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.

α-Defensin HD5 Stabilizes Human Papillomavirus 16 Capsid/Core Interactions.

BACKGROUND: Human papillomavirus (HPV) is linked to nearly all cases of cervical cancer. Despite available vaccines, a deeper understanding of the immune response to HPV is needed. Human α-defensin 5 (HD5), an innate immune effector peptide, blocks infection of multiple sero-types of HPV, including high-risk HPV16. While a common mechanism of α-defensin anti-viral activity against nonenveloped viruses such as HPV has emerged, there is limited understanding of how α-defensins bind to viral capsids to block infection. METHODS: We have used cryo-electron microscopy (cryoEM), mass spectrometry (MS) crosslinking and differential lysine modification studies, and molecular dynamics (MD) simulations to probe the interaction of HPV16 pseudovirions (PsVs) with HD5. RESULTS: CryoEM single particle reconstruction did not reveal HD5 density on the capsid surface. Rather, increased density was observed under the capsid shell in the presence of HD5. MS studies indicate that HD5 binds near the L1 and L2 capsid proteins and specifically near the C-terminal region of L1. MD simulations indicate that favorable electrostatic interactions can be formed between HD5 and the L1 C-terminal tail. CONCLUSIONS: A model is presented for how HD5 affects HPV16 structure and cell entry. In this model, HD5 binds to disordered regions of L1 and L2 protruding from the icosahedrally ordered capsid. HD5 acts to cement interactions between L1 and L2 and leads to a closer association of the L2/genome core with the L1 capsid. This model provides a structural rationale for our prior observation that HD5 interferes with the separation of L1 from the L2/genome complex during cell entry.

Negative-Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging.

Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative-stain image analysis to ascertain domain level molecular structure (10 to 20 Å) more quickly and efficiently than possible by atomic resolution cryo-EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto-picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head-domain epitope. By comparing to the cryo-EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative-stain method. © 2019 The Authors.

Immunoelectron Microscopy of Viral Antigens.

Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmission electron microscopy provides structural information, identity of components within a sample may be confounding. Immunoelectron microscopy allows for identification and visualization of antigens and their relative positions within a particulate sample. This allows for simple qualitative analysis of samples including whole virus, viral components, and viral-like particles. This article describes methods for immunogold labeling of viral antigens in a liquid suspension, with examples of immunogold-labeled influenza virus glycoproteins, and also discusses the important considerations for sample preparation and determination of morphologies. Together, these methods allow for understanding the antigenic makeup of viral particulate samples, which have important implications for molecular virology and vaccine development. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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.

Bioinspired Shielding Strategies for Nanoparticle Drug Delivery Applications.

Nanoparticle delivery systems offer advantages over free drugs, in that they increase solubility and biocompatibility. Nanoparticles can deliver a high payload of therapeutic molecules while limiting off-target side effects. Therefore, delivery of an existing drug with a nanoparticle frequently results in an increased therapeutic index. Whether of synthetic or biologic origin, nanoparticle surface coatings are often required to reduce immune clearance and thereby increase circulation times allowing the carriers to reach their target site. To this end, polyethylene glycol (PEG) has long been used, with several PEGylated products reaching clinical use. Unfortunately, the growing use of PEG in consumer products has led to an increasing prevalence of PEG-specific antibodies in the human population, which in turn has fueled the search for alternative coating strategies. This review highlights alternative bioinspired nanoparticle shielding strategies, which may be more beneficial moving forward than PEG and other synthetic polymer coatings.

Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs.

Platform technologies based on plant virus nanoparticles (VNPs) and virus-like particles (VLPs) are attracting the attention of researchers and clinicians because the particles are biocompatible, biodegradable, noninfectious in mammals, and can readily be chemically and genetically engineered to carry imaging agents and drugs. When the Physalis mottle virus (PhMV) coat protein is expressed in Escherichia coli, the resulting VLPs are nearly identical to the viruses formed in vivo. Here, we isolated PhMV-derived VLPs from ClearColi cells and carried out external and internal surface modification with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries, respectively. The uptake of dye-labeled particles was tested in a range of cancer cells and monitored by confocal microscopy and flow cytometry. VLPs labeled internally on cysteine residues were taken up with high efficiency by several cancer cell lines and were colocalized with the endolysosomal marker LAMP-1 within 6 h, whereas VLPs labeled externally on lysine residues were taken up with lower efficiency, probably reflecting differences in surface charge and the propensity to bind to the cell surface. The infusion of dye and drug molecules into the cavity of the VLPs revealed that the photosensitizer (PS), Zn-EpPor, and the drugs crystal violet, mitoxantrone (MTX), and doxorubicin (DOX) associated stably with the carrier via noncovalent interactions. We confirmed the cytotoxicity of the PS-PhMV and DOX-PhMV particles against prostate cancer, ovarian and breast cancer cell lines, respectively. Our results show that PhMV-derived VLPs provide a new platform technology for the delivery of imaging agents and drugs, with preferential uptake into cancer cells. These particles could therefore be developed as multifunctional tools for cancer diagnosis and therapy.

Cryo-electron tomography investigation of serum albumin-camouflaged tobacco mosaic virus nanoparticles.

Nanoparticles offer great potential in drug delivery and imaging, but shielding strategies are necessary to increase circulation time and performance. Structure-function studies are required to define the design rules to achieve effective shielding. With several formulations reaching clinical testing and approval, the ability to assess and detail nanoparticle formulations at the single particle level is becoming increasingly important. To address this need, we use cryo-electron tomography (cryo-ET) to investigate stealth-coated nanoparticles. As a model system, we studied the soft matter nanotubes formed by tobacco mosaic virus (TMV) coated with human serum albumin (SA) stealth proteins. Cryo-ET and subtomogram averaging allow for visualization of individual SA molecules and determination of their orientations relative to the TMV surface, and also for measurement of the surface coverage provided by added stealth proteins. This information fills a critical gap in the understanding of the structural morphology of stealth-coated nanoparticles, and therefore cryo-ET may play an important role in guiding the development of future nanoparticle-based therapeutics.

Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays.

The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe.

Multiple Administrations of Viral Nanoparticles Alter in Vivo Behavior-Insights from Intravital Microscopy.

Multiple administrations of nanoparticle-based formulations are often a clinical requirement for drug delivery and diagnostic imaging applications. Steady pharmacokinetics of nanoparticles is desirable to achieve efficient therapeutic or diagnostic outcomes over such repeat administrations. While clearance through mononuclear phagocytic system is a key determinant of nanoparticle persistence in vivo, multiple administrations could potentially result in altered pharmacokinetics by evoking innate or adaptive immune responses. Plant viral nanoparticles (VNPs) represent an emerging class of programmable nanoparticle platform technologies that offer a highly organized proteinaceous architecture and multivalency for delivery of large payloads of drugs and molecular contrast agents. These very structural features also render them susceptible to immune recognition and subsequent accelerated systemic clearance that could potentially affect overall efficiency. While the biodistribution and pharmacokinetics of VNPs have been reported, the biological response following repeat administrations remains an understudied area of investigation. Here, we demonstrate that weekly administration of filamentous plant viruses results in the generation of increasing levels of circulating, carrier-specific IgM and IgG antibodies. Furthermore, PVX specific immunoglobulins from the serum of immunized animals quickly form aggregates when incubated with PVX in vitro. Such aggregates of VNP-immune complexes are also observed in the mouse vasculature in vivo following repeat injections when imaged in real time using intravital two-photon laser scanning microscopy (2P-LSM). The size of aggregates diminishes at later time points, coinciding with antibody class switching from IgM to IgG. Together, our results highlight the need for careful in vivo assessment of (viral) nanoparticle-based platform technologies, especially in studying their performance after repeat administration. We also demonstrate the utility of intravital microscopy to aid in this evaluation.

Silica-coated Gd(DOTA)-loaded protein nanoparticles enable magnetic resonance imaging of macrophages.

The molecular imaging of in vivo targets allows non-invasive disease diagnosis. Nanoparticles offer a promising platform for molecular imaging because they can deliver large payloads of imaging reagents to the site of disease. Magnetic resonance imaging (MRI) is often preferred for clinical diagnosis because it uses non-ionizing radiation and offers both high spatial resolution and excellent penetration. We have explored the use of plant viruses as the basis of for MRI contrast reagents, specifically Tobacco mosaic virus (TMV), which can assemble to form either stiff rods or spheres. We loaded TMV particles with paramagnetic Gd ions, increasing the ionic relaxivity compared to free Gd ions. The loaded TMV particles were then coated with silica maintaining high relaxivities. Interestingly, we found that when Gd(DOTA) was loaded into the interior channel of TMV and the exterior was coated with silica, the T1 relaxivities increased by three-fold from 10.9 mM-1 s-1 to 29.7 mM-1s-1 at 60 MHz compared to uncoated Gd-loaded TMV. To test the performance of the contrast agents in a biological setting, we focused on interactions with macrophages because the active or passive targeting of immune cells is a popular strategy to investigate the cellular components involved in disease progression associated with inflammation. In vitro assays and phantom MRI experiments indicate efficient targeting and imaging of macrophages, enhanced contrast-to-noise ratio was observed by shape-engineering (SNP > TMV) and silica-coating (Si-TMV/SNP > TMV/SNP). Because plant viruses are in the food chain, antibodies may be prevalent in the population. Therefore we investigated whether the silica-coating could prevent antibody recognition; indeed our data indicate that mineralization can be used as a stealth coating option to reduce clearance. Therefore, we conclude that the silica-coated protein-based contrast agent may provide an interesting candidate material for further investigation for in vivo delineation of disease through macrophage imaging.

Detection and imaging of aggressive cancer cells using an epidermal growth factor receptor (EGFR)-targeted filamentous plant virus-based nanoparticle.

Molecular imaging approaches and targeted drug delivery hold promise for earlier detection of diseases and treatment with higher efficacy while reducing side effects, therefore increasing survival rates and quality of life. Virus-based nanoparticles are a promising platform because their scaffold can be manipulated both genetically and chemically to simultaneously display targeting ligands while carrying payloads for diagnosis or therapeutic intervention. Here, we displayed a 12-amino-acid peptide ligand, GE11 (YHWYGYTPQNVI), on nanoscale filaments formed by the plant virus potato virus X (PVX). Bioconjugation was used to produce fluorescently labeled PVX-GE11 filaments targeted toward the epidermal growth factor receptor (EGFR). Cell detection and imaging was demonstrated using human skin epidermoid carcinoma, colorectal adenocarcinoma, and triple negative breast cancer cell lines (A-431, HT-29, MDA-MB-231), all of which upregulate EGFR to various degrees. Nonspecific uptake in ductal breast carcinoma (BT-474) cells was not observed. Furthermore, co-culture experiments with EGFR(+) cancer cells and macrophages indicate successful targeting and partitioning toward the cancer cells. This study lays a foundation for the development of EGFR-targeted filaments delivering contrast agents for imaging and diagnosis, and/or toxic payloads for targeted drug delivery.