Production, purification, crystallization and structure determination of<i>H-1 Parvovirus</i>

Halder, Sujata; Nam, Hyun Joo; Govindasamy, Lakshmanan; Vogel, Michael; Dinsart, Christiane; Salomé, Nathalie; McKenna, Robert; Agbandje‐McKenna, Mavis

doi:10.1107/s1744309112045563

Cited by 12 publications

(21 citation statements)

References 33 publications

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“…Capsid residues controlling receptor attachment and transduction efficiency are also known for several AAV serotypes (90)(91)(92). In the four antigenic structures reported, several of the predicted epitope and occluded residues (Table 1) are close to or overlap residues controlling these functions.…”

Section: Discussionmentioning

confidence: 99%

Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

Tseng

Gurda

Chipman

et al. 2015

J Virol

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114

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The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ϳ11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2-and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions. IMPORTANCEThe adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that report therapeutic efficacy. However, a large percentage of the population has preexisting AAV capsid antibodies and therefore must be excluded from clinical trials or vector readministration. This report represents our continuing efforts to understand the antigenic structure of the AAVs, specifically, to obtain a picture of "polyclonal" reactivity as is the situation in humans. It describes the structures of four AAV-antibody complexes determined by cryoelectron microscopy and image reconstruction, increasing the number of mapped epitopes to four and three, respectively, for AAV1 and AAV5, two vectors currently in clinical trials. The results presented provide information essential for generating antigenic escape vectors to overcome a critical challenge remaining in the optimization of this highly promising vector delivery system. T he adeno-associated viruses (AAVs), single-stranded DNA packaging viruses belonging to the Parvoviridae family, are promising vectors for gene delivery. There are over 100 AAV genomic isolates, and 13 human and nonhuman serotypes h...

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Section: Discussionmentioning

confidence: 99%

Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

Tseng

Gurda

Chipman

et al. 2015

J Virol

Self Cite

114

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Section: Methodsmentioning

confidence: 99%

“…H-1PV virions and empty capsids were produced in NB324K cells, purified by CsCl gradient ultracentrifugation, and crystallized as previously described (40). For the virions, X-ray diffraction data collection on crystals grown in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 8 mM CaCl 2 · 2H 2 O, and 3% polyethylene glycol (PEG) 8000, processing of the data, and structure determination to 2.7-Å resolution by the molecular replacement method have also been reported (40). For the empty capsids, a total of 251 usable X-ray diffraction images were collected from a single crystal, grown under conditions similar to those for the virions, at the X29 beamline ( ϭ 1.0895 Å) of the National Synchrotron Light Source (NSLS; Brookhaven National Laboratory).…”

Section: Methodsmentioning

confidence: 99%

“…There are two capsids per unit cell related by a 2 1 screw axis, with one particle occupying a crystallographic asymmetric unit. The data collection and processing statistics for the empty capsids are summarized in Table 1, along with the data previously reported for the virions (40).…”

Section: Methodsmentioning

confidence: 99%

“…The structure determination of the H-1PV virions by the molecular replacement method is reported elsewhere (40). For the empty capsid structure determination, the orientation and position of capsids in the P2 1 unit cell were determined by molecular replacement procedures utilizing rotation and translation function searches conducted with a 60-mer C␣ model of the MVM VP2 crystal structure (Protein Data Bank [PDB] accession number 1z14 [31]) using the AMoRe program (43).…”

Section: Methodsmentioning

confidence: 99%

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Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

Halder

Nam

Govindasamy

et al. 2013

J Virol

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bThe structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7-and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an ␣-helix and an eightstranded anti-parallel ␤-barrel with large loop regions between the strands. The ␤-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ϳ12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. H -1 parvovirus (H-1PV) is a member of the rodent subgroup of the Parvovirus genus of the single-stranded DNA (ssDNA) Parvoviridae (1). H-1PV was first isolated from rats transplanted with HEP-1, a human liver adenocarcinoma cell line (2), and also from aborted human fetuses (3). Recombinant vectors based on H-1PV and a number of other rodent parvoviruses, including minute virus of mice (MVM) and LuIII, are promising candidates for antitumor delivery vectors, particularly for cytoreductive and immunogene therapy approaches (reviewed in references 4 and 5). The inherent oncotropism of these autonomous parvoviruses is based on their dependence on cellular proliferation factors expressed during the S phase and the differentiated state of the host cell (6, 7). The rodent parvoviruses display oncopreferential cytotoxic activity in vitro and also possess an oncosuppressive potential, inhibiting the formation of spontaneous and chemical or virus-induced tumors in vitro and in vivo (8-13). These viruses can also persistently infect their natural hosts, do not integrate their genome into cellular chromosomes, and are not associated with human disease (reviewed in reference 4).Recombinant rodent parvovirus vectors targeted for tumor therapy utilize a double-edged strategy that takes advantage of their inherent oncotropism and selective cytotoxicity plus their ability to deliver therapeutic genes that code for ...

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A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

Leuchs

Frehtman

Riese

et al. 2017

Appl Microbiol Biotechnol

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The rodent protoparvovirus H-1PV, with its oncolytic and oncosuppressive properties, is a promising anticancer agent currently under testing in clinical trials. This explains the current demand for a scalable, good manufacturing practice-compatible virus purification process yielding high-grade pure infectious particles and overcoming the limitations of the current system based on density gradient centrifugation. We describe here a scalable process offering high purity and recovery. Taking advantage of the isoelectric point difference between full and empty particles, it eliminates most empty particles. Full particles have a significantly higher cationic charge than empty ones, with an isoelectric point of 5.8–6.2 versus 6.3 (as determined by isoelectric focusing and chromatofocusing). Thanks to this difference, infectious full particles can be separated from empty particles and most protein impurities by Convective interaction media® diethylaminoethyl (DEAE) anion exchange chromatography: applying unpurified H-1PV to the column in 0.15 M NaCl leaves, the former on the column and the latter in the flow through. The full particles are then recovered by elution with 0.25 M NaCl. The whole large-scale purification process involves filtration, single-step DEAE anion exchange chromatography, buffer exchange by cross-flow filtration, and final formulation in Visipaque/Ringer solution. It results in 98% contaminating protein removal and 96% empty particle elimination. The final infectious particle concentration reaches 3.5E10 plaque forming units (PFU)/ml, with a specific activity of 6.8E11 PFU/mg protein. Overall recovery is over 40%. The newly established method is suitable for use in commercial production.

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Production, purification, crystallization and structure determination ofH-1 Parvovirus

Cited by 12 publications

References 33 publications

Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

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