Chimeric Capsid Research Articles

Epitope Insertion at the N-Terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T=3 Capsid Protein Leads to Larger T=4 Capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.

Chimeric Capsid Protein as a Nanocarrier for siRNA Delivery: Stability and Cellular Uptake of Encapsulated siRNA

For the efficient cytoplasmic delivery of siRNA, we designed a chimeric capsid protein composed of a capsid shell, integrin targeting peptide, and p19 RNA binding protein. This recombinant protein assembled into a macromolecular container-like structure with capsid shell and provided a nanocarrier for siRNA delivery. Our capsid nanocarriers had dual affinity both for siRNA within the interior and integin receptors on the exterior, and the capsid shell structure allowed the encapsulated siRNAs to be protected from the external nucleases, leading to the enhanced stability of siRNA in serum conditions. The capsid nanocarriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. Moreover, RGD peptides on the exterior surface of the capsid shell enabled the capsid nanocarriers to deliver siRNA into the cytosol of the target cells. Here, we demonstrated the superior efficiency of our siRNA/capsid nanocarrier complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhancing the stability of siRNA as well as to achieving targeted siRNA delivery.

A Retroviral Chimeric Capsid Protein Reveals the Role of the N-Terminal β-Hairpin in Mature Core Assembly

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.

Papillomavirus capsid proteins mutually impact structure

We studied a panel of mutant viruses containing wild-type and chimeric capsid HPV16 and HPV18 proteins. The mutant capsid protein expression, genome amplification, and episomal maintenance were comparable with the wild-type virus. However, the chimeric viruses varied in their titers from wild-type. We show that the intertypical mutant chimeric capsid viruses, that L2 affects the structure of L1 and that L1 affects the structure of L2 in the virion. These effects were measured using a panel of conformation-dependent neutralizing L1 MAbs and an L2 capsid surface peptide derived neutralizing antibody. These data suggest that variation of one capsid gene not only affects its own structure and antigenicity, but also affects the structure and antigenicity of the other capsid protein. Implications of our data suggest that for the continued effectiveness of a vaccine, variation in both capsid proteins need to be considered and not just the protein the vaccine is directed against.

Recurrent isolation of poliovirus 3 strains with chimeric capsid protein Vp1 suggests a recombination hot-spot site in Vp1

Five oral poliovirus vaccine (OPV) strains carrying an intertypic PV3/PV2 recombination in VP1 capsid protein were isolated during poliovirus surveillance. These five PV3 strains had altogether four diverse recombination crossover points near the 3′ end of the VP1 coding region. The complete antigenic site IIIa was replaced by PV2-specific amino acids in four of the studied PV3 strains. Low overall number of nucleotide substitutions in VP1 indicated that the predicted replication time, “age”, of the PV3 strains was short, 6 months or less. The nucleotide 472-T in the 5′ non-coding region, associated to the attenuated phenotype of PV3/Sabin, was reverted to wild-type C in all studied PV3/PV2 recombinant strains. Three of the PV3 strains had at least a tripartite genome deduced from the partial 3D polymerase-coding region sequences. Our results suggest that there exists a PV3/PV2 recombination hot-spot site in the 3′ partial region of the VP1 capsid protein and that the recombination may occur within weeks or a few months after the administration of OPV.

Isolation of a recombinant type 3/type 2 poliovirus with a chimeric capsid VP1 from sewage in Shandong, China

The genetic and phenotypic characterization of poliovirus strain P3/Jinan/1/09, isolated from sewage sample in Jinan city, Shandong province, China, was described. The strain had a Sabin type 3/type 2/type 3 recombinant genome, with the first crossover site located in capsid VP1 coding region between nucleotide positions 3293 and 3294 (numbering according to Sabin 3), and the second crossover site in 3D region between positions 6374 and 6378. The recombinant had introduced six Sabin 2-derived amino acids into the carboxyl terminus of Sabin 3 VP1 capsid protein. The complete genome of the isolate revealed eight nucleotide substitutions in Sabin 3 region with two substitutions resulting in amino acid alteration, and two missense substitutions in the Sabin 2 region. An estimation based on the evolution rate of the P1 coding region of Sabin 3 background suggested that evolution time of strain P3/Jinan/1/09 might be 76–80 days. The person who excreted the recombinant was not known and no evidence was obtained for its circulation in population via acute flaccid paralysis surveillance. The virus showed Sabin 3 serological characterization in neutralization test, and it did not lose temperature sensitivity phenotype at 40 °C. The significance of environmental surveillance and the presence of natural capsid recombinant poliovirus strain in the context of the global polio eradication initiative are discussed.

Norwalk virus-like particles as vaccines

Noroviruses (NoV) cause the great majority of epidemic nonbacterial gastroenteritis in humans. Expression of the capsid protein in recombinant systems, including insect and plant cells, yields assembly of virus-like particles (VLPs) that mimic the antigenic structure of authentic virions, and are relatively acid- and heat-stable. Norwalk virus (NV), the prototype NoV, has been studied extensively, and Norwalk virus-like particles (NVLPs) produced in insect cells and plants are immunogenic in mice and humans when delivered orally, stimulating the production of systemic and mucosal anti-NV antibodies. NVLPs are also highly immunogenic when delivered intranasally, provoking antibodies at levels similar to orally delivered VLP at much lower doses. Oral and nasal delivery of NVLPs efficiently produces antibodies at distal mucosal sites, which suggests that NVLPs could be used to deliver heterologous peptide antigens by production of genetic fusion chimeric capsid proteins. Examination of norovirus VLP surface structures and receptor binding motifs facilitates identification of potential sites for insertion of foreign peptides that will minimally affect the efficiency of VLP assembly and receptor binding. Thus, there is strong potential to use norovirus VLPs as vaccine-delivery vehicles.

Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle

Reengineering the receptor footprints of adeno-associated virus (AAV) isolates may yield variants with improved properties for clinical applications. We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism. These features of AAV2i8 suggest that it is well suited to translational studies in gene therapy of musculoskeletal disorders.

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

Directed evolution of adeno-associated virus for glioma cell transduction.

Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.

AAV-mediated gene therapy for metabolic diseases: dosage and reapplication studies in the molybdenum cofactor deficiency model

In a mouse model for molybdenum cofactor deficiency as an example for an inherited metabolic disease we have determined the dosage of recombinant AAV necessary to rescue the lethal deficiency phenotype. We demonstrated long-term expression of different expression cassettes delivered in a chimeric AAV capsid of serotype 1/2 and compared different routes of application. We then studied the effect of double and triple injections at different time points after birth and found a short neonatal window for non-response of the immune system. Exposition with rAAV capsids within this window allows transgene expression after a second rAAV transduction later. However, exposition within this window does not trigger immunotolerance to the viral capsid, which limits rAAV-mediated refurbishment of the transgene to only one more application outside this permissive window.

DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

Characterization of the Relationship of AAV Capsid Domain Swapping to Liver Transduction Efficiency

Recombinant adeno-associated virus (AAV) vectors show promise for use in gene therapy. For liver-targeted gene transfer in animals, AAV vectors pseudotyped with the AAV serotype 8 (AAV8) capsid have definite advantages over the widely used but less efficient serotype AAV2, even though the capsid amino acid sequences are 82% conserved. To demonstrate the mechanism behind the higher liver transduction efficiency associated with AAV8 capsids, we adopted a domain-swapping strategy that would generate 27 chimeric capsid genes containing exchanged domains between AAV2 and AAV8. The resulting chimeric capsids were then used to package AAV genomes with a liver-specific human coagulation factor IX (hFIX) expression cassette. By comparing the transduction efficiencies between vectors pseudotyped with chimeric, AAV2 and AAV8 capsids, we found that the more efficient liver transduction achieved by AAV8 was closely related to the components of its interstrand Loop IV domain, particularly the subloops 1 and 4. These subloops are exposed on opposite sides of a threefold proximal peak on the virion surface, which may function as a critical structural determinant for AAV transduction. Because a single specific peptide component could not explain all the observed differences in the transduction parameters, we suggest that important subloop regions require interaction with other portions of the capsid for their functioning.

Transient expression of the ectodomain of matrix protein 2 (M2e) of avian influenza A virus in plants

We have previously reported an expression system based on the capsid protein gene (CP) of cucumber mosaic virus (CMV) placed under transcriptional control of a potato virus X (PVX)-based vector. PVX-expressed CMV CP formed virus-like particles, which served as carriers for heterologous antigens of the Newcastle disease virus (NDV). In this work, we applied our expression tool toward the development of plant-derived vaccine candidate against avian influenza A virus. Twenty-three amino acid-long extracellular domain of the viral M2 protein (M2e) was engineered into the internal motif 5 of CMV CP and the recombinant gene then was transiently expressed in plants through a PVX vector. Chimeric CMV capsids reacted with specific antibodies produced to synthetic M2e epitope of the H5N1 strain of the virus. In addition, CMV CP-M2e protein was expressed to high levels in Escherichia coli bacterial cells and was recognized by antibodies to both CMV and M2e. This initial study demonstrates the feasibility of using plant virus-based vectors for expression of antigenic epitopes of H5N1 avian influenza in plants.

Versatile RHDV virus‐like particles: Incorporation of antigens by genetic modification and chemical conjugation

Virus-like particles have proved to be excellent molecular scaffolds, yet the individual characteristics and immune responses generated against each VLP requires the development of a wide range of capsids for use as vaccines, molecular delivery vessels, and nanoscale templates. Here we describe the development of Rabbit haemorrhagic disease virus (RHDV)-like particles as a rapidly versatile molecular workbench, overcoming limitations imposed by established genetic antigen incorporation procedures with chimeric VLP. Production of the RHDV capsid protein in a baculovirus system led to the self-assembly of VLP which were recovered at over 99% purity and manipulated both genetically and chemically. Fusion of small peptide sequences to RHDV VLP was well tolerated, forming chimeric capsids that enhanced the presentation of foreign peptide to hybridoma T helper cells 700-fold. Rapid and simple conjugation techniques employing the hetero-bifunctional chemical linker sulfo-SMCC enabled both small peptides and whole proteins to be conjugated to the surface of RHDV VLP, overcoming limitations imposed on VLP formation and yield experienced with chimeric VLP. Administration of VLP/ovalbumin conjugate provoked high titre ovalbumin-specific antibody in mice, demonstrating the immune stimulatory properties of the capsid were conferred to conjugated foreign antigen. VLP facilitated delivery of conjugated antigen to dendritic cells, eliciting proliferative responses in naïve TCR transgenic T helper cells that were at least 10-fold greater than ovalbumin antigen delivered alone.

Comparison of properties of particles of Cucumber mosaic virus and Tomato aspermy virus based on the analysis of molecular surfaces of capsids

The plant RNA viruses Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) (genus Cucumovirus) have similar icosahedral particles, the crystal structures of which have been reported recently. Similarity in particle structure agrees with reports of stable capsids assembled from their capsid proteins and of viable recombinant viruses with chimeric capsid proteins derived from CMV and TAV. However, differences between the cucumoviruses have been reported for physicochemical properties. Here, structural and electrostatic features of the molecular surfaces are studied to investigate their relationship with these observations. Two coat-protein recombinants with structures modelled by taking CMV and TAV as templates were also included in the analysis. Results show that there exists an external region of negative electrostatic potential that has arisen from strictly conserved charged residues situated near the external HI loop of the subunits in the capsomers. This negative domain surrounds the fivefold and quasi-sixfold axes and locates above regions of positive potential that extend to cover, nearly homogeneously, the inner surface of capsids, where interaction with encapsidated RNA occurs. Differences between the outer electrostatic distributions in CMV and TAV explain the distinct response of both viruses to variations in physicochemical conditions required for particle stability and are essential to rationalize the biological activity of the coat-protein recombinants, in spite of their seemingly distinct electrostatic characteristics.

743. Directed Evolution of Adeno-Associated Virus (AAV) by DNA Shuffling Yields Enhanced Gene Delivery Vectors

AAV serotypes have provided alternative capsid backbone for optimizing vector transduction in diverse tissue targets. With these available vector substrates, many cell types are still poorly or not infected at all. In order to increase the efficiency in targeting specific cell types, we introduce an in vitro recombination method, (DNA shuffling) as originally described by Shaffer and colleagues (Nat Bio Feb. 2006), combined with high throughput screening strategy. Briefly AAV Cap genes were randomly fragmented and reassembled using cycles of denaturation, annealing and extension by PCR. Following this reassembly reaction, PCR amplification with specific primers was used to generate full-length Cap chimeras for cloning into psub-201 (library of 2.0|[times]|106 independent clones). The library containing recombinat cap genes were packaged into AAV particles made up partially of AAV-2 cap (as described by Kleinschmidt and colleagues) by cotransfecting of pXR2 along with the AAV plasmid library into 293 cells. Then 293T cells were infected with AAV library transfer shuttles at low multiplicity of infection, and cells were superinfected with Ad dl309 to generate high titer of chimeric particles with respective mutant capsid genome. Hamster melanoma CS1 cells, previously shown to be restricted for AAV transduction (Summerford et. Al., Nat Med. 1999), were tested with chimeric AAV library for premissive clone. After 5 cycles of infection chimeric capsid DNA was isolated and sequenced. A single chimeric virus was obtained containing genome sequences from AAV1/AAV2/AAV8/AAV9, (AAV-1829). This chimeric capsid genome was constructed into an AAV packaging plasmid and GFP transduction assays were carried out. On CS1 cells, AAV-1829 yielded much higher GFP positive cells (87.5%) compared to contributing parental vectors (10-60% or less). In vivo analysis are on-going, however our preliminary studies demonstrate that DNA shuffling may be a significant tool for deriving optimize AAV capsid backbones that are tissue tropic.

Potential for broad-spectrum protection against feline calicivirus using an attenuated myxoma virus expressing a chimeric FCV capsid protein

It has previously been demonstrated that recombinant myxoma viruses expressing FCV capsid protein are capable of eliciting protective responses against virulent FCV challenge, following vaccination, in cats. An attempt was made to produce a bivalent myxoma recombinant expressing the capsid protein genes of both FCV strains F9 and LS015. The FCV capsid protein genes were inserted into the myxoma growth factor gene (MGF) locus, and the serine protease inhibitor (SERP 2) gene locus. Subsequent recombination between myxoma-FCV viruses resulted in a recombinant expressing a chimeric form of the capsid protein. Nonetheless, cats immunised with this myxoma-FCV recombinant demonstrate high levels of serum neutralising antibodies against both F9 and LS015 strains. Such a chimeric vaccine may provide effective protection against a wide range of FCV strains.

AAV Hybrid Serotypes: Improved Vectors for Gene Delivery

In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus.