Methods of viral neutralizing antibody epitope mapping

Abstract

Disclosed herein are methods of high-throughput mapping of viral neutralizing antibody epitopes. Also disclosed are in vitro immunoprecipitation-based adeno-associated virus Barcode-Seq-based methods of mapping viral neutralizing antibody epitopes. In some embodiments, a method of high-throughput mapping of viral NtAb conformational epitopes can be utilized, which may comprise HP scanning of mutant viral libraries, immunoprecipitation (IP), and/or next-generation sequencing (NGS) technology. In some embodiments, a method of identifying one or more dominant epitopes in a viral vector may comprise contacting a mutant capsid of a virus with serum from a subject previously exposed to the virus and immunoprecipitating serum immunoglobulins from the serum. In various embodiments, the viral vector may be an AAV vector.

Claims

1. An AAV9 viral vector comprising, an antibody neutralizing mutation in amino acids 453-457 in an AAV9 capsid.

2. The vector of claim 1, wherein the antibody neutralizing mutation is a mutation to an alanine.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The embodiments disclosed herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. These drawings depict only typical embodiments, which will be described with additional specificity and detail through use of the accompanying drawings in which:

(2) FIG. 1A depicts a map of the DNA-barcoded AAV genome containing a pair of 12 nucleotide-long DNA barcodes (lt-VBC and rt-VBC) downstream of the AAV2 pA. Each virus barcode (VBC) can be PCR-amplified separately.

(3) FIG. 1B is a representation of double alanine (AA) scanning mutagenesis of the AAV9 capsid.

(4) FIG. 1C is a representation of hexapeptide (HP) scanning mutagenesis of the AAV2R585E capsid at a two amino acid interval.

(5) FIG. 1D is a representation of a procedure for AAV Barcode-Seq analysis. PCR products obtained from each sample are indexed with sample-specific barcodes attached to the PCR primers. This allows multiplexed ILLUMINA sequencing. Phenotypic Difference (PD) values provide information about a spectrum of phenotypes (receptor binding, transduction, tropism, blood clearance, reactivity to NtAbs, blood-cerebrospinal fluid barrier (BCSFB) penetrability, etc.) for each serotype or mutant.

(6) FIG. 2A is a graph showing pharmacokinetic profiles of 117 HP scanning AAV2R585E mutants following intravenous injection of AAV2R585E-HP-VBCLib in anti-AAV1 NtAb-positive mice.

(7) FIG. 2B is a graph showing pharmacokinetic profiles of 117 HP scanning AAV2R585E mutants following intravenous injection of AAV2R585E-HP-VBCLib in anti-AAV9 NtAb-positive mice.

(8) FIG. 2C is a graph showing pharmacokinetic profiles of 117 HP scanning AAV2R585E mutants following intravenous injection of AAV2R585E-HP-VBCLib in naïve mice. For each of FIGS. 2A, 2B, and 2C, blood AAV concentrations of each AAV mutant relative to those of AAV2R585E were determined by AAV Barcode-Seq. Only the results of AAV2R585E, 451-16000, and 451-00009 are highlighted with the patterned lines, as indicated. The results of all the other 115 HP mutants are shown with gray lines. The 451-16000 and 451-00009 exhibited significantly accelerated blood clearance only in anti-AAV1 and anti-AAV9 NtAb-harboring animals, respectively. This was not observed in the naïve animals. In the 451-16000 and 451-00009 mutants, the native AAV2R585E sequence 451-PSGTTT-456 (SEQ ID NO:3) located at the 3-fold spike was replaced with QSGSAQ (AAV1) (SEQ ID NO:1) and GSGQN (AAV9) (SEQ ID NO:2), respectively. This indicates that QSGSAQ (SEQ ID NO:1) and GSGQN (SEQ ID NO: 2) are anti-AAV1 and anti-AAV9 capsid antibody epitopes, respectively.

(9) FIG. 3A is a representation of an RNA barcode-expressing recombinant AAV (rAAV). Clone-specific DNA barcodes (lt-VBC and rt-VBC) are transcribed into RNA under the control of the U6 promoter.

(10) FIG. 3B is a graph depicting a scatter plot showing a linear correlation between relative DNA and RNA quantities determined by AAV DNA/RNA Barcode-Seq. HEK293 cells were infected with two different AAV libraries containing 25 rAAV2 clones mixed at an equal amount or at approximately a 1:3:10:30:100 ratio, and harvested 48 hours post-infection. Each clone was tagged with a clone-specific barcode. Relative DNA and RNA quantifies of each clone in the same HEK293 cell sample were determined by ILLUMINA barcode sequencing read numbers and plotted.

(11) FIG. 4 depicts AAV2R585E and AAV9 HP scanning mutants included in the DNA/RNA-barcoded dsAAV-U6-VBCLib-1. The amino acid sequences around the highest peak of the AAV capsids derived from AAV2R585E, devoid of HP mutations and AAV2R585E-HP mutants, are aligned to the left and those derived from wild-type AAV9 and AAV9-HP mutants are aligned to the right. Bold letters and hyphens indicate amino acid mutations and deletions compared to the parental sequences, respectively. The name of each mutant is given to the amino acid sequences based on the naming system as follows. The left three digits indicate the first amino acid position of the hexapeptide based on AAV2 VP1 (left panel) and AAV9 VP1 (right panel). The right five digits indicate AAV serotype from which each hexapeptide is derived: 10000, AAV1; 06000, AAV6; 00700, AAV7; 00080, AAV8; 00009, AAV9; and 00002, AAV2. When a hexapeptide amino acid sequence is shared with multiple serotypes, the right five digits have more than one positive integer.

(12) FIG. 5A is a graph plotting the averages of the relative quantities of two different clones carrying the same HP mutation in an IP precipitate.

(13) FIG. 5B is a graph plotting the averages of the relative quantities of two different clones carrying the same HP mutation in an IP supernatant. For each of FIGS. 5A and 5B, four 8-week-old C57BL/6 male mice (Mouse 1, 2, 3 and 4) were injected intravenously with AAV1-CMV-lacZ vector at a dose of 1×10.sup.11 vector genomes (vg) per mouse. Serum samples containing anti-AAV1 NtAbs were collected 3 weeks post-injection. 20 μl of PROTEIN A/G PLUS-AGAROSE beads were first coated with sample immunoglobulins by incubating the beads with 25 μl of serum samples at 4° C. for one hour, and then reacted with 1×10.sup.9 vg of DNA/RNA-barcoded dsAAV-U6-VBC-Lib-1 at 4° C. overnight. This library contained 72 AAV clones composed of 24 HP mutants and two reference controls (AAV2R585E and wild-type AAV9, 15 clones each). Viral genomic DNAs were extracted from agarose beads-bound and unbound AAV particles in the IP precipitates and the IP supernatants, respectively, and subjected to the AAV Barcode-Seq analysis (id.). The relative quantity of each clone (two clones per mutant) determined by ILLUMINA sequencing read numbers was normalized with the ILLUMINA sequencing read numbers of the reference control AAV2R585E. The Y-axis shows Phenotypic Difference (PD) values (id.) of each mutant relative to the control AAV2R585E in antibody-positive sera, normalized with PD values obtained with naïve mouse serum. Plotted are the averages of the relative quantities of two different clones carrying the same HP mutation. Arrows indicate mutants harboring the heterologous peptides that bind to anti-AAV1 antibody, and therefore, represent anti-AAV1 antibody epitopes.

(14) FIG. 6 is a graph wherein the Y-axis shows Phenotypic Difference (PD) values (id.) of each mutant relative to the control wild-type AAV9. An arrow indicates a mutant harboring the heterologous peptide that binds to anti-AAV2 antibody, and therefore, represents an anti-AAV2 antibody epitope. Four 8-week-old C57BL/6 male mice (Mouse 1, 2, 3 and 4) were injected intravenously with AAV2-CMV-lacZ vector at a dose of 1×10.sup.11 vg/mouse. Serum samples containing anti-AAV2 NtAbs were collected three weeks post-injection. The subsequent experimental procedure is the same as that for FIGS. 5A and 5B.

(15) FIG. 7 is a graph wherein the Y-axis shows Phenotypic Difference (PD) values (id.) of each mutant relative to the control AAV2R585E. An arrow indicates a mutant harboring the heterologous peptide that binds to anti-AAV7 antibody, and therefore, represents an anti-AAV7 antibody epitope. Three 8-week-old C57BL/6 male mice (Mouse 1, 2, and 3) were injected intravenously with AAV7-CMV-lacZ vector at a dose of 1×10.sup.11 vg/mouse. Serum samples containing anti-AAV7 NtAbs were collected three weeks post-injection. The subsequent experimental procedure is the same as that for FIGS. 5A and 5B.

(16) FIG. 8 is a graph wherein the Y-axis shows Phenotypic Difference (PD) values (id.) of each mutant relative to the control AAV2R585E. An arrow indicates a mutant harboring the heterologous peptide that binds to anti-AAV9 antibody, and therefore, represents an anti-AAV9 antibody epitope. Four 8-week-old C57BL/6 male mice (Mouse 1, 2, 3 and 4) were injected intravenously with AAV9-CMV-lacZ vector at a dose of 1×10.sup.11 vg/mouse. Serum samples containing anti-AAV9 NtAbs were collected three weeks post-injection. The subsequent experimental procedure is the same as that for FIGS. 5A and 5B.

(17) FIG. 9 is a graph depicting anti-AAV2 capsid mouse monoclonal antibody (A20) epitope identification by the magnetic beads-based IP-Seq analysis in conjunction with the dsAAV9-HP-U6-VBCLib-2 library. 1×10.sup.9 vg of the dsAAV9-HP-U6-VBCLib-2 library was reacted with Pierce Protein A/G Magnetic Beads coated with the A20 antibody, and subjected to immunoprecipitation. Subsequently, viral genomic DNAs were extracted from magnetic beads-bound and unbound AAV particles in the IP precipitates (A20_IP) and the IP supernatants (A20_Sup), respectively, and a demographic change of the library composition in the IP precipitates (A20_IP) and the IP supernatants (A20_Sup) was determined by the AAV Barcode-Seq analysis. The relative quantity of each clone (2 clones per mutant) determined by Illumina barcode sequencing read numbers was normalized with the Illumina sequencing read numbers of the reference control AAV9. The Y-axis shows Phenotypic Difference (PD) values of each mutant relative to the control AAV9. The X-axis shows different AAV strains. They are AAV9, AAV2R585E, AAV2, 009-00002, . . . , 718-00002 from the left to the right. Due to space limitations, not all the AAV2 strains are labeled. Plotted are the averages of the relative quantities of two different clones carrying the same HP mutation. An arrow indicates the mutant 261-00002 harboring a heterologous AAV2-derived peptide that binds to the A20 antibody (261-SSQSGA-266 (SEQ ID NO:50)). Another peak to the leftmost represents AAV2R585E and AAV2.

(18) FIGS. 10A, 10B, 10C, and 10D are graphs depicting anti-AAV2 mouse polyclonal antibody epitope identification by the magnetic beads-based IP-Seq analysis in conjunction with the dsAAV9-HP-U6-VBCLib-2 library. Four 8-week-old C57BL/6 male mice (Mouse 1, 2, 3 and 4 in FIGS. 10A, 10B, 10C, and 10D, respectively) were injected intravenously with AAV2-CMV-lacZ vector at a dose of 1×10.sup.11 vg/mouse. Serum samples containing anti-AAV2 neutralizing antibodies were collected 3 weeks post-injection and subjected to the IP-Seq analysis for epitope mapping. Demographic change of the library composition in the IP precipitates (Mousel, 2, 3 and 4_IP) and the IP supernatants (Mousel, 2, 3, and 4_Sup) were determined by the AAV Barcode-Seq analysis. The relative quantity of each clone (2 clones per mutant) determined by IIlumina barcode sequencing read numbers was normalized with the Illumina sequencing read numbers of the reference control AAV9. The Y-axis shows Phenotypic Difference (PD) values of each mutant relative to the control AAV9. The X-axis shows different AAV strains. They are AAV9, AAV2R585E, AAV2, 009-00002, . . ., 718-00002 from the left to the right. Due to space limitations, not all the AAV2 strains are labeled. Plotted are the averages of the relative quantities of two different clones carrying the same HP mutation. Gray arrows, in FIGS. 10A-10D, indicate the two hexapeptide mutants containing the dominant epitope, 513-RDSLVNPG-520 (SEQ ID NO:52). The thick black arrow, in FIG. 10D, indicates the same epitope identified for the A20 antibody. Thin black arrows, in FIGS. 10A-10C, also indicate mutants that may contain epitopes. The peak to the leftmost represents AAV2R585E and AAV2.

DETAILED DESCRIPTION

(19) It will be readily understood that the embodiments, as generally described herein, are exemplary. The following more detailed description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. Moreover, the order of steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified.

(20) The term “viral vector” as used herein means any vector that comprises or derives from components of a given virus and is suitable to infect mammalian cells, including human cells, of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo. The term “viral vector” may be used to refer to a viral particle (or virion) comprising at least a nucleic acid molecule encoding a protein of interest.

(21) The term “AAV vector” as used herein means any vector that comprises or derives from components of AAV and is suitable to infect mammalian cells, including human cells, of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo. The term “AAV vector” may be used to refer to an AAV type viral particle (or virion) comprising at least a nucleic acid molecule encoding a protein of interest.

(22) Additionally, the AAVs disclosed herein may be derived from various serotypes, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single-stranded or self-complementary). In particular embodiments, the AAV vectors disclosed herein may comprise desired proteins or protein variants. A “variant” as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both.

(23) Methods of producing AAV vectors as disclosed herein are well known in the art, including methods, for example, using packaging cells, auxiliary viruses or plasm ids, and/or baculovirus systems (see, e.g., Samulski et al., J Virol 63, 3822 (1989); Xiao et al., J Virol 72, 2224 (1998); Inoue et al., J Virol 72, 7024 (1998); W01998/022607; and W02005/072364).

(24) Methods of producing pseudotyped AAV vectors are also known (see, e.g., W000/28004), as well as various modifications or formulations of AAV vectors, to reduce their immunogenicity upon in vivo administration (see, e.g., W001/23001; W000/73316; W004/112727; W005/005610; and W099/06562). In some embodiments, AAV vectors may be prepared or derived from various serotypes of AAVs which may be mixed together or mixed with other types of viruses to produce chimeric (e.g., pseudotyped) AAV viruses.

(25) In some embodiments, a method of high-throughput mapping of viral NtAb conformational epitopes can be utilized, which may comprise HP scanning of mutant viral libraries, immunoprecipitation (IP), and/or next-generation sequencing (NGS) technology.

(26) As discussed, NtAb epitope mapping can be used in the development of new vaccines and drugs for the prevention and treatment of infectious diseases. NtAb epitope mapping can also be used for the development of novel gene delivery vectors. Identification of and knowledge regarding NtAb epitopes may help in the genetic engineering of viral components of novel vectors that can evade, or better evade, the host immune response, as the host immune response can be a significant obstacle in effective in vivo gene therapy. In various embodiments, the present disclosure can overcome the limitations of currently available methods for NtAb epitope mapping (e.g., X-ray co-crystallography, cryoelectron microscopy, synthetic peptide array, phage display, etc.) and may effectively identify conformational epitopes of viral antigens in a high-throughput manner.

(27) NtAbs may recognize mostly conformational epitopes as opposed to linear epitopes. X-ray diffraction and cryoelectron microscopic analyses of co-crystallized antigen-antibody complexes can identify conformational NtAb epitopes. However, it may be technically challenging to apply this method to conformational epitope mapping of multiple samples due to its low-throughput, time-intensive, and cost-consuming nature. Synthetic peptide array-based methods can adopt a high-throughput format but they may primarily identify linear epitopes and may be inefficient in identifying conformational epitopes. Phage display approaches have some ability to identify conformational epitopes but may not necessarily be able to present antigens in their native three dimensional protein structures. In contrast, the methods disclosed herein can express epitopes in the context of native tertiary and quaternary structures of viral proteins and identify conformational epitopes of multiple samples at one time in a high-throughput manner.

(28) In various embodiments, the methods disclosed herein can use DNA/RNA-barcoded HP scanning libraries in which platform viral proteins that are not neutralized by NtAbs of interest carry HPs derived from homologous viral proteins that are neutralized by the NtAbs of interest for which one may wish to identify conformational epitopes. HPs can be expressed in the platform viral proteins with appropriately juxtaposed amino acids in native-like tertiary and quaternary structures. Examples of HP scanning libraries include, but are not limited to, the AAV2R585E-HP and AAV9-HP libraries.

(29) Conventional epitope mapping methods such as peptide array- or phage display-based systems can map epitopes of one antigen or one antibody at a time. The methods of the present disclosure can make it possible to map epitopes of multiple antibodies against multiple antigens at one time by an NGS-based method such as, but not limited to, multiplexed ILLUMINA sequencing technology.

(30) In certain embodiments, the present method may comprise the following steps: 1) incubation of samples and a HP scanning library; 2) IP; 3) viral DNA extraction; 4) PCR amplification of viral DNA barcodes; 5) multiplexed ILLUMINA barcode sequencing; and/or 6) data analysis.

(31) An in vitro IP-based AAV Barcode-Seq method can be used to identify anti-AAV antibody epitopes as opposed to the previously reported in vivo approach described above. Also, the highest peak around the amino acid positions 452-457 in the capsid may be a potential dominant epitope for antibodies against various AAV serotypes.

(32) In some embodiments, IP conditions may be optimized using a recombinant AAV9 vector, anti-AAV9 mouse sera, and protein A/G agarose beads. Additionally, a DNA-barcoded AAV HP scanning capsid mutant library comprising 78 AAV clones can be produced, which comprises nineteen AAV2R585E-derived HP mutants and five AAV9-derived HP mutants that span a 14-amino-acid region in and flanking the vicinity of the highest peak of the AAV1, AAV2, AAV7, AAV8, and AAV9 capsids. Using this library, and anti-AAV1, anti-AAV2, anti-AAV7, anti-AAV8, anti-AAV9, and naïve mouse sera collected from four mice per serotype/native, IP can be performed and AAV library genomes from both immunoprecipitates and supernatants may be recovered, which may then be subjected to the AAV Barcode-Seq analysis. Mutants harboring AAV1-, AAV2-, AAV7-, AAV8-, and AAV9-derived peptides spanning eight amino acids within the vicinity of the highest peak, but not other mutants or the control AAV2R585E or AAV9, can be found to be captured by the corresponding anti-AAV serotype sera of some, if not all, of the immunized mice, indicating that the identified peptides constitute a dominant epitope. Thus, a combination of AAV Barcode-Seq with IP can map anti-AAV antibody epitopes in a high-throughput manner.

(33) In some embodiments, a method of identifying one or more dominant epitopes in a viral vector may comprise contacting a mutant capsid of a virus with serum from a subject previously exposed to the virus and immunoprecipitating serum immunoglobulins from the serum. In various embodiments, the viral vector may be an AAV vector. In certain embodiments, the mutant capsid of the virus may be an AAV mutant capsid. In some embodiments, the mutant capsid of the virus of the disclosed method may be included in a mutant capsid library, wherein the capsids in the mutant capsid library are barcoded.

(34) In some embodiments, an AAV1 viral vector may comprise an antibody neutralizing mutation in amino acids 452-457 in the AAV1 capsid (452-QSGSAQ-457) (SEQ ID NO:1). In other embodiments, an AAV9 vector may comprise an antibody neutralizing mutation in amino acids 453-457 in the AAV9 capsid (453-GSGQN-457) (SEQ ID NO: 2). In certain embodiments, the antibody neutralizing mutation of either the AAV1 viral vector or the AAV9 viral vector may comprise a mutation to an alanine.

(35) A dsAAV9-HP-U6-VBCLib-2 HP-scanning mutant capsid library containing a total of 338 AAV clones has been created. These clones are composed of 153 AAV9-HP mutants, AAV2, AAV2R585E and AAV9. The IP-Seq (Immunoprecipitation followed by AAV Barcode-Seq) has been optimized using Protein A/G magnetic beads. An epitope in the AAV2 capsid that is recognized by the mouse monoclonal antibody against intact AAV2 particles (A20) has been mapped by IP-Seq. Epitopes in the AAV2 capsid have been mapped that are recognized by the mouse polyclonal antibodies developed in mice immunized by intravenous injection of an AAV2 vector. Strategies for the creation of anti-AAV neutralizing antibody-escaping AAV capsid mutants have been developed based on the new IP-Seq data.

AAV Barcode-Seq

(36) AAV Barcode-Seq, an NGS-based method that allows the characterization of phenotypes of hundreds of different AAV strains (i.e., naturally occurring serotypes and laboratory-engineered mutants) in a high-throughput manner with significantly reduced time and effort and using only a small number of subjects (e.g., tissue cultures and experimental animals), has recently been established (Adachi K et al., Nat Commun 5, 3075 (2014)). Using this approach, biological aspects including, but not limited to, blood clearance rate, transduction efficiency, tissue tropism, and reactivity to anti-AAV NtAbs can be assessed. FIGS. 1A-1D schematically depict the AAV Barcode-Seq approach. The principle of this approach is as follows. When a library stock comprising many different AAV strains is applied to certain types of samples (e.g., cells), the composition of the AAV population would in theory not change between the original input library and the library recovered from the samples if each of the AAV strains had exactly the same biological properties in a given context. However, if some strains show a different biological property (e.g., faster blood clearance or more efficient cellular internalization) compared to the others, there would be a change in the population composition between the input library (i.e., the library stock) and the output library (i.e., the library recovered from the samples). The basic method consists of a bioinformatic comparison between the input and output libraries using a similar principle as that employed in RNA-Seq (Wang Z et al., Nat Rev Genet 10, 57-63 (2009)). This method allows the quantification of phenotypic differences between different AAV strains as a function of strain demographics. Such an analysis becomes possible by tagging each AAV strain with a unique short DNA barcode and applying ILLUMINA barcode sequencing to the resulting population (Smith AM et al., Genome Res 19, 1836-1842 (2009)).

In Vivo-Based Anti-AAV NtAb Epitope Mapping Using AAV2R585E Hexapeptide Scanning Libraries

(37) Construction has been completed of a total of 452 hexapeptide (HP) scanning AAV2R585E capsid mutants that carry all the AAV1-, AAV6-, AAV7-, AAV8-, and AAV9-specific HPs that are not present in the AAV2 capsid (see Table 1). In Adachi K et al., Nat Commun 5, 3075 (2014), AAV2R585E-HP-VBCLib-1 and 2 libraries were produced containing a total of 117 capsid-forming HP mutants, the libraries were injected intravenously into anti-AAV1 or AAV9 NtAb-harboring C57BL/6 mice (n=3) or naive mice (n=2) at 1×10.sup.13 vg/kg, and relative blood concentrations of each mutant were determined at one, 10, 30 and 60 minutes post-injection by AAV Barcode-Seq. Because there is no or minimal serologic cross-reactivity between AAV2 and any of AAV1, AAV6, AAV7, AAV8, and AAV9 (Gao G et al., J Virol 78, 6381-6388 (2004)), only AAV2R585E mutants with a HP containing an antibody epitope would be neutralized, and therefore would be cleared faster than other mutants in the same immunized animal or faster than the same mutant in naïve animals. By taking this approach, 452-QSGSAQ-457 (SEQ ID NO:1) and 453-GSGQN-457 (SEQ ID NO:2) were identified as epitopes for mouse anti-AAV1 and AAV9 NtAbs developed by viral immunization (see FIGS. 2A-2C). Taking the same approach, 449-RTQSNPGGTAG-459 (SEQ ID NO:4) was identified as a mouse anti-AAV7 NtAb epitope. These observations establish that injection of AAV2R585E-HP scanning libraries into anti-AAV NtAb positive mice combined with AAV Barcode-Seq can identify anti-AAV NtAb epitopes effectively.

Establishment of a Universal AAV DNA/RNA Barcode-Seq System Expressing RNA Barcodes

(38) A universal Barcode-Seq system expressing RNA barcodes, termed AAV DNA/RNA Barcode-Seq, has been devised. In this system, AAV libraries are produced in which each viral particle contains a DNA genome that is devoid of the rep and cap genes but is transcribed into an RNA barcode unique to its own capsid. To show proof-of-principle of this new method, two libraries of 25 recombinant AAV2 viral clones mixed at defined ratios were constructed, HEK293 cells were infected with each library in duplicate, and the cells were harvested at 48 hours post-infection. In these libraries, each viral clone carried the dsAAV-U6-VBCLib genome expressing RNA containing a pair of clone-specific 12 ribonucleotides transcribed from the corresponding DNA barcode sequences placed downstream of the human U6 snRNA promoter (see FIG. 3A). ILLUMINA sequencing of DNA-PCR and reverse-transcription (RT)-PCR barcode amplicons from total DNA and RNA extracted from the same library-infected cells showed that viral genome expression could be determined by Barcode-Seq in at least a 2-log dynamic range with a Pearson's correlation coefficient of 0.98 (see FIG. 3B). This RNA barcode system, AAV DNA/RNA Barcode-Seq, has been employed for anti-AAV NtAb epitope mapping.

AAV Libraries for Anti-AAV NtAb Epitope Mapping Created Based on the Universal AAV DNA/RNA Barcode-Seq System

(39) In this new system, DNA/RNA-barcoded dsAAV-U6-VBCLib libraries packaged with HP scanning mutants can be produced. Such HP mutants can be AAV2R585E-HP scanning mutants for anti-AAVx NtAb epitope mapping (x=any strains other than AAV2 that do not cross-react with anti-AAV2 NtAb) and AAV9-HP scanning mutants for anti-AAV2 NtAb epitope mapping. The structure of AAV2R585E-HP mutants is shown in FIG. 1C. AAV9-HP mutants are those in which AAV9 HPs are replaced with those derived from the AAV2 capsid. All of them are HP scanning libraries that contain the dsAAV-U6-VBCLib genome (see FIG. 3A). DNA/RNA-barcoded dsAAV2R585E-HP-U6-VBCLib libraries for anti-AAV1, anti-AAV6, anti-AAV7, anti-AAV8, and anti-AAV9 NtAb epitope mappings and DNA/RNA-barcoded dsAAV9-HP-U6-VBCLib libraries for anti-AAV2 NtAb epitope mapping can be created. The former set of libraries can comprise a total of 452 AAV2R585E-HP scanning mutants, and the latter set of libraries can comprise 153 AAV9-HP mutants. These mutants, in theory, can cover all the potential hexapeptide epitopes of AAV1, AAV2, AAV6, AAV7, AAV8, and AAV9. Each library will contain two clones per mutant plus 15 clones each of the reference controls, AAV9 and AAV2R585E.

Immunoprecipitation (IP)-Seq Based Anti-AAV Antibody Epitope Mapping

(40) The IP-Seq based method does not require animals and is capable of mapping antibody epitopes of multiple samples at one time using multiplexed ILLUMINA sequencing. Differentiation between NtAb epitopes and non-NtAb epitopes may be achieved by integrating an AAV RNA Barcode-Seq-based neutralization antibody assay into a system as detailed below in the section “AAV RNA Barcode-Seq-based analysis of the neutralizing ability of anti-AAV antibodies with defined epitopes.”

(41) The procedure for IP-Seq based anti-AAV antibody epitope mapping can be as follows. First, 25 μl of serum samples (containing anti-AAV NtAbs) and 20 μl of PROTEIN A/G PLUS-AGAROSE (SANTA CRUZ sc-2003) can be incubated in a total volume of 100 μl in PBS in 1.5 ml tubes at 4° C. for 1 hour on a rotation device. After washing with PBS, a DNA/RNA-barcoded dsAAV-U6-VBCLib library and the agarose beads coated with immunoglobulins can be mixed in a total volume of 100 μl PBS, and may then be incubated at 4° C. overnight on a rotation device. On the next day, a standard IP procedure may be followed, the supernatants and immunoprecipitates can be collected, and viral genome DNA can be extracted using a WAKO DNA Extraction Kit following Proteinase K treatment of the samples. The subsequent procedure may be similar to that used for AAV Barcode-Seq as described in Adachi K et al., Nat Commun 5, 3075 (2014). Briefly, left and right viral clone-specific barcodes (lt-VBC and rt-VBC in FIGS. 1A-1D) may be PCR-amplified using viral genome DNA recovered from the IP supernatants and precipitates. The PCR primers can be indexed with sample-specific DNA barcodes. All the PCR amplicons may then be mixed into a pool and the pool may be subjected to ILLUMINA sequencing. The ILLUMINA sequencing data may be bioinformatically analyzed to detect demographic changes of the AAV library in each sample. The principle of the method is that viral clones with higher avidity to sample immunoglobulins than others can be detected as clones that are decreased or depleted in the supernatants while enriched in the precipitates by ILLUMINA barcode sequencing. Such clones may likely carry epitopes for anti-AAV antibodies under investigation, and the epitopes targeted by the antibodies may likely be the heterologous peptides incorporated into the capsid of particular AAV clones showing a demographic change. 1×10.sup.7, 1×10.sup.8, and 1×10.sup.9 vg per 1.5 ml tube have been used and it has been found that this range of the virus quantity may give clear results as described below.

(42) To show proof-of-principle, a DNA/RNA-barcoded dsAAV-U6-VBCLib-1 library exhibiting low diversity was produced. This library was designed to identify anti-AAV1, AAV2, AAV6, AAV7, AAV8, and AAV9 antibody epitopes at the highest peak around the amino acid positions 452-457 in the capsid. Based on data obtained in the in vivo-based epitope mapping study as described above, it was hypothesized that this region is a potential dominant epitope for antibodies against various AAV serotypes. Therefore, it was assumed that targeting this region in the proof-of-principle experiments may have a higher success rate in finding anti-AAV antibody epitopes for various AAV serotypes. This library was composed of 78 AAV clones, which included 19 AAV2R585E-derived HP mutants and five AAV9-derived HP mutants that spanned a 14-amino-acid region in and flanking the vicinity of the highest peak of the AAV1, AAV2, AAV7, AAV8, and AAV9 capsids (see FIG. 4) in addition to 15 clones each of reference control AAV strains, AAV9, and AAV2R585E devoid of HP mutations. Using this library and anti-AAV1, anti-AAV2, anti-AAV7, anti-AAV8, anti-AAV9 mouse sera collected from 3-4 mice per serotype, the IP-Seq-based epitope mapping procedure described above was performed. The mice from which anti-AAV sera was collected had been immunized by intravenous injection of 1×10.sup.11 vg of AAV-CMV-lacZ vector packaged with the corresponding serotype capsids. The data presented below were obtained when 1×10.sup.9 vg per tube was used for IP-Seq. The IP-Seq procedure was also performed using native mouse sera to control nonspecific binding of the AAV library to the immunoglobulin-coated agarose beads.

(43) FIGS. 5A and 5B show the results of IP-Seq using anti-AAV1 mouse sera collected from four mice. Mouse 1 exhibited >10 fold enrichment of 451-16000 and 453-16000 in the IP precipitations, and Mouse 3 also showed >10 fold enrichment of 453-16000 in the IP precipitation. Consistent with this observation, reduction of 451-16000 and 453-16000 in the supernatant, although not dramatic, was found in the Mouse 1 serum. Mouse 2 and Mouse 4 exhibited a lesser degree of enrichment of 453-16000 in the precipitation. The reduction of these mutants in the supernatants was not detected in the Mouse 2, 3, and 4 sera. These observations indicate that, in Mouse 1 and 3 and perhaps Mouse 2 and 4, an anti-AAV1 antibody epitope resides within the heterologous peptides contained in 451-16000 and 453-16000 (i.e., 452-QSGSAQNK-459 (SEQ ID NO:5)). The fact that the heterologous peptides in these mutants are derived from the AAV1 capsid may also lend support to this conclusion. In addition, this conforms to the result obtained by the in vivo-based epitope mapping by AAV Barcode-Seq (Adachi K et al., Nat Commun 5, 3075 (2014)). The sensitivity of IP-Seq may be increased by decreasing the amount of AAV library added to the IP reaction. For example, the use of 1×10.sup.7 vg or 1×10.sup.8 vg per tube may identify epitopes more effectively and clearly in both the IP supernatants and the IP precipitations. A preliminary experiment using 1×10.sup.7 vg and 1×10.sup.8 vg per tube of an AAV library has supported this prediction.

(44) FIG. 6 shows the results of IP-Seq using anti-AAV2 mouse sera collected from four mice. All the AAV2R585E mutants were found enriched in the IP precipitations. In Mouse 2, 453-00002 was enriched by >10 fold in the precipitates, demonstrating that the AAV2-derived heterologous peptide in this mutant, 451-PSGTTT-456 (SEQ ID NO:3), may be an epitope for anti-AAV2 antibodies developed in Mouse 2.

(45) FIG. 7 shows the results of IP-Seq using anti-AAV7 mouse sera collected from three mice. 451-00700 was significantly enriched in the IP precipitates in Mouse 1 and 2. This demonstrates that AAV7-derived heterologous peptide in the 451-00700 mutant, 453-NPGGTAG-459 (SEQ ID NO:6), may be an epitope for anti-AAV7 antibodies developed in Mouse 1 and 2.

(46) FIG. 8 shows the results of IP-Seq using anti-AAV9 mouse sera collected from four mice. 451-00009 was significantly enriched in the IP precipitates in Mouse 1 and 3. This demonstrates that AAV9-derived heterologous peptide in the 451-00009 mutant, 453-GSGQN-457 (SEQ ID NO:2), may be an epitope for anti-AAV9 antibodies developed in Mouse 1 and 3. This also conforms to the result obtained by the in vivo-based epitope mapping by AAV Barcode-Seq (Adachi K et al., Nat Commun 5, 3075 (2014)). Regarding the IP-Seq analysis of anti-AAV8 mouse sera, no epitopes could be detected at the sensitivity of the assay used.

(47) In summary, a series of proof-of-concept experiments demonstrates that the IP-Seq using AAV capsid hexapeptide scanning libraries is a means to map anti-AAV antibody epitopes, presumably including conformational epitopes, effectively and in a high-throughput manner. Although the AAV library used for this preliminary set of experiments contained only 24 hexapeptide (HP) scanning mutants, 452 AAV2R585E-HP mutants were created to look for anti-AAV1, anti-AAV6, anti-AAV7, anti-AAV8, and anti-AAV9 antibody epitopes. A total of 153 AAV9-HP mutants to cover the entire region of AAV2 VP1 capsid protein can also be created. Additionally, the same approach can be exploited for epitope mapping of antibodies against other AAV serotypes or capsid-engineered mutants. This method should also have a potential to be adapted to antiviral antibody epitope mapping for any viruses other than AAV.

AAV RNA Barcode-Seq-Based Analysis of the Neutralizing Ability of Anti-AAV Antibodies with Defined Epitopes

(48) AAV DNA/RNA Barcode-Seq may be used to assess the neutralizing ability of anti-AAV antibodies that recognize defined epitopes. The principle of this new assay system is as follows. A DNA/RNA-barcoded dsAAV-U6-VBCLib library that has been prepared for IP-Seq can be pre-incubated with samples under investigation (serum samples, purified monoclonal/polyclonal antibodies, etc.) at 37° C. for one hour or pre-incubated with a naive animal serum devoid of anti-AAV NtAbs (e.g., naïve mouse serum) as a control. The mixture can then be applied to a reporter cell line in vitro in duplicate or in triplicate. Two to three days after AAV library infection, total RNA can be recovered from cells and reverse-transcribed using an AAV genome-specific RT primer. Then clone-specific viral RNA barcodes may be PCR-amplified and subjected to AAV Barcode-Seq (i.e., multiplexed ILLUMINA barcode sequencing followed by data analysis). When the ILLUMINA sequencing data are compared between the samples and the control, AAV clones that are neutralized by anti-AAV antibodies can be identified as a relative decrease of ILLUMINA sequencing reads among all the AAV clones in the library used for the analysis. By combining the heterologous peptide information of each AAV clone and the AAV Barcode-Seq results, it may be determinable whether or not an anti-AAV antibody, or anti-AAV antibodies, that recognizes a defined antibody epitope on the capsid can neutralize the virus and impair the virus infectivity. This assay complements IP-Seq because IP-Seq by itself may not be able to differentiate NtAb epitopes from non-neutralizing antibody epitopes, although the in vivo-based epitope mapping approach is capable of differentiation between these two types of anti-AAV antibodies. The reporter cells should be selected carefully because in vitro transduction efficiencies significantly vary depending on cell types and AAV strains. For example, HEK293 cells can be appropriate for anti-AAV2 antibody epitopes and Chinese Hamster Ovary (CHO) Lec2 cells can be appropriate for anti-AAV9 antibody epitopes.

(49) TABLE-US-00001 TABLE 1 Hexapeptide scanning AAV2R585E-derived mutants Name of mutant.sup.1 Amino acid substitutions in addition to R585E 441-00700 S446A 441-16000 S446N 443-00009 R447K 445-00009 R447K/N449I/T450N 445-00080 N449Q 445-00700 S446A/N449Q/T450S 445-16000 S446N/N449Q/T450N 447-00009 R447K/N449I/T450N/P451G/S452 447-00080 N449Q/P451T/S452G 447-00700 N449Q/T450S/P451N/S452G 447-16000 N449Q/T450N/P451Q 449-00009 N449I/T450N/P451G/S452/G453S/T454G 449-00700 N449Q/T450S/P451N/S452G 449-16000 N449Q/T450N/P451Q/T454S 451-00009 P451G/S452/G453S/T454G/T455Q/T456N 451-00080 P451T/S452G/T455A/T456N 451-00700 P451N/S452G/T455A/T456G 451-16000 P451Q/T454S/T455A/T456Q 453-00009 G453S/T454G/T455Q/T456N/S458Q 453-00080 T455A/T456N/Q457T/S458Q 453-00700 T455A/T456G/Q457N/S458R 453-16000 T454S/T455A/T456Q/Q457N/S458K 455-00009 T455Q/T456N/S458Q/R459T 455-00080 T455A/T456N/Q457T/S458Q/R459T 455-00700 T455A/T456G/Q457N/S458R/R459E 455-16000 T455A/T456Q/Q457N/S458K/R459D 457-00009 S458Q/R459T/Q461K 457-00080 Q457T/S458Q/R459T/Q461G 457-00700 Q457N/S458R/R459E 457-16000 Q457N/S458K/R459D/Q461L 459-00009 R459T/Q461K/Q464V 459-00080 R459T/Q461G 459-00700 R459E/S463Y 459-16000 R459D/Q461L/Q464R 461-00009 Q461K/Q464V 461-00080 Q461G/A465G 461-00700 S463Y/A465G 461-16000 Q461L/Q464R/A465G/G466S 463-00009 Q464V/A467P 463-00080 A465G/A467P/S468N 463-00700 S463Y/A465G/A467P 463-16000 Q464R/A465G/G466S/A467P/S468A 465-00009 (R585E.9-4.sup.2) A467P/D469N/I470M 465-00080 A465G/A467P/S468N/D469T/I470M 465-00700 A465G/A467P/D469T/I470M 465-16000 A465G/G466S/A467P/S468A/D469G/I470M 467-00009 (R585E.9-5.sup.2) A467P/D469N/I470M/R471A/D472V 467-00080 A467P/S468N/D469T/I470M/R471A/D472N 467-00700 A467P/D469T/I470M/R471A/D472E 467-16000 A467P/S468A/D469G/I470M/R471S/D472V 469-00009 D469N/I470M/R471A/D472V/S474G 469-00080 D469T/I470M/R471A/D472N/S474A 469-00700 D469T/I470M/R471A/D472E/S474A 469-16000 D469G/I470M/R471S/D472V/S474P 471-00009 R471A/D472V/S474G 471-00080 R471A/D472N/S474A/R475K 471-00700 R471A/D472E/S474A/R475K 471-16000 R471S/D472V/S474P/R475K 473-00009 S474G/W477Y/L478I 473-00780 S474A/R475K 473-16000 S474P/R475K 475-00009 W477Y/L478I 475-16780 R475K 477-00009 W477Y/L478I/C482S 479-00009 C482S 479-00700 Y483F 571-00009 Q575S 571-00780 Q575E 571-16000 Q575R/Y576F 573-00009 Q575S/S578Q 573-00780 Q575E/S578I 573-16000 Q575R/Y576F/S578T 575-00009 Q575S/S578Q/S580A 575-00080 Q575E/S578I/S580A 575-16000 Q575R/Y576F/S578T/S580A 577-00009 S578Q/S580A 577-00080 S578I/S580A/T581D 577-00700 S578I/T581S 577-16000 S578T/S580A/T581V 579-00009 S580A/L583H 579-00080 S580A/T581D 579-00700 T581S 579-06000 S580A/T581V 579-10000 S580A/T581V/L583F 581-00009 L583H/E585S/G586A 581-00080 T581D/E585Q/G586Q 581-00700 T581S/E585A/G586A 581-06000 T581V/E585S/G586S 581-10000 T581V/L583F/E585S/G586S 583-00009 L583H/E585S/G586A/N587Q/R588A 583-00080 E585Q/G586Q/R588T 583-00700 E585A/G586A/R588T 583-06000 E585S/G586S/N587S/R588T 583-10000 L583F/E585S/G586S/N587S/R588T 585-00009 (2i9.sup.3) E585S/G586A/N587Q/R588A 585-00080 (2i8.sup.3) E585Q/G586Q/R588T/Q589A/A590P 585-00700 (2i7.sup.3) E585A/G586A/R588T/Q589A 585-16000 (2i1.sup.3) E585S/G586S/N587S/R588T/Q589D/A590P 587-00009 N587Q/R588A/A591Q 587-00080 R588T/Q589A/A590P/A591Q/T592I 587-00700 R588T/Q589A/A591Q 587-16000 N587S/R588T/Q589D/A590P 589-00009 A591Q/A593G/D594W 589-00080 Q589A/A590P/A591Q/T592I/A593G/D594T 589-00700 Q589A/A591Q/A593Q/D594V 589-16000 Q589D/A590P/A593G 591-00009 A591Q/A593G/D594W/N596Q 591-00080 A591Q/T592I/A593G/D594T 591-00700 A591Q/A593Q/D594V 591-16000 A593G/N596H 593-00009 A593G/D594W/N596Q/T597N 593-00080 A593G/D594T/T597S 593-00700 A593Q/D594V/T597N 593-06000 A593G/N596H/T597V/Q598M 593-10000 A593G/N596H/T597A/Q598M 595-00009 N596Q/T597N/V600I 595-00080 T597S/V600A 595-00700 T597N/V600A 595-06000 N596H/T597V/Q598M/V600A 595-10000 N596H/T597A/Q598M/V600A 597-00009 T597N/V600I 597-06000 T597V/Q598M/V600A 597-10000 T597A/Q598M/V600A 599-00009 V600I 599-16780 V600A 485-00089 K490T 487-00080 K490T/S492T 487-16000 S492K 491-00009 S492V/A493T/D494Q 491-00080 S492T/A493G/D494Q 493-00009 A493T/D494Q 493-00080 A493G/D494Q 493-00700 A493D/D494Q 493-16000 A493T 495-16780 E499N/Y500F 497-00009 Y500F/S501A 497-16000 E499N/Y500F/S501T 499-00009 Y500F/S501A/T503P 501-00009 S501A/T503P/T506S 501-00080 S501A/G504A/A505G 503-00080 G504A/A505G 503-16000 T506S 505-00080 A505G 505-16000 T506S/H509N 507-16000 H509N 509-00780 D514N 509-16000 H509N/D514E 513-16000 D514E/L516I/V517I 515-00080 V517A 523-00009 D528E 523-00780 S525T 525-00009 D528E/D529G 525-06000 E530K 527-00009 D528E/D529G/E531D/K532R 527-00080 K532R 527-00700 E531D/K532R 527-06000 E530K/E531D 527-10000 E531D 529-00009 D529G/E531D/K532R 531-00080 K532R/Q536S 533-00009 Q536L 533-00700 Q536S 533-16000 Q536M 535-16000 Q536M/L540M 537-16000 L540M 543-00009 S547T/E548G 543-00080 G546N/S547A/E548A 543-00700 Q545T/S547A/E548T 543-16000 Q545E/G546S/S547A/E548G 545-00009 S547T/E548G/K549R/T550D 545-00080 G546N/S547A/E548A/K549R/T550D 545-00700 Q545T/S547A/E548T/K549/T550N 545-16000 Q545E/G546S/S547A/E548G/K549A 549-00009 K549R/T550D/I554A 549-00080 K549R/T550D/V552A/I554Y 549-00700 K549/T550N/N551K/V552T/D553T/I544L 549-16000 K549A/T550S/V552T/D553A/I554L 551-00009 I554A/E555D 551-00080 V552A/I554Y/E555S/K556D 551-00700 N551K/V552T/D553T/I554L/K556N 551-16000 V552T/D553A/I554L/E555D/K556N 555-00009 E555D 555-00080 E555S/K556D/I559L 555-00700 K556N/M558L/I559M 555-16000 E555D/K556N 557-00009 D561N 557-00080 I559L/D561S 557-00700 M558L/I559M/D561N 561-00080 D561S/R566K 561-16000 R566K 563-00700 T567P 563-16000 R566K/T567A 489-00080 K490T/S492T/A493G/D494Q 489-00700 S492L/A493D/D494Q 495-00009 Y500F 499-00080 E499N/Y500F/S501A/G504A 501-00700 S501A 501-16000 S501T/T506S 503-00009 T503P/T506S/K507S/Y508W 505-00009 T506S/K507S/Y508W/H509A 507-00009 K507S/Y508W/H509A 509-00009 H509A/D514N 515-16000 L516I/V517I 531-00009 E531D/K532R/Q536L 531-00700 E531D/K532R/Q536S 531-16000 E531D/Q536M 533-00080 Q536S/S537N 561-00009 D561N/R566K 623-00789 H627N 635-00009 L639M 651-00089 N656D 653-16700 S658P 711-00080 T713A/D715N 713-00089 T713A/D715N/N717E 717-16000 V719L/S721T 489-00009 K490T/S492V/A493T/D494Q 489-16000 S492K/A493T 515-00009 V517M 537-00080 S537N/V539I 355-00009 Q359E 367-16789 V372I 377-00009 N382D 381-00700 A386S 405-00089 T410Q 405-00700 T410E 407-00080 T410Q/S412T 409-00009 T410Q/T414E 409-00700 T410E/T414S 411-00009 T414E 411-00080 S412T 411-00700 T414S 413-00009 T414E/D417N 413-10000 D417E 415-00009 D417N 513-00009 D514N/V517M 513-00080 D514N/V517A 519-00080 P521I 519-16000 P521T 521-00080 P521I/S525T 521-00700 P521V/S525T 535-00009 Q536L/V539S 535-00080 Q536S/S537N/V539I 537-00009 V539S 539-00080 V539I 541-00080 G546N 541-00700 Q545T 541-16000 Q545E/G546S 547-00080 S547A/E548A/K549R/T550D/V552A 547-00700 S547A/E548T/K549/T550N/N551K/V552T 547-16000 S547A/E548G/K549A/T550S/V552T 553-00080 I554Y/E555S/K556D 553-00700 D553T/I554L/K556N/M558L 559-00700 I559M/D561N 567-16000 T567A 603-00780 D608N 637-10000 H641N 653-00089 N656D/S658P 655-00009 N656D/S658P/T660A 655-16000 S658P/T659A/T660E 657-00009 S658P/T660A/S662N 657-00700 S658P/T659E/T660V/S662T 659-00009 T660A/S662N/A663K/A664D 659-00080 S662N/A663Q/A664S 659-00700 T659E/T660V/S662T/A663P 659-16000 T659A/T660E/A664T 661-00080 S662N/A663Q/A664S/F666L 661-00700 S662T/A663P 661-16000 A664T 663-00009 A663K/A664D/F666L/A667N 663-00080 A663Q/A664S/F666L/A667N 663-00700 A663P 665-00089 F666L/A667N 667-00089 A667N 693-16000 I698V 699-00700 Y704F 701-00089 N705Y 701-00700 Y704F/N705E 701-16000 N705A 703-00009 N705Y/V708N 703-00080 N705Y/V708T 703-00700 Y704F/N705E/S707Q/V708T 703-16000 N705A/V708A 705-00080 N705Y/V708T/N709S 705-00700 N705E/S707Q/V708T/N709G 707-00009 V708N/D711E 707-00700 S707QA/708T/N709G 709-00009 D711E/T713A 709-00700 N709G/T713A 711-00009 D711E/T713A/D715N 711-00700 T713A/T716S 711-16000 T716N 713-00700 T713A/T716S/N717Q 715-00089 D715N/N717E 715-00700 T716S/N717Q 715-16000 T716NA/V719L 717-00089 N717E 717-00700 N717Q 729-16000 N734P 009-16789 T14N 019-16700 Q21E/K24D 025-16789 P29A 035-00009 A35N/E36Q/R37Q/K39Q 037-00700 R37Q/H38K/K39Q/D41N/S42G 063-16789 E67A 101-16780 K105Q 131-00009 P135A/V136A 137-00700 G141A 161-00780 A162K 161-16000 A162T 185-16780 Q190E 193-00089 L198V 195-00009 L198V/T200S 197-00009 L198V/T200S/N201L 197-00700 G197S/L198V/T200S/N201G 149-00780 V151Q/E152R/153S 149-16009 V151Q 155-00009 S157A/T159I 155-00780 S157T/T159I 017-16789 Q21E 019-00089 Q21E/K24A 023-00089 K24A 027-00009 P29A/P31Q 027-16780 P29A/P31K 029-00009 P29A/P31Q/P34A 031-00009 P31Q/P34A/A35N/E36Q 031-16780 P31K/P34A/A35N/E36Q 033-00009 P34A/A35N/E36Q/R37Q 033-16780 P34A/A35N/E36Q/R37Q/H38K 035-16780 A35N/E36Q/R37Q/H38K/K39Q 037-16080 R37Q/H38K/K39Q/D41N/S42G 039-00700 K39Q/D41N/S42G 039-16080 K39Q/S42G 041-00700 D41N/S42G 041-16080 S42G 051-00009 F56G 077-16789 R81Q 081-16709 R81Q/D84K/S85A 085-16789 S85A 087-16780 K92R 121-00009 V125L 125-06000 L129F 131-16780 P135GA/136A 143-00780 H148P 143-16009 H148Q 147-16009 H148Q/V151Q 159-00009 T159I/A162S/Q164A 161-00009 A162S/Q164A 163-00009 Q164A/R168K 163-16000 R168K 175-16780 A179S/D180E 183-00009 L188I 185-00009 L188I/Q190E 193-00700 G197S/L198V 195-00080 L198V/T200P 195-16000 S196A/G197A/L198V/T200P 197-16000 G197A/L198V/T200P/N201T 199-00009 T200S/N201L 199-00700 T200S/N201G/M203V 201-00080 T205A 201-00700 N201G/M203V/T205A 201-16000 N201T/T205S 203-16009 T205S/S207G 207-00009 S207G/M211V 209-00009 M211V 219-00089 N223S 229-00009 T233Q 231-00009 T233Q/M235L 231-16780 M235L 257-00009 262N 257-00080 S262N 257-00700 262E 259-00700 S262E/Q263T/S264A 261-16000 Q263A/265T 265-00009 A266S 267-00009 H271A 267-00780 S267T/H271T 269-00780 H271T 305-00700 R310K 307-00080 N312S 307-00700 R310K/N312R 311-00700 N312R 323-00009 Q325D/D327N 323-00080 D327E 325-00009 Q325D/D327N/T329V/T330K 325-00080 D327E/T330K 325-16700 Q325T/T329V 327-00009 D327N/T329V/T330K 329-00009 T329V/T330K 329-00080 T330K 339-16000 T344S 343-00009 E347D 511-16000 D514E/L516I 553-16000 D553A/I554L/E555D/K556N 655-00700 S658P/T659E/T660V 657-00080 S658P/S662N 661-00009 S662N/A663K/A664D/F666L 707-00080 V708T/N709S 707-16000 V708A 709-00080 N709S/T713A 517-00080 V517A/P521I 517-00700 P521V 517-16000 V517I/P521T 721-16000 S721T 155-16000 T159I 157-00009 S157A/T159I/A162S 157-16000 T159I/A162T 023-16700 K24D 029-16780 P29A/P31K/P34A 037-00009 R37Q/K39Q/D41N/S42A 039-00009 K39Q/D41N/S42A 041-00009 D41N/S42A 079-00080 R81Q/D84Q 079-16709 R81Q/D84K 081-00080 R81Q/D84Q/S85A 083-16709 D84K/S85A 147-00780 H148P/V151Q/E152R/153S 175-00009 A179T/D180E 189-16000 Q190E/A194T 191-16000 A194T/S196A 193-16000 A194T/S196A/G197A/L198V 195-00700 G197S/L198V/T200S 199-00080 T200P 199-16000 T200P/N201T 201-00009 N201L/T205S 203-00080 T205A/S207G 207-16780 S207G 219-16700 S224A 259-00009 262N/Q263T 259-16000 Q263A 261-00009 262N/Q263T/A266S 321-00009 Q325D 335-00780 V340I 339-00700 V340I/T344S 083-00080 D84Q/S85A 159-00780 T159I/A162K 203-00700 M203V/T205A/S207G 259-00080 S262N/263G/Q264T 261-00700 S262E/Q263T/S264A/A266S 263-00009 Q263T/265G/A266S 263-00080 Q263T/267G/S270T 263-00700 Q263T/S264A/A266S/S267T 265-00700 A266S/S267T 321-16700 Q325T 261-00080 S262N/263G/Q264T/267G 265-00080 S267T 327-16700 T329V 487-00009 K490T/S492V 487-00700 S492L 497-00780 E499N/Y500F/S501A 153-00009 S157A 153-00780 S157T 157-00780 S157T/T159I/A162K .sup.1The following system is used to name the hexapeptide scanning AAV2R585E mutants. The left three digits indicate the first amino acid position of the hexapeptide based on AAV2 VP1. The right five digits indicate AAV serotype from which each hexapeptide is derived: 10000, AAV1; 06000, AAV6; 00700, AAV7; 00080, AAV8; and 00009, AAV9. When a hexapeptide amino acid sequence is shared with multiple serotypes, the right five digits have more than one positive integer. .sup.2Alternative names used in Adachi K et al., Nat Commun 5, 3075 (2014). .sup.3Alternative names used in Asokan et al., Nature Biotechnology 28, 79-83 (2010)

EXAMPLES

(50) The following examples are illustrative of disclosed methods. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other examples of the disclosed method would be possible without undue experimentation.

Example 1—Development of an In Vitro IP-Based AAV Barcode-Seq Method to Identify Anti-AAV Antibody Epitopes

(51) IP conditions were optimized using a recombinant AAV9 vector, anti-AAV9 mouse sera, and protein A/G agarose beads. Then, a DNA-barcoded AAV HP scanning capsid mutant library comprising 78 AAV clones was produced, which included nineteen AAV2R585E-derived HP mutants and five AAV9-derived HP mutants that spanned a 14-amino-acid region in and flanking the vicinity of the highest peak of the AAV1, AAV2, AAV7, AAV8, and AAV9 capsids. Using this library and anti-AAV1, anti-AAV2, anti-AAV7, anti-AAV8, anti-AAV9, and naive mouse sera collected from four mice per serotype/native, IP was performed and AAV library genomes were recovered from both immunoprecipitates and supernatants, which were then subjected to the AAV Barcode-Seq analysis. As a result, it was found that mutants harboring AAV1-, AAV2-, AAV7-, AAV8-, and AAV9-derived peptides spanning eight amino acids within the vicinity of the highest peak, but not other mutants or the control AAV2R585E or AAV9, were clearly captured by the corresponding anti-AAV serotype sera of some, if not all, of the immunized mice, indicating that the identified peptides constitute a dominant epitope. Thus, these results demonstrated that a combination of AAV Barcode-Seq with IP can be a substantially easy and effective approach to map anti-AAV antibody epitopes in a high-throughput manner.

Example 2—Generation of an HP Scanning AAV Mutant Library Covering the Entire AAV2 Region

(52) Similar to Example 1, 153 HP scanning AAV9 mutants that cover the entire region of AAV2 can be created.

Example 3—Generation of a dsAAV9-HP-U6-VBCLib-2 Library Containing a Total of 338 AAV Clones

(53) To map anti-AAV2 antibody epitopes, a total of 153 AAV helper plasmids expressing the AAV2 Rep protein and various AAV9 capsid mutant proteins each of which contained a different hexapeptide region derived from the AAV2 capsid (AAV9-HP scanning mutants) were constructed. Using these AAV helper plasmids, a DNA/RNA-barcoded dsAAV-U6-VBCLib library packaged with the AAV9-HP scanning mutants was produced. This library, termed dsAAV9-HP-U6-VBCLib-2, contained all the AAV9-HP mutants listed in Table 2 (2 clones per mutant). It also contained AAV2 (2 clones) and the two reference controls, AAV2R585E and AAV9 (15 clones each). The titer of this library was 2.8×10.sup.13 vector genomes (vg)/ml.

(54) TABLE-US-00002 TABLE 2 Hexapeptide scanning AAV9-derived mutants Name of mutant.sup.4 Amino acid substitutions 009-00002 N14T 017-00002 E21Q 019-00002 E21Q/A24K 023-00002 A24K 025-00002 A29P 027-00002 A29P/Q31P 029-00002 A29P/Q31P/A34P 031-00002 Q31P/A34P/N35A/Q36E 033-00002 A34P/N35A/Q36E/Q37R 035-00002 N35A/Q36E/Q37R/Q39K 037-00002 Q37R/Q39K/N41D/A42S 039-00002 Q39K/N41D/A42S 041-00002 N41D/A42S 051-00002 G56F 063-00002 A67E 077-00002 Q81R 079-00002 Q81R/K84D 081-00002 Q81R/K84D/A85S 083-00002 K84D/A85S 085-00002 A85S 121-00002 L125V 131-00002 A135P/A136V 143-00002 Q148H 147-00002 Q148H/Q151V 149-00002 Q151V 153-00002 A157S 155-00002 A157S/I159T 157-00002 A157S/I159T/S162A 159-00002 I159T/S162A/A164Q 161-00002 S162A/A164Q 163-00002 A164Q/K168R 165-00002 K168R 175-00002 T179A/E180D 183-00002 I188L 185-00002 I188L/E190Q 189-00002 E190Q 193-00002 V198L 195-00002 V198L/S200T 197-00002 V198L/S200T/L201N 199-00002 S200T/L201N 201-00002 L201N/S205T 203-00002 S205T/G207S 207-00002 G207SA/211M 209-00002 V211M 219-00002 S223N 229-00002 Q233T 231-00002 Q233T/L235M 235-00002 L235M 257-00002 N262S/S263 259-00002 N262S/S263/T264Q 261-00002 N262S/S263/T264Q/G267/G268A 264-00002 T264Q/G267/S268A 267-00002 S268A 269-00002 A273H 323-00002 D327Q 325-00002 D327Q/N329D 327-00002 D327Q/N329D/V331T/K332T 329-00002 N329D/V331T/K332T 331-00002 V331T/K332T 345-00002 D349E 357-00002 E361Q 369-00002 I374V 379-00002 D384N 407-00002 Q412T 411-00002 Q412T/E416T 413-00002 E416T 415-00002 E416T/N419D 417-00002 N419D 445-00002 K449R 447-00002 K449R/I451N/N452T 449-00002 K449R/I451N/N452T/G453PS 451-00002 1451N/N452T/G453PS/S454G/G455T 453-00002 G453PS/S454G/G455T/Q456T/N457T 454-00002 S454G/G455T/Q456T/N457T/Q459S 456-00002 Q456T/N457T/Q459S/T460R 458-00002 Q459S/T460R/K462Q 460-00002 T460R/K462Q/V465Q 462-00002 K462Q/V465Q 464-00002 V465Q/P468A 466-00002 P468A/N470D/M471I 468-00002 P468A/N470D/M471I/A472R/V473D 470-00002 N470D/M471I/A472R/V473D/G475S 472-00002 A472R/V473D/G475S 474-00002 G475S/Y478W/I479L 476-00002 Y478W/I479L 478-00002 Y478W/I479L/S483C 480-00002 S483C 486-00002 T491K 488-00002 T491K/V493S 490-00002 T491K/V493S/T494A/Q495D 492-00002 V493S/T494A/Q495D 494-00002 T494A/Q495D 496-00002 F501Y 498-00002 F501Y/A502S 500-00002 F501Y/A502S/P504T 502-00002 A502S/P504T/S507T 504-00002 P504T/S507T/S508K/W509Y 506-00002 S507T/S508K/W509Y/A510H 508-00002 S508K/W509Y/A510H 510-00002 A510H/N515D 512-00002 N515D 514-00002 N515D/M518V 516-00002 M518V 524-00002 E529D 526-00002 E529D/G530D 528-00002 E529D/G530D/D532E/R533K 530-00002 G530D/D532E/R533K 532-00002 D532E/R533K/L537Q 534-00002 L537Q 536-00002 L537Q/S540V 538-00002 S540V 544-00002 T548S/G549E 546-00002 T548S/G549E/R550K/D551T 550-00002 R550K/D551T/A555I 552-00002 A555I/D556E 556-00002 D556E 558-00002 N562D 562-00002 N562D/K567R 564-00002 K567R 572-00002 S576Q 574-00002 S576Q/Q579S 576-00002 S576Q/Q579S/A581S 578-00002 Q579S/A581S 580-00002 A581S/H584L 582-00002 H584L/S586R/A587G 584-00002 H584L/S586R/A587G/Q588N/A589R 586-00002 S586R/A587G/Q588N/A589R 588-00002 Q588N/A589R/Q592A 590-00002 Q592A/G594A/W595D 592-00002 Q592A/G594A/W595D/Q597N 594-00002 G594A/W595D/Q597N/N598T 596-00002 Q597N/N598T/I601V 598-00002 N598T/I601V 600-00002 I601V 624-00002 N628H 636-00002 M640L 652-00002 D657N 654-00002 D657N/P659S 656-00002 D657N/P659S/A661T 658-00002 P659S/A661T/N663S 660-00002 A661T/N663S/K664A/D665A 662-00002 N663S/K664A/D665A/L667F 664-00002 K664A/D665A/L667F/N668A 666-00002 L667F/N668A 668-00002 N668A 702-00002 Y706N 704-00002 Y706N/N709V 708-00002 N709V/E712D 710-00002 E712D/A714T 712-00002 E712D/A714T/N716D 714-00002 A714T/N716D/E718N 716-00002 N716D/E718N 718-00002 E718N .sup.4The following system is used to name the hexapeptide scanning AAV9 mutants. The left three digits indicate the first amino acid position of the hexapeptide based on AAV9 VP1. The right five digits indicate AAV serotype from which each hexapeptide is derived: 10000, AAV1; 06000, AAV6; 00700, AAV7; 00080, AAV8; and 00009, AAV9; and 00002, AAV2. When a hexapeptide amino acid sequence is shared with multiple serotypes, the right five digits have more than one positive integer.

Example 4—Optimization of the IP-Seg Procedure Using Protein A/G Magnetic Beads

(55) In preliminary IP-Seg experiments, a traditional protein A/G agarose beads-based method for immunoprecipitation of anti-AAV capsid antibody-binding AAV particles was used. In this set of experiments, the IP procedure was optimized using magnetic beads, which have become more favorable than agarose beads in various aspects such as easy handling and faster rate of binding. During the course of the optimization using AAV2 particles and Pierce Protein A/G Magnetic Beads (Thermo Scientific, Product No. 88804), it was found that a significant fraction of input AAV2 viral particles in the IP reaction tubes can bind nonspecifically to the magnetic beads. To prevent this nonspecific binding, a series of blocking reagents was tested including 1%, 2%, 4%, and 8% bovine serum albumin (BSA, Sigma, A3294-500G) in PBS (BioWhittaker, 17-516F) and ethanolamine (Sigma-Aldrich, E0135)/glycine (Sigma Life Science, G8898-1KG) solution. The ethanolamine/glycine solution was prepared with 50 mM Tris, 200 mM glycine, 1% Tween-20 (Sigma, P5927), 200 mM ethanolamine, pH 10.6. As a result, it was found that 2% BSA in PBS yielded the best blocking efficiency. Since buffer stringency could affect the IP procedure, low stringency buffer (PBS), medium stringency buffer (1% Triton X-100 (Sigma, T8532) in TBS, pH 7.4) and high stringency buffer (RIPA buffer) in the presence of 2% BSA was tested. It was found that low stringency IP buffer (PBS) had the lowest level of AAV particles nonspecifically bound to magnetic beads. Therefore, the subsequent experiments were done using 2% BSA in PBS as the IP buffer for IP-Seq. Various combinations of temperature and incubation time were compared at each step (at 37° C. for 1 hour vs. at 4° C. overnight), and no significant difference was found. Based on these observations in the optimization experiments, the IP-Seq procedure was established as follows:

(56) (1) Wash 0.20 mg (20 μL) of Pierce Protein A/G Magnetic Beads (Thermo Scientific, Product No. 88804) with 1 mL PBS.

(57) (2) Incubate with rotation the washed Pierce Protein A/G Magnetic Beads and an anti-AAV antibody-containing sample in 500 μL PBS at 37° C. for 1 hour. In the experiments described here, the antibody-containing samples were either mouse monoclonal A20 antibody (the antibody against intact AAV2 particles, 500 ng (10 μL) per IP reaction) or sera from the mice immunized with intravenous injection of 1×10.sup.11 vg of AAV2-CMV-lacZ (20 μL per IP reaction). However, any samples containing anti-AAV antibody including anti-AAV antibody-positive human sera can be analyzed using the IP-Seq method described herein.

(58) (3) Discard the PBS containing the sample.

(59) (4) Block nonspecific binding by incubating the magnetic beads with 500 μL of PBS containing 2% BSA at 37° C. for 1 hour.

(60) (5) Discard the blocking buffer.

(61) (6) Incubate the BSA-treated magnetic beads with 1×10.sup.9 vg of a DNA/RNA-barcoded dsAAV-U6-VBCLib library in 350 μL of PBS containing 2% BSA at 37° C. for 1 hour. The amount of input viral particles can be in a range from 5×10.sup.7 vg to 1×10.sup.9 vg.

(62) (6) Save the supernatant for the AAV Barcode-Seq analysis.

(63) (7) Wash the magnetic beads with 500 μL of PBS twice.

(64) (8) Extract DNA from the supernatant and the magnetic beads with Proteinase K treatment (Proteinase K from Ambion) and Wako DNA Extractor Kit (Wako Chemicals, Richmond, USA).

(65) (9) Resuspend the dried DNA pellets in 10-20 μL of TE.

(66) (10) Amplify virus DNA barcodes using 1/10 of the above-described DNA preparation.

(67) (11) Combine PCR products and subject them to Illumina sequencing.

Example 5—Epitope Mapping of a Mouse Monoclonal Antibody Against Intact AAV2 Capsid

(68) A20 may be the most widely used, commercially available mouse monoclonal antibody against intact AAV2 capsid. This antibody is available from American Research Product Inc. (Catalog No. 03-61055). In order to map A20 antibody epitopes on the AAV2 capsid, IP was performed using 500 ng of A20 antibody and 1×10.sup.9 vg of dsAAV9-HP-U6-VBCLib-2. Viral DNA recovered from the IP supernatant and magnetic beads were subjected to the AAV Barcode-Seq analysis. In brief, Pierce Protein A/G Magnetic Beads were first coated with the A20 antibody at 37° C. for 1 hour, blocked with PBS/2% BSA at 37° C. for 1 hour, and then reacted with 1×10.sup.9 vg of dsAAV9-HP-U6-VBCLib-2 at 37° C. for 1 hour. This library contained 338 AAV clones composed of 153 AAV9-HP mutants, AAV2 and two reference controls (AAV2R585E and wild-type AAV9, 15 clones each). These AAV9-HP mutants were created to identify anti-AAV2 antibody epitopes by scanning the entire AAV2 capsid region with a set of AAV2 capsid protein-derived hexapeptides. Two of the 153 AAV9-HP mutants, 584-00002 and 586-00002 (see Table 2), could not be produced at levels sufficient for the downstream analysis; therefore, they are not included in the dataset. As expected, AAV2 and AAV2R585E bound to A20 efficiently, resulting in substantial enrichment and reduction of AAV2 and AAV2R585E viral genomes in the IP fraction and the supernatant, respectively (see FIG. 9). There is a clear peak on 261-00002 showing more than 30-fold enrichment in the IP fraction (see FIG. 9). This mutant carries 261-SSQSGA-266 (SEQ ID NO:50) of AAV2 capsid in place of 261-SNSTSGGS-268 (SEQ ID NO:51) of AAV9 capsid; therefore, 261-SSQSGA-266 (SEQ ID NO:50) should include an A20 antibody epitope. This finding is in keeping with the previous cryo-electron microscopy study showing that S261, Q263, and S264 are among the amino acids found in the A20 binding footprint (McCraw DM et al., Virology 431 (1-2), 40-49 (2012)). No other epitopes were identified by this approach.

Example 6—Epitope Mapping of Mouse Polyclonal Antibodies Against AAV2 Capsid

(69) The same magnet beads-based IP-Seg analysis for epitope mapping was applied to anti-AAV2 antibody-positive sera collected from 4 C57BL/6 male mice. The serum samples used for this analysis were the same as those used for the data presented in FIG. 6, for which a traditional agarose beads-based immunoprecipitation was used. Briefly, 8-week-old C57BL/6 male mice (Mouse 1, 2, 3 and 4) were injected intravenously with AAV2-CMV-lacZ vector at a dose of 1×10.sup.11 vg/mouse. Serum samples containing anti-AAV2 neutralizing antibodies were collected 3 weeks post-injection. 20 μL of each serum sample was then subjected to the magnetic beads-based IP-Seg analysis for epitope mapping using the dsAAV9-HP-U6-VBCLib-2 library, as described above. In the preliminary agarose beads-based IP-Seg analysis using the dsAAV-HP-U6-VBCLib-1 that contained only 5 AAV9-HP mutants, 451-PSGTTT-456 (SEQ ID NO:3) was identified as an epitope of polyclonal anti-AAV2 antibodies developed in Mouse 2 (see FIG. 6). This was reproduced in this new IP-Seg procedure (see FIG. 10B). In addition, the absence of this epitope in Mouse 3 and 4 (see FIGS. 10C and 10D), and a weak reactivity to this epitope in Mouse 1 (see FIG. 10A) was also reproduced. By scanning the entire AAV2 capsid region with hexapeptides, a dominant epitope that was found in all the mice could be identified, 513-RDSLVNPG-520 (SEQ ID NO:52) of the AAV2 capsid, based on the observation that there is a peak at 514-00002 (513-RDSLVN-518 (SEQ ID NO:53)) and 516-00002 (515-SLVNPG-520 (SEQ ID NO:54)) (see FIGS. 10A-D). Other epitopes, 325-QNDGTT-330 (SEQ ID NO:55) (based on a peak at 327-00002 in Mouse 3, see FIG. 10C) and 261-SSQSGA-266 (SEQ ID NO:50) (based on a peak at 261-00002 in Mouse 4, see FIG. 10D) could also be identified. The latter epitope is the same as that for the A20 mouse monoclonal antibody and a modest peak at this position was also found in Mouse 3 (see FIG. 10C). Moreover, modest peaks were also found at 486-00002 and 588-00002 in Mouse 2 (see FIG. 10B), indicating that 485-QQRVSK-490 (SEQ ID NO:56) and 587-NRQAAT-592 (SEQ ID NO:57) are epitopes.

Example 7—Development of Anti-AAV Neutralizing Antibody-Escaping AAV Capsid Mutants

(70) The IP-Seg analysis of anti-AAV antibody-positive mouse sera has revealed that 513-RDSLVNPG-520 (SEQ ID NO:52) may be the most dominant epitope for anti-AAV2 antibodies. The RDSLVNPG (SEQ ID NO:52) is an evolutionarily conserved region across different AAV serotypes and variants, and therefore this region may likely be the dominant epitope for anti-AAV antibodies. In addition, this study indicated that the same topological region around 453-456 is found to be a common epitope across different AAV strains; i.e., 452-QSGSAQNK-459 (SEQ ID NO:5) in the AAV1 capsid, 451-PSGTTT-456 (SEQ ID NO:3) in the AAV2 capsid, 453-NPGGTAG-459 (SEQ ID NO:6) in the AAV7 capsid and 453-GCGQN-457 (SEQ ID NO:58) in the AAV9 capsid. Thus, introduction of amino acid mutations in the RDSLVNPG (SEQ ID NO:52)-corresponding regions and/or in the vicinity of the 453-456 region, or swapping the amino acids in these regions, may offer an effective approach to develop anti-AAV neutralizing antibody-escaping AAV mutants. In addition, other epitope motifs that have been identified so far and that may be identified using the method described herein may be the targets for capsid mutagenesis aimed at creating novel anti-AAV neutralizing antibody-escaping AAV capsid mutants.

(71) It will be apparent to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.