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.

Commercial influenza vaccines vary in HA-complex structure and in induction of cross-reactive HA antibodies.

Influenza virus infects millions of people annually and can cause global pandemics. Hemagglutinin (HA) is the primary component of commercial influenza vaccines (CIV), and antibody titer to HA is a primary correlate of protection. Continual antigenic variation of HA requires that CIVs are reformulated yearly. Structural organization of HA complexes have not previously been correlated with induction of broadly reactive antibodies, yet CIV formulations vary in how HA is organized. Using electron microscopy to study four current CIVs, we find structures including: individual HAs, starfish structures with up to 12 HA molecules, and novel spiked-nanodisc structures that display over 50 HA molecules along the complex's perimeter. CIV containing these spiked nanodiscs elicit the highest levels of heterosubtypic cross-reactive antibodies in female mice. Here, we report that HA structural organization can be an important CIV parameter and can be associated with the induction of cross-reactive antibodies to conserved HA epitopes.

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.

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.

An essential RNA element resides in a central region of hepatitis E virus ORF2.

Hepatitis E virus (genus Hepevirus, family Hepeviridae) is one of the most important causes of acute hepatitis in adults, particularly among pregnant women, throughout Asia and Africa where mortality rates can be 20-30 %. Hepatitis E virus has a single-stranded positive-sense RNA genome that contains three translated ORFs. The two 3' ORFs are translated from a subgenomic RNA. Functional RNA elements have been identified in and adjacent to the genomic 5' and 3' UTRs and in and around the intergenic region. Here we describe an additional RNA element that is located in a central region of ORF2. The RNA element is predicted to fold into two highly conserved stem-loop structures, ISL1 and ISL2. Mutations that disrupt the predicted structures, without altering the encoded amino acid sequence, result in a drastic reduction in capsid protein synthesis. This indicates that the RNA element plays an important role in one of the early steps of virus replication. The structures were further investigated using a replicon that expresses Gaussia luciferase in place of the capsid protein. Single mutations in ISL2 severely reduced luciferase expression, but a pair of compensatory mutations that were predicted to restore the ISL2 structure, restored luciferase expression to near-WT levels, thus lending experimental support to the predicted structure. Nonetheless the precise role of the ISL1+ISL2 element remains unknown.

Cross-species infections of cultured cells by hepatitis E virus and discovery of an infectious virus-host recombinant.

The RNA virus, hepatitis E virus (HEV) is the most or second-most important cause of acute clinical hepatitis in adults throughout much of Asia, the Middle East, and Africa. In these regions it is an important cause of acute liver failure, especially in pregnant women who have a mortality rate of 20-30%. Until recently, hepatitis E was rarely identified in industrialized countries, but Hepatitis E now is reported increasingly throughout Western Europe, some Eastern European countries, and Japan. Most of these cases are caused by genotype 3, which is endemic in swine, and these cases are thought to be zoonotically acquired. However, transmission routes are not well understood. HEV that infect humans are divided into nonzoonotic (types 1, 2) and zoonotic (types 3, 4) genotypes. HEV cell culture is inefficient and limited, and thus far HEV has been cultured only in human cell lines. The HEV strain Kernow-C1 (genotype 3) isolated from a chronically infected patient was used to identify human, pig, and deer cell lines permissive for infection. Cross-species infections by genotypes 1 and 3 were studied with this set of cultures. Adaptation of the Kernow-C1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene.

Release of genotype 1 hepatitis E virus from cultured hepatoma and polarized intestinal cells depends on open reading frame 3 protein and requires an intact PXXP motif.

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.

Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles.

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

ORF3 protein of hepatitis E virus is not required for replication, virion assembly, or infection of hepatoma cells in vitro.

A subclone of Huh-7 cells that could be relatively efficiently transfected and infected with hepatitis E virus was identified. Following transfection, infectious virus was produced but remained predominantly cell associated. Intracellular virus, recovered by lysis of transfected cells, infected naïve cells. This in vitro-produced virus appeared to be antigenically identical to virus isolated from clinical samples. Lysates from cells transfected with mutant viral genomes unable to synthesize ORF3 protein contained infectious virions that were similar in number, thermostability, and sedimentation characteristics to those in lysates transfected with wild-type viral genomes. Therefore, in contrast to its requirement in vivo, ORF3 protein is not required for infection of Huh-7 cells or production of infectious virus in vitro.

A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus.

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay.

Monolayers of Hep G2/C3A cells were inoculated with genotype 1 Hepatitis E virus (HEV) mixed with either anti-HEV or an appropriate control. After 5 or 6 days, cell monolayers were stained with anti-HEV and infected cells were identified by immunofluorescence microscopy and counted. Anti-HEV from vaccinated or infected rhesus monkeys neutralized the virus, as did mAbs that recognized epitopes on the C terminus of a recombinant vaccine protein. Antibodies were broadly cross-reactive, since convalescent serum from animals infected with any one of the four mammalian genotypes all neutralized the genotype 1 virus.

myotilin Mutation found in second pedigree with LGMD1A.

Limb-girdle muscular dystrophy 1A (LGMD1A [MIM 159000]) is an autosomal dominant form of muscular dystrophy characterized by adult onset of proximal weakness progressing to distal muscle weakness. We have reported elsewhere a mutation in the myotilin gene in a large, North American family of German descent. Here, we report the mutation screening of an additional 86 families with a variety of neuromuscular pathologies. We have identified a new myotilin mutation in an Argentinian pedigree with LGMD1 that is predicted to result in the conversion of serine 55 to phenylalanine (S55F). This mutation has not been found in 392 control chromosomes and is located in the unique N-terminal domain of myotilin, only two residues from the T57I mutation reported elsewhere. Both T57I and S55F are located outside the alpha-actinin and gamma-filamin binding sites within myotilin. The identification of two independent pedigrees with the same disease, each bearing a different mutation in the same gene, has long been the gold standard for establishing a causal relationship between defects in a gene and the resultant disease. As a description of the second known pedigree with LGMD1A, this finding constitutes that gold standard of proof that mutations in the myotilin gene cause LGMD1A.