Structural and antigenic characterization of the avian adeno-associated virus capsid.

IMPORTANCE: 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 Receptor-Binding: Flexible Domains and Alternative Conformations through Cryo-Electron Tomography of Adeno-Associated Virus 2 (AAV2) and AAV5 Complexes.

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near-universal entry receptor, AAVR, and structures detected by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now ß-strand A is the minor conformer and, for the major conformer, partially ordered N termini near the 2-fold axis join the canonical capsid jellyroll fold at the ßA-ßB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors.

Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington's disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-Å resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity.IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit wide-spread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the high-resolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-Å resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors.

Atomic structure of a rationally engineered gene delivery vector, AAV2.5.

AAV2.5 represents the first structure-guided in-silico designed Adeno-associated virus (AAV) gene delivery vector. This engineered vector combined the receptor attachment properties of AAV serotype 2 (AAV2) with the muscle tropic properties of AAV1, and exhibited an antibody escape phenotype because of a modified antigenic epitope. To confirm the design, the structure of the vector was determined to a resolution of 2.78 Šusing cryo-electron microscopy and image reconstruction. The structure of the major viral protein (VP), VP3, was ordered from residue 219 to 736, as reported for other AAV structures, and the five AAV2.5 residues exchanged from AAV2 to AAV1, Q263A, T265 (insertion), N706A, V709A, and T717N, were readily interpretable. Significantly, the surface loops containing these residues adopt the AAV1 conformation indicating the importance of amino acid residues in dictating VP structure.

Intra-family differences in efficacy of inactivation of small, non-enveloped viruses.

The use of specific model viruses for validating viral purification process steps and for assessing the efficacies of viral disinfectants is based, in part, on the assumption that viral susceptibilities to such treatments will be similar for different members, including different genera, within a given viral family. This assumption is useful in cases where cell-based infectivity assays or laboratory strains for the specific viruses of interest might not exist. There are some documented cases, however, where exceptions to this assumption exist. In this paper, we discuss some of the more striking cases of intra-family differences in susceptibilities to inactivation steps used for downstream viral purification steps in biologics manufacture (e.g. heat inactivation, low pH, and guanidinium hydrochloride inactivation) and to specific viral disinfectants (e.g. alcohols, hydrogen peroxide, and quaternary ammonium-containing disinfectants) that might be employed for facility/equipment disinfection. The results suggest that care should be taken when extrapolating viral inactivation susceptibilities from specific model viruses to different genera or even to different members of the same genus. This should be taken into consideration by regulatory agencies and biologics manufacturers designing viral clearance and facility disinfection validation studies, and developers and evaluators of viral disinfectants.

Viral clearance capacity by continuous Protein A chromatography step using Sequential MultiColumn Chromatography.

In response to the strong demand of biological protein therapeutics, such as monoclonal antibodies (MAbs), continuous downstream process was developed to deliver these molecules while maintaining desired product consistency and quality attributes, and providing manufacturing efficiency and flexibility. Viral safety is a critical quality attribute for biopharmaceuticals, such as MAbs. Evaluation of the viral clearance by the downstream process is a key component of risk mitigation. Protein A chromatography is typically used as an initial capture step for MAbs and efficient for the removal of process-related impurities like Host Cell Proteins (HCP). This step can also contribute to the clearance of potential viral contaminants. Murine Minute Virus (MMV)-spiking experiments were performed at small scale to investigate the impact on the viral clearance efficiency of the way the Protein A chromatography step is carried out, whether in batch or multicolumn mode. Protein A chromatography step using Novasep Sequential MultiColumn Chromatography (SMCC) technology demonstrated no statistical difference in the viral reduction with reduction factor (RF) of 3.7 log10 (vs. RF of 3.8 log10 for batch). The experiments showed also similar viral distribution over the purification cycles and columns. Data confirmed that the viral clearance capacity by the continuous Protein A chromatography step using SMCC technology is maintained and efficient.

Selection of an aptamer against Muscovy duck parvovirus for highly sensitive rapid visual detection by label-free aptasensor.

Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in ducks. This study investigated a novel aptamer-based, label-free aptasensor detection of MDPV. In this study, we developed an ssDNA aptamer using the filtration partition and lambda exonuclease method with an affinity-based monitor and counter-screening process. After 15 rounds of SELEX (systematic evolution of ligands by exponential enrichment), the ssDNA aptamer Apt-10, which specifically bound to MDPV with high affinity (Kd = 467nM) was successfully screened, and the aptamer was also found to be good specific to MDPV. The selected Apt-10 aptamer can be used to distinguish MDPV and goose parvovirus (GPV). Three-dimensional structural analysis of the Apt-10 aptamer indicated that it folded into a compact stem-loop motif, which was related to its high affinity. Finally, a label-free detection method based on unmodified gold nanoparticles and Apt-10 aptamer was developed for MDPV determination. The concentration of Apt-10 aptamer at 5µM was optimal for MDPV determination in the label-free aptasensor. Excellent linearity was acquired and the lowest detection limit was 1.5 or 3 EID50 (50% egg infection dose) of MDPV, respectively, depending upon spectrophotometry or the naked eye were used. These results show the potential of the aptamer for the rapid detection of MDPV and antiviral research.

Precision Fluorescent Labeling of an Adeno-Associated Virus Vector to Monitor the Viral Infection Pathway.

Adeno-associated virus 2 (AAV2) is a common vehicle for the delivery of a variety of therapeutic genes. A better understanding of the process of infection of AAV2 will advance our knowledge of AAV2 biology and allow for the optimization of AAV2 capsids with favorable transduction profiles. However, the precise fluorescent labeling of an AAV2 vector for probing virus tracking without affecting the nature of the virus remains a challenge. In this study, a lab-synthesized azide-moieties on the viral capsid at modifiable sites is precisely displayed. Upon bioorthogonal copper-less click reaction, fluorophores are subsequently conjugated to AAV2 vectors for visualization of particles. Using this principle, the authors demonstrate that it can be used for visibly studying the cell entry, and intracellular trafficking of AAV2 particles, enabling the monitoring of the intracellular dynamics of AAV2 infection. This study provides new insights into the precision labeling of AAV2 particles with important implications for a better understanding of the molecular mechanism of therapeutic gene delivery.