral genomic RNA segments of different lengths into pleomorphic influenza virions. RNPs also function in viral RNA transcription and replication. Different RNP segments have varying lengths, but all must be incorporated into virions during assembly and then released during viral entry for productive infection cycles. RNP structures serve varied functions in the viral replication cycle, therefore understanding their molecular organization and flexibility is essential to understanding these functions. Here, we show using electron tomography and image analyses that isolated RNP filaments are not rigid helical structures, but instead display variations in lengths, curvatures, and even tolerated kinks and local unwinding. Additionally, we observed NP rings within RNP preparations, which were commonly composed of 5, 6, or 7 NP molecules and were of similar widths to filaments, suggesting plasticity in NP-NP interactions mediate RNP structural polymorphism. To demonstrate that NP alone could generate rings of variable oligomeric state, we performed 2D single particle image analysis on recombinant NP and found that rings of 4 and 5 protomers dominated, but rings of all compositions up to 7 were directly observed with variable frequency. This structural flexibility may be needed as RNPs carry out the interactions and conformational changes required for RNP assembly and genome packaging as well as virus uncoating.

Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms.

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.

Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20.

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 8.5Å 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.