Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.

Several members of the Protoparvovirus genus, capable of infecting humans, have been recently discovered, including cutavirus (CuV) and tusavirus (TuV). To begin the characterization of these viruses, we have used cryo-electron microscopy and image reconstruction to determine their capsid structures to ~2.9 Å resolution, and glycan array and cell-based assays to identify glycans utilized for cellular entry. Structural comparisons show that the CuV and TuV capsids share common features with other parvoviruses, including an eight-stranded anti-parallel β-barrel, depressions at the icosahedral 2-fold and surrounding the 5-fold axes, and a channel at the 5-fold axes. However, the viruses exhibit significant topological differences in their viral protein surface loops. These result in three separated 3-fold protrusions, similar to the bufaviruses also infecting humans, suggesting a host-driven structure evolution. The surface loops contain residues involved in receptor binding, cellular trafficking, and antigenic reactivity in other parvoviruses. In addition, terminal sialic acid was identified as the glycan potentially utilized by both CuV and TuV for cellular entry, with TuV showing additional recognition of poly-sialic acid and sialylated Lewis X (sLeXLeXLeX) motifs reported to be upregulated in neurotropic and cancer cells, respectively. These structures provide a platform for annotating the cellular interactions of these human pathogens.

AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.

Adeno-associated virus (AAV) are classified as non-enveloped ssDNA viruses. However, AAV capsids embedded within exosomes have been observed, and it has been suggested that the AAV membrane associated accessory protein (MAAP) may play a role in envelope-associated AAV (EA-AAV) capsid formation. Here, we observed and selected sufficient homogeneous EA-AAV capsids of AAV2, produced using the Sf9 baculoviral expression system, to determine the cryo-electron microscopy (cryo-EM) structure at 3.14 Å resolution. The reconstructed map confirmed that the EA-AAV capsid, showed no significant structural variation compared to the non-envelope capsid. In addition, the Sf9 expression system used implies the notion that MAAP may enhance exosome AAV encapsulation. Furthermore, we speculate that these EA-AAV capsids may have therapeutic benefits over the currently used non-envelope AAV capsids, with advantages in immune evasion and/or improved infectivity.

The capsid structures of most Adeno-associated virus (AAV) serotypes, already assigned to an antigenic clade, have been previously determined. This study reports the remaining capsid structures of AAV7, AAV11, AAV12, and AAV13 determined by cryo-electron microscopy and three-dimensional image reconstruction to 2.96, 2.86, 2.54, and 2.76 Å resolution, respectively. These structures complete the structural atlas of the AAV serotype capsids. AAV7 represents the first clade D capsid structure; AAV11 and AAV12 are of a currently unassigned clade that would include AAV4; and AAV13 represents the first AAV2-AAV3 hybrid clade C capsid structure. These newly determined capsid structures all exhibit the AAV capsid features including 5-fold channels, 3-fold protrusions, 2-fold depressions, and a nucleotide binding pocket with an ordered nucleotide in genome-containing capsids. However, these structures have viral proteins that display clade-specific loop conformations. This structural characterization completes our three-dimensional library of the current AAV serotypes to provide an atlas of surface loop configurations compatible with capsid assembly and amenable for future vector engineering efforts. Derived vectors could improve gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting.

Adeno-associated viruses (AAVs) are non-enveloped ssDNA icosahedral T = 1 viruses used as vectors for clinical gene delivery. Currently, there are over 200 AAV-related clinical trials and six approved biologics on the market. As such new analytical methods are continually being developed to characterize and monitor the quality and purity of manufactured AAV vectors, these include ion-exchange chromatography and Direct Mass Technology. However, these methods require homogeneous analytical standards with a high molecular weight standard comparable to the mass of an AAV capsid. Described here is the design, production, purification, characterization, and the cryo-electron microscopy structure of an AAV1-VP3-only capsid that fulfills this need as a calibrant to determine capsid mass, charge, homogeneity, and transgene packaging characteristics.

Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB), thereby enabling the delivery of therapeutic genes to the central nervous system. Here, the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08-, 2.75-, and 3.39-Å resolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% identical to AAV8. However, the capsid structures of all three viruses are similar, with only minor differences observed in the previously described surface variable regions, suggesting that specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket, supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus The structural characterization of these viruses provides a platform for future vector engineering efforts toward improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity, or receptor retargeting.IMPORTANCE Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. The closely related AAVrh.39 has also shown promise in vivo As recently attained for other AAV biologics, e.g., Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain U.S. Food and Drug Administration approval for commercialization in the near future. This study characterized the capsid structures of these clinical vectors at atomic resolution using cryo-electron microscopy and image reconstruction for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for central nervous system delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.

The giant tiger prawn (Penaeus monodon) is a decapod crustacean widely reared for human consumption. Currently, viruses of two distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated and cloned from Vietnamese P. monodon specimens, designated Penaeus monodon metallodensovirus (PmMDV). This is the first member of a third divergent lineage shown to infect penaeid decapods. PmMDV has a transcription strategy unique among invertebrate PVs, using extensive alternative splicing and incorporating transcription elements characteristic of vertebrate-infecting PVs. The PmMDV proteins have no significant sequence similarity with other PVs, except for an SF3 helicase domain in its nonstructural protein. Its capsid structure, determined by cryoelectron microscopy to 3-Å resolution, has a similar surface morphology to Penaeus stylirostris densovirus, despite the lack of significant capsid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV folds its VP without incorporating a βA strand and displayed unique multimer interactions, including the incorporation of a Ca2+ cation, attaching the N termini under the icosahedral fivefold symmetry axis, and forming a basket-like pentamer helix bundle. While the PmMDV VP sequence lacks a canonical phospholipase A2 domain, the structure of an EDTA-treated capsid, determined to 2.8-Å resolution, suggests an alternative membrane-penetrating cation-dependent mechanism in its N-terminal region. PmMDV is an observed example of convergent evolution among invertebrate PVs with respect to host-driven capsid structure and unique as a PV showing a cation-sensitive/dependent basket structure for an alternative endosomal egress.

Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10-30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.

Adeno-associated viruses (AAVs) are being developed as vectors for the treatment of genetic disorders. However, pre-existing antibodies present a significant limitation to achieving optimal efficacy for the AAV gene delivery system. Efforts aimed at engineering vectors with the ability to evade the immune response include identification of residues on the virus capsid important for these interactions and changing them. Here K531 is identified as the determinant of monoclonal antibody ADK6 recognition by AAV6, and not the closely related AAV1. The AAV6-ADK6 complex structure was determined by cryo-electron microscopy and the footprint confirmed by cell-based assays. The ADK6 footprint overlaps previously identified AAV antigenic regions and neutralizes by blocking essential cell surface glycan attachment sites. This study thus expands the available repertoire of AAV-antibody information that can guide the design of host immune escaping AAV vectors able to maintain capsid functionality.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.

Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enable the externalization of the capsid protein (VP) N termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits the phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0), and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together, these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, in both the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N termini, presumably in prelude to the externalization of VP1u at pH 4.0, which is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates that AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N terminus externalization. IMPORTANCE There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1 to -3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and is used as a gene therapy vector. This is partly because AAV9 is capable of crossing the blood-brain barrier and readily transduces a wide array of tissues, including the central nervous system. In this study, we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering the pH.

Bufavirus strain 1 (BuV1), a member of the Protoparvovirus genus of the Parvoviridae, was first isolated from fecal samples of children with acute diarrhea in Burkina Faso. Since this initial discovery, BuVs have been isolated in several countries, including Finland, the Netherlands, and Bhutan, in pediatric patients exhibiting similar symptoms. Towards their characterization, the structures of virus-like particles of BuV1, BuV2, and BuV3, the current known genotypes, have been determined by cryo-electron microscopy and image reconstruction to 2.84, 3.79, and 3.25 Å, respectively. The BuVs, 65-73% identical in amino acid sequence, conserve the major viral protein, VP2, structure and general capsid surface features of parvoviruses. These include a core β-barrel (βB-βI), α-helix A, and large surface loops inserted between these elements in VP2. The capsid contains depressions at the icosahedral 2-fold and around the 5-fold axes, and has three separated protrusions surrounding the 3-fold axes. Structure comparison among the BuVs and to available parvovirus structures revealed capsid surface variations and capsid 3-fold protrusions that depart from the single pinwheel arrangement of the animal protoparvoviruses. These structures provide a platform to begin the molecular characterization of these potentially pathogenic viruses.

Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated N-acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. IMPORTANCE Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

Bocaparvoviruses are emerging pathogens of the Parvoviridae family. Human bocavirus 1 (HBoV1) causes severe respiratory infections and HBoV2 to HBoV4 cause gastrointestinal infections in young children. Recent reports of life-threatening cases, lack of direct treatment or vaccination, and a limited understanding of their disease mechanisms highlight the need to study these pathogens on a molecular and structural level for the development of therapeutics. Toward this end, the capsid structures of HBoV1, HBoV3, and HBoV4 were determined to a resolution of 2.8 to 3.0 Å by cryo-electron microscopy and three-dimensional image reconstruction. The bocaparvovirus capsids, which display different tissue tropisms, have features in common with other parvoviruses, such as depressions at the icosahedral 2-fold symmetry axis and surrounding the 5-fold symmetry axis, protrusions surrounding the 3-fold symmetry axis, and a channel at the 5-fold symmetry axis. However, unlike other parvoviruses, densities extending the 5-fold channel into the capsid interior are conserved among the bocaparvoviruses and are suggestive of a genus-specific function. Additionally, their major viral protein 3 contains loops with variable regions at their apexes conferring capsid surface topologies different from those of other parvoviruses. Structural comparisons at the strain (HBoV) and genus (bovine parvovirus and HBoV) levels identified differences in surface loops that are functionally important in host/tissue tropism, pathogenicity, and antigenicity in other parvoviruses and likely play similar roles in these viruses. This study thus provides a structural framework to characterize determinants of host/tissue tropism, pathogenicity, and antigenicity for the development of antiviral strategies to control human bocavirus infections.IMPORTANCE Human bocaviruses are one of only a few members of the Parvoviridae family pathogenic to humans, especially young children and immunocompromised adults. There are currently no treatments or vaccines for these viruses or the related enteric bocaviruses. This study obtained the first high-resolution structures of three human bocaparvoviruses determined by cryo-reconstruction. HBoV1 infects the respiratory tract, and HBoV3 and HBoV4 infect the gastrointestinal tract, tissues that are likely targeted by the capsid. Comparison of these viruses provides information on conserved bocaparvovirus-specific features and variable regions resulting in unique surface topologies that can serve as guides to characterize HBoV determinants of tissue tropism and antigenicity in future experiments. Based on the comparison to other existing parvovirus capsid structures, this study suggests capsid regions that likely control successful infection, including determinants of receptor attachment, host cell trafficking, and antigenic reactivity. Overall, these observations could impact efforts to design antiviral strategies and vaccines for HBoVs.

Parvoviruses (family Parvoviridae) are currently defined by a linear monopartite ssDNA genome, T = 1 icosahedral capsids, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. We report the discovery of a parvovirus with a bipartite genome, Acheta domesticus segmented densovirus (AdSDV), isolated from house crickets (Acheta domesticus), in which it is pathogenic. We found that the AdSDV harbors its NS and VP cassettes on two separate genome segments. Its vp segment acquired a phospholipase A2-encoding gene, vpORF3, via inter-subfamily recombination, coding for a non-structural protein. We showed that the AdSDV evolved a highly complex transcription profile in response to its multipartite replication strategy compared to its monopartite ancestors. Our structural and molecular examinations revealed that the AdSDV packages one genome segment per particle. The cryo-EM structures of two empty- and one full-capsid population (3.3, 3.1 and 2.3 Å resolution) reveal a genome packaging mechanism, which involves an elongated C-terminal tail of the VP, "pinning" the ssDNA genome to the capsid interior at the twofold symmetry axis. This mechanism fundamentally differs from the capsid-DNA interactions previously seen in parvoviruses. This study provides new insights on the mechanism behind ssDNA genome segmentation and on the plasticity of parvovirus biology.

Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new cap, genes resulting in hundreds of natural and engineered AAV capsid variants, while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3' end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. IMPORTANCE A major by-product of all adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Bocaparvovirus is an expansive genus of the Parvovirinae, with a wide range of vertebrate hosts. This study investigates Canine minute virus (CnMV), Rat bocavirus (RBoV), and Porcine bocavirus 1 (PBoV1). Both CnMV and PBoV1 have been found in gastrointestinal infections in their respective hosts, with CnMV responsible for spontaneous abortions in dogs, while PBoV has been associated with encephalomyelitis in piglets. The pathogenicity of the recently identified RBoV is currently unknown. To initiate the characterization of these viruses, their capsids structures were determined by cryo-electron microscopy at resolutions ranging from 2.3 to 2.7 Å. Compared to other parvoviruses, the CnMV, PBoV1, and RBoV capsids showed conserved features, such as the channel at the fivefold symmetry axis. However, major differences were observed at the two- and threefold axes. While CnMV displays prominent threefold protrusions, the same region is more recessed in PBoV1 and RBoV. Furthermore, the typical twofold axis depression of parvoviral capsids is absent in CnMV or very small in PBoV and RBoV. These capsid structures extend the structural portfolio for the Bocaparvovirus genus and will allow future characterization of these pathogens on a molecular level. This is important, as no antivirals or vaccines exist for these viruses.

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma, for the treatment of spinal muscular atrophy and are being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of preexisting neutralizing antibodies in 40 to 80% of the general population. These preexisting antibodies can reduce therapeutic efficacy through viral neutralization and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction were used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs), ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound to or near the icosahedral 3-fold axes, HL2368 bound to the 2/5-fold wall, and HL2372 bound to the region surrounding the 5-fold axes. Pseudoatomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis, followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding preexisting circulating neutralizing antibodies. IMPORTANCE The use of recombinant adeno-associated viruses (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna and Zolgensma, based on serotypes AAV2 and AAV9, respectively. However, high-titer anti-AAV neutralizing antibodies in the general population exempt patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by preexisting neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with preexisting AAV antibodies.

We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs) from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i) low isoelectric point of a capsid and (ii) relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP) column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP) grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.