WO2012031760A1 - Parvovirus mutated structural proteins comprising cross - protective b - cell epitopes of a hpv l2 protein as well as products and methods relating thereto - Google Patents

Parvovirus mutated structural proteins comprising cross - protective b - cell epitopes of a hpv l2 protein as well as products and methods relating thereto Download PDF

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WO2012031760A1
WO2012031760A1 PCT/EP2011/004528 EP2011004528W WO2012031760A1 WO 2012031760 A1 WO2012031760 A1 WO 2012031760A1 EP 2011004528 W EP2011004528 W EP 2011004528W WO 2012031760 A1 WO2012031760 A1 WO 2012031760A1
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hpv
preferably
protein
aav
l2
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John Nieland
Markus HÖRER
Mirko Ritter
Florian Sonntag
Jürgen KLEINSCHMIDT
Kerstin Lux
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Medigene Ag
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/58Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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    • C12N2750/14134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The present invention relates to parvovirus mutated structural proteins comprising insertions of cross-protective B-cell epitopes of an HPV L2 protein, multimeric structures, nucleic acids, cells, compositions and vaccines relating to such proteins, as well as their manufacture.

Description

PARVOVIRUS MUTATED STRUCTURAL PROTEINS COMPRISING CROSS - PROTECTIVE B - CELL EPITOPES OF A HPV L2 PROTEIN AS WELL AS PRODUCTS AND METHODS RELATING THERETO

The present invention relates to parvovirus mutated structural proteins comprising insertions of cross- protective B-cell epitopes of an HPV L2 protein, multimeric structures, nucleic acids, cells, compositions and vaccines relating to such proteins, as well as their manufacture.

Cervical cancer is the second most common cancer of women worldwide. Persistent high-risk human papillomavirus (HPV) infection has been identified as a necessary event for the development of this type of cancer. Fifteen of more than 120 different HPV types described so far are involved in the development of cancer of the cervix or other anogenital cancers.

Oncogenic HPV infection causes approx. 5% of all cancer deaths globally. Its impact is greatest for women who are currently not reached by effective cervical cancer screening programs (e.g. Pap testing) because approx. 80% of cervical cancer occur in low-resource settings in the developing world, and this malignancy accounts for the vast majority of cancer-related deaths attributable to HPV infection (Parkin, 2006, Parkin and Bray, 2006). Attempts to prevent infection with the most prevalent HPV genotypes - HPV 16 and HPV 18 - led to the development of two prophylactic vaccines; Gardasil™ targeting HPV 6, 1 1 , 16 and 18 and Cervarix™ targeting HPV 16 and 18. They induce neutralizing antibodies by immunization with virus-like particles (VLPs) assembled from the major capsid protein LI of the individual HPV genotypes. However, the use of these vaccines is highly controversial as these commercially available vaccines protect only against the HPV type from which the LI peptide is derived - although there is some evidence for partial cross- protection against HPV types 31 and 45. Accordingly, it is unclear whether other genotypes will step in for HPV 16 and 18 resulting in only limited success in the long term in avoiding cervical cancer.

Further, Gardasil™ and Cervarix™ are too expensive for population-wide implementation for those patients that have no access to screening. Furthermore, nearly one third of cervical cancer is caused by oncogenic HPV types not included in current HPV vaccines, which means that screening is to be maintained. Therefore, vaccination costs add upon the screening costs and do not relieve the health system from screening costs. Accordingly, it is still an unsolved problem to develop an affordable and effective HPV vaccine, which means that the HPV vaccine must protect against a wide range of malignant genotypes and needs to be simple in manufacturing. Additionally, manufacture of present vaccines is expensive as VLPs for present genotypes are individually manufactured and then mixed. Whereas VLPs have proven to be a very useful vaccine basis, this concept in the end limits the possibility to include further genotypes due to increasing costs for vaccinations. Therefore, HPV VLP-based vaccines are not seen as the solution to generate better be 5 cross-protective vaccines against a wide range of HPV types.

Further, present vaccines require refrigeration for storage and transport. As most of the cancer patients suffering from cervical cancer are found in the developing world, future vaccines should therefore be stable at room temperature, meaning that they can be transported and/or stored at room temperature, 10 ideally as a dry powder. Preferably this includes that such vaccines even are stable for a short time (e.g.

6 hours) at high temperatures such as 50°C (stress conditions).

There are a number of proposed second generation vaccines that address some of the above problems. Whereas peptides of the HPV L2 protein can induce antibodies that neutralize a broad range of HPV 15 genotypes, L2 is antigenically subdominant to LI in the virus capsid (Pastrana et al., 2005). Still, cross- neutralizing epitopes were identified (Roden et al., 2000) that can potentially be used in different kinds of vaccination approaches using the epitopes in a different context or carrier.

Slupetzky et al. (2007) describe the generation of two different vaccine constructs, where HPV 16 L2 69- 20 81 or HPV 16 L2 108-120 are inserted within an immunodominat surface loop of HPV LI of bovine papillomavirus type 1. Whereas L2 69-81 was partially neuralizing, 108-120 failed to do so. Also, the authors report that serum titers were still low compared to anti-Ll titers obtained with VLP vaccines (page 2010, second paragraph).

25 Alphs et al. (2008) describe a synthetic lipopeptide vaccine containing HPV 16 L2 17-36. Whereas neutralizing antibodies were obtained also for types other than HPV 16 titers were rather low (e.g. 1 10 for HPV45) making it unlikely that a long-lasting protection can be achieved by this approach (as discussed for higher titers by Jagu et al., see below).

30 Jagu et al. (2009) disclose a systematic approach to identify cross-reacting L2 epitopes. Concatenated multitype HPV L2 fusion proteins have been used in vaccination protocols in rabbits and mice. However, the authors themselves conclude that weaker immune responses to multitype L2 vaccines as compared to LI VLPs raise concerns about the longevity of the response and the (unsolved) problem of a stronger adjuvant. Further, these titers were only achieved by the use of Freund's adjuvant, a strong

35 adjuvant that is not practicable for clinical use (Alphs et al., 2008, page 5851 , left column, last paragraph). Further, rabbits were vaccinated 5 times - a frequency which is most likely not suitable for clinical use (page 784, left column, third paragraph). Caldeira Jdo et al. (2010) describe the generation of PP7 VLP based vaccine against a broadly cross- neutralizing epitope from HPV L2 epitope of HPV16, 17-31 (QLYKTCKQAG TCPPD; SEQ ID NO: l ). However, it is generally not preferred to use highly immunogenic scaffolds such as bacterial proteins or phages in humans, as this might result in sporadic, but highly problematic post-vaccine autoimmune reactions in previously healthy susceptible individuals (Salemi and D'Amelio, 2010). Additionally, it is generally a technical problem to purify bacterially expressed proteins from endotoxins (Lopes et al., 2010). Rubio et al. (2009) have used bacterial thioredoxin as a scaffold to insert HPV 16 L2 peptides. The authors were able to show a robust immunogenicity to all tested peptides and induced strong neutralizing as well as cross-neutralizing responses. Besides the general concerns for using bacterial proteins for vaccination of humans the authors point to the problem that cross-reactivity to HPV31 was weak. These constructs also were tested in combination with Freund's adjuvant, which is not practicable for clinic use (see above).

Although the above discussed approaches are important advances in the field, there is still no solution to problems of sufficient protections against a wide range of (malignant) HPV genotypes, strong, long- lasting immune responses without adjuvants or in combination with an adjuvant practicable for clinical use, cost-effectiveness, transport and/or storage at room temperature, avoidance of endotoxins and minimizing the potential induction of auto-immune diseases. Accordingly, it was an object of the present invention to provide an HPV vaccine that preferably overcomes one or more of the above disadvantages, namely in that it is cross-protective, induces sufficient cross-protective antibody responses alone or in combination with an adjuvant practicable for clinical use, is stable at room temperature, minimizes the induction of auto-immune diseases and/or is affordable for developing world countries.

The object is solved by providing parvoviral mutated structural proteins which comprises an insertion containing at least one cross-protective B-cell epitope of a human papillomavirus (HPV) L2 protein, wherein the parvovirus structural protein is capable of forming a multimeric structure, and wherein the B-cell epitope is located at the surface of the protein. Surprisingly, these multimeric structures are able to induce high titers of cross-protective antibodies, they are based on a backbone structure of a virus that humans are familiar with (most humans are infected with the AAV2 which is not considered to have any known role in disease), manufacture of only one VLP is cost efficient and they are stable and therefore suitable also for countries of the developing world.

We have previously described a VLP platform based on adenovirus-associated virus (AAV), more specifically on the human serotype AAV2 (WO 2008145401 ). This platform has been successfully used to display known epitopes on the surface of the VLPs made from VPl, VP2 and VP3 proteins and generate robust immune responses without Freund's adjuvants. We were now able to adapt this platform to HPV L2 epitopes as follows. Detailed description of the invention

The following definitions explain how the defined terms are to be interpreted in the context of the products, methods and uses of the present invention.

"AA" is used as abbreviation for amino acid(s), "nt" is used as abbreviation for nucleotide(s).

"AAVLP" is used as an abbreviation for virus-like particles derived from AAV. These can be generally composed of VPl , VP2 and/or VP3.

The term "about" means according to the invention a general error range of ± 20%, especially ± 10%, in particular ± 5%.

According to this invention "parvovirus" or "parvoviral" relates to a member of the family of Parvoviridae wherein the wildtype expresses VPl , VP2 and VP3 as capsid proteins. The family of Parvoviridae contains several genera divided between 2 subfamilies Parvovirinae (Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus) and Densovirinae (Densovirus, Iteravirus, Brevidensovirus, Pefudensovirus and Contravirus) (Fields: Virology, fourth edition 2001, Volume 2, chapters 69 and 70, Lippincott Williams Wilkins, Philadelphia; http://virus.stanford.edu/parvo/ parvovirus; http://www.ncbi.nlm.nih.gov/ICTVdb/ Ictv/fs _parvo.htm#SubFamilyl). The wildtype capsid is assembled of the three structural proteins VPl, VP2 and VP3 that form the 60 subunits of the AAV capsid in a ratio of 1 : 1 :8 (Kronenberg et al., 2001). Hence, the term "VP3" stands for virus protein 3. The naturally occurring parvoviral particle is composed of the icosahedral capsid that encloses the single stranded DNA genome. Preferred parvoviruses are the Dependoviruses, including AAV.

In the context of this invention the term "serotype" stands for the kind of virus of a group of closely related viruses distinguished by their characteristic set of antigens. Thus, the serotype is characterized by serologic typing (testing for recognizable antigens on the virus surface). Accordingly, the AAV can be selected from any serotype, particularly evolved from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1 1 to AAV 12 and AAV 13, in particular from AAV2. Parvoviral particles consisting "essentially of VP3" or "essentially only VP3" means that the capsid is assembled to at least 98%, preferably at least 99%, more preferably at least 99,6% and essentially at least 99,8% of VP3. This means that only 1/50, preferably 1/100, more preferably 1/250 and essentially only 1/500 or less of the proteins assembling the capsid are N-terminally extended versions of VP3 or completely different proteins. In a preferred embodiment the capsid is assembled to at least 98%, preferably at least 99%, more preferably at least 99.6% and essentially at least 99.8% of VP3, meaning that only 1/50, preferably 1/100, more preferably 1/250 and essentially only 1/500 or less of the proteins assembling the capsid are N-terminally extended versions of VP3 or different parvoviral proteins. It is especially preferred that the parvoviral capsid consists only of one type of protein according to this invention, which is VP3 in its wild type sequence or a mutated form of it as defined herein. Such parvoviral particles do not contain any of the functional Rep proteins, particularly Rep40, Rep52, Rep68 and Rep78. Preferably, such VP3 does not contain a heterologous nuclear localization signal.

"Extended versions of VP3" comprise in general N-terminal extensions by several AAs. These N- terminal extensions represent the 3 ' part of the sequence coding for VP2 but not for VP3, since the AAV capsid genes are encoded by overlapping sequences of the same ORF using different start. Thus, N- terminally extended VP3 is identical to N-terminally truncated VP2 meaning that parts of VP2 can be present within the N-terminal extension of VP3 but no complete and intact wildtype VP2 protein is expressed as e.g. given by Ruffing et al. (1994) and accessible from NCBI (number of entree: NC_001401 ). According to this invention the particles consist essentially of VP3 (as defined) and therefore extended versions of VP3 are very rare, whereas naturally occurring particles comprise VP1. VP2. VP3 in a ratio of 1 : 1 :8 (Kronenberg et al., 2001 ).

"Mutations" are changes to the nucleotide sequence of the genetic material of an organism. Such mutations may lead to a change of the encoded protein and therefore may have varying effects depending on where they occur and whether they alter the structure and/or function of the encoded protein. Structurally, mutations can be classified as point mutations, insertions adding one or more extra nt into the DNA/AA into the protein or deletions removing one or more nt/AA. An "insertion" of nt/AA is generally speaking an insertion of at least one heterologous nt/AA into the sequence of - for this invention - a parvovirus protein. 'Heterologous' in this context means heterologous as compared to the virus, from which the parvovirus protein is derived. Exemplified for a parvovirus structural protein, the inserted AAs can simply be inserted between two given AAs of the parvovirus structural protein. An insertion of AAs can also go along with a deletion of given AAs of the parvovirus structural protein at the site of insertion, leading to a complete substitution (e.g. 10 given AAs are substituted by 10 or more inserted AAs) or partial substitution (e.g. 10 given AAs are substituted by 8 inserted AAs) of AAs of the parvovirus structural protein. A "B-cell epitope" is the part of a macromolecule that is recognized by the immune system, specifically by antibodies or B-cells. A B-cell epitope can be both a linear AA sequence and a structural epitope being the surface of the macromolecule which can be build by a secondary structure of AAs or in combination with other organic substances.

"Cross-protective" with respect to HPV B-cell epitopes means that vaccination with one or more B-cell epitopes derived from one HPV genotype induces protective antibody titers in the vaccinated subject that protect the vaccinated subject against infection by a different HPV genotype. Assays for determining cross-protectiveness are described for example in Jagu et al. (2009), page 784 and Table 1, Rubio et al. (2009), page 1951 and Fig. 4. Within the present invention we speak of "cross-protective" if, with respect to a specific HPV genotype, in vitro neutralizing titers after vaccination with a Montanide ISA 51 (Seppic), Montanide ISA 720 (Seppic) or an alumn based adjuvant and preferably without an adjuvant of at least 500, preferably of at least 1.000, more preferably of at least 5.000, especially of at least 10.000 are reached. In one embodiment, cross-protective titers with Monatanide ISA 51 of at least 500, preferably of at least 1.000, more preferably of at least 5.000, especially of at least 10.000 are reached. In a further embodiment cross-protective titers with Monatanide ISA 720 of at least 500, preferably of at least 1.000, more preferably of at least 5.000, especially of at least 10.000 are reached. In a further embodiment cross-protective titers with an alumn based adjuvants of at least 500, preferably of at least 1.000, more preferably of at least 5.000, especially of at least 10.000 are reached.

A "Rep-independent promoter" is a promoter which can be activated in the absence of the Rep protein, whereas in the context of this invention Rep stands for the non-structural protein(s) encoded by a parvovirus, particularly Rep40, Rep52, Rep68 and Rep78 as described by Muzyczka and Berns (2001). These promoters include for example heterologous constitutive promoters and inducible promoters.

In a first aspect, this invention relates to a parvovirus mutated structural protein which comprises an insertion containing at least one cross-protective B-cell epitope of a human papillomavirus (HPV) L2 protein, wherein the parvovirus structural protein is capable of forming a multimeric structure, and wherein the B-cell epitope is located at the surface of the multimeric structure. The location of the inserted B-cell epitope on the surface of the multimeric structure formed by the parvoviral structural protein in combination with the presence of T-helper epitopes provided by the parvovirus mutated structural protein are efficiently recognized by B-cells and lead to the generation of antibodies. The fact that the at least one B-cell epitope is inserted into the surface of the multimeric structure in a defined way with a fixed structure is especially suitable for generating reproducible results.

The structural protein can be derived from adeno-associated virus (AAV), Goose parvovirus, Duck parvovirus, Snake parvovirus, feline panleukopenia virus, canine parvovirus, B19 or minute virus of mice (MVM) and may be mutated as described herein. Due to the high conservation of genome organization amongst the parvoviruses, the invention can easily be transferred to other parvovirus members. Preferred parvoviruses are those that share the general capsid assembly from viral proteins VP1, VP2 and VP3 and therefore enable VLP production from VP3 only according to this invention (VP3 AAVLPs). Presently known viruses of this subgroup are adeno-associated virus (AAV), Goose parvovirus, Duck parvovirus, and Snake parvovirus. Preferably AAV is selected from the group consisting of bovine AAV (b-AAV), canine AAV (CAAV), mouse AAV 1, caprine AAV, rat AAV, avian AAV (AAAV), AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1 1, AAV12, and AAV13, especially AAV2. The human immune system in general is well adapted to AAV2 capsid proteins as the largest fraction of the human population is infected with this virus that is not associated with any disease. Further, AAV2 as a gene therapy vector has been tested in large number of human patients and appeared not to be associated to immunological complications. Accordingly, compared to other backbones aiming at putting B-cell epitopes into a multimeric structure, AAV2 has the enormous advantage that the backbone itself, for most of the vaccinated humans, will not generate an unprecedented immune reaction that may cause autoimmune diseases in vaccinated humans. As papilloma viruses are also causing considerable problems in domestic animals, the skilled artisan can easily choose an appropriate parvovirus for specific domestic or farm animals e.g. chose the bovine AAV for HPV vaccines designated for cattle.

In one embodiment of the present invention the parvovirus mutated structural protein is VP3. Surprisingly, it has been found that multimeric structures useful as vaccines can be generated that are based upon multimeric structures consisting essentially of VP3. Whereas multimeric structure based vaccines containing VP1 , VP2 and VP3 have been previously described by us (WO 2008/145401), clinical development of vaccines based on multimeric structures is simplified for products based on a single active compound/protein and being as pure as possible. With respect to e.g. VLPs this is a problem in general as viruses are often composed of more than one protein and are capable of packaging specifically viral DNA or unspecifically DNA from the host cell. Accordingly, it is desirable to obtain "pure" VLPs that contain as few different proteins as possible and preferably no nucleic acid. Further, vaccines containing VP1 , VP2 and VP3 are generally produced in the presence of the parvoviral Rep protein. Rep does not only represent a further protein that is attached to VLPs but also is held responsible for packaging of virus genomes and unspecific DNA into preformed capsids (King et al., 2001 ). Packaging of DNA is to be avoided as VLPs potentially can enter cells of a patient and thereby transfect such contaminating DNA, which may cause all sorts of unwanted effects. We have surprisingly found that vaccines can be made consisting essentially of VP3 if VP3 is co-expressed with the novel protein AAP as described in (Sonntag et al., 2010) and in the examples herein. Whereas Hoque et al. (Hoque et al., 1999a, Hoque et al., 1999b) described particle formation by analyzing a series of deletion mutants of VP2 that started expression at different sites 5' of the VP3 start codon, they identified a region necessary for nuclear transfer of VP3 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of VLP formation correlated well. They observed that viral particles were formed as long as a region between amino acid 29 and 34 in the cds of VP2 or in other words in the 5' extension of VP3, was present. From the amino acid motif of this region which is PARKRL (SEQ ID NO: 2) they concluded that it functions as a nuclear localization signal 5 (NLS) which is important for the translocation of VP3 into the nucleus. However, due to the method for mutant construction used by them, all constructs started with an ATG start codon directly at the 5' end of the coding sequence. Since in general the "position effect" (Kozak, 2002) will cause the first (most upstream) ATG start codon of a transcript to initiate translation, the main protein to be expressed and generating the particle will be N-terminally extended VP3 - or N-terminally deleted VP2. Only a minor 10 part of translation (if at all) will start at the further downstream ATG start codon of VP3. Accordingly, Hoque et al. failed to produce particles consisting essentially of VP3. Alternatively, capsids also could be obtained if the NLS of simian virus 40 (SV40) large T antigen was fused to the N-terminus of the VP3 protein (NLSsv4Q-VP3). This fusion protein was described to form VLPs indicating that the VP2- specific region located on the N-terminal side of the protein is not structurally required. Due to this 15 finding the authors reasoned that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins, which again is a prerequisite for VLP

g

formation. However, we could not quantify capsid assembly in detectable amounts (>10 capsids/ml, see example 8) using the NLSsv4Q-VP3 fusion construct. Accordingly, the method of Hoque et al. is not suitable for the generation of large amount of pure VLPs suitable for vaccination purposes for the 20 market.

Therefore, the problem to provide particles useful as a vaccine based on essentially VP3 and methods of producing the same was solved by expressing in a cell VP3 from a VP3 coding sequence (cds) of the parvoviral structural protein VP3 (VP3 cds) under control of a Rep-independent promoter. Additionally, 25 in this method the polypeptide designated "assembly activating protein" (AAP) (Sonntag et al., 2010) is

5 6

expressed, which allows for high yields, e.g. approximately about 10 , preferably about 10 , and more

7

preferably about 10 virus particles to be formed per cell. Surprisingly and in line with its function of encoding a polypeptide, the sequence encoding AAP can be provided either in cis or in trans to assemble capsids consisting essentially of VP3. Virus particle titers can be quantified from lysates of transfected

30 cells (see above) in their undiluted form or in a dilution using a commercially available titration ELISA kit which is based on the binding of the monoclonal antibody A20 to the viral capsid in an assembled state to measure the virus concentration. As already described above, if the antibody A20 does not bind to the capsid of e.g. a different virus serotype, particle titers can be visualized by electron microscopy and quantified by counting (Mittereder et al., 1996, Grimm and leinschmidt, 1999, Grimm et al.,

35 1999). To analyze protein expression and estimate its amount cell lysates of identical portions of transfected cells can be processed for SDS-PAGE. Upon gel electrophoresis and transfer to a nitro- cellulose membrane, proteins can be probed using binders specific to the target protein (e.g. monoclonal antibodies B l, A69, anti-GFP). The amount of protein translation can be estimated from the amount of binders that specifically bind to the protein. These complexes can be visualized and quantified by e.g. immunohistochemical staining, immunofluorescent staining or radioactive labeling.

In contrast to the state of the art these VLPs do not contain a heterologous NLS or a VP2 protein or a truncated variant thereof. Upon epitope insertion at one or several of the preferred sites in the VP3, particles could be efficiently assembled that presented epitopes for vaccine development. With this

1 1 12 13

method 10 , preferably about 10 , and more preferably about 10 virus particles are formed per ml crude lysate and therefore yields are sufficient for a commercially viable product.

Homologous polypeptides of AAP of AAV2 can be identified for different parvoviruses. Such an alignment of predicted AAP protein sequences derived from ORF2 of the cap gene of different parvoviruses are shown in Figure 2. Accordingly, the AAP preferably is characterized in that it is a polypeptide comprising the amino acid sequence of AAV2, AAVl, or the amino acid sequence of AAV5. Sequences of AAP can be taken from Table 1.

Table 1: NCBI entry numbers of AAP encoding nucleotide and protein sequences from different parvoviruses.

No. of nt entree at

Parvovirus Length ofORF2 /nt Length o/AAP/AA

NCBI

AAV l NC_002077 678 225

AAV2 NC_001401 627 208

AAV3b AF028705 627 208

AAV4 NC_001829 597 198

AAV5 NC_006152 681 226

AAV6 AF028704 678 225

AAV 7 NC_006260 681 226

AAV 8 NC_006261 684 227

AAV9 AY530579 681 226

AAV 10 AY631965 606 201

AAV 1 1 AY631966 594 197

AAV12 DQ813647 621 206

AAV 13 EU285562 627 208

b-AAV (bovine) NC_005889 600 199 Avian AAV ATCC VR-865 AY186198 789 262

Avian AAV strain DA-1 AY629583 723 240

Mouse AAV1 DQ100362 534 177

Caprine AAV1 isolate AAV-

AY724675 581 226

Go. l

Rat AAV 1 DQ100363 756 251

Goose parvovirus strain DB3 EU088102 639 212

Duck parvovirus strain 90-

AY382892 693 230

0219

Snake parvovirus 1 AY349010 600 199

Parvoviruses other than AAV2 also encode functional AAP and make use of the same mechanism for capsid assembly. Further, AAP and VP3 are in principal interchangeable between different parvoviruses, especially between closely related viruses where VP3 and AAP can be exchanged mutually trans-complementing each other regarding VP3 particle assembly without substantial loss of yield in particle production.

Preferably the cross-protective B-cell epitope is derived from a HPV L2 protein of a malignant HPV genotype, preferably selected from HPV 16, HPV 18, HPV31, HPV33, HPV35, HPV 39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73 and HPV82, more preferably selected from HPV 16, HPV 18, HPV31, HPV45, HPV52 and HPV58, especially HPV16 and HPV31, and/or of a benign genotype, preferably selected from HPV1 , HPV2, HPV5, HPV6, HPV8, HPV 1 1 and HPV63, especially selected from HPV5, HPV6 and HPV1 1. Whereas a large number of HPV types is associated with some kind of disease like warts or other skin diseases, it is especially the group of malignant HPV or so-called "high-risk" genotypes which cause lesions that may progress to precancerous lesions (e.g. cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN)) and invasive cancer. These genotypes are responsible for nearly all cases of cervical cancer as well as other less frequent forms of epithelial cancers (anal cancer, vulvar cancer and penile cancer, oropharyngeal squamous-cell carcinoma). Within this group the combination of HPV16 and HPV31 is especially preferred, as HPV 16 is the most frequent malignant genotype, whereas HPV31 is a malignant genotype with a relatively low homology within the L2- terminus compared to other HPV genotypes.

Within the benign genotypes, which are not associated with cancer, those genotypes associated with genital warts (condylomata acuminate), a highly contagious, painful and disfiguring disease. Here, HPV5, 6 and 1 1 are most abundant and therefore especially preferred. In one embodiment the parvovirus mutated structural protein of the invention contains one or more cross-protective B-cell epitope which is derived from the N-terminal 200 amino acids of HPV L2 protein, preferably from the N-terminal 100 amino acids of HPV L2 protein, more preferably from amino acids 10 to 40 of HPV L2 protein, especially from amino acids 17-36 of HPV L2 protein. In an especially preferred embodiment, these cross-protective B-cell epitiopes are derived from HPV 16 and HPV31. A number of vaccination attempts have been made with vaccines comprising N-terminal peptides from papillomavirus L2 proteins (see Table 2) with the drawbacks as outlined above.

Table 2: HPV L2 epitopes

Figure imgf000012_0001

B-cell epitopes generally have a length of at least 9 AA. Accordingly, the parvovirus mutated structural protein of the invention preferably contains an insertion of at least 9, preferably at least 15, especially at least 20 amino acids from the HPV L2 protein. It is preferred according to this invention that the insertion(s) is (are) inserted into one or more positions selected from the group consisting of 1-261, 1-266, 1-381 , 1-447, 1-448, 1-453, 1-459, 1-471, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591 , 1-657, 1-664, 1-713 and 1-716, preferably 1-261 , 1-453, 1-534, 1-570, I- 573 and 1-587, more preferably 1-453, 1-534 and 1-587, especially 1-453 and 1-587. The used nomenclature I-### refers to the insertion site with ### naming the AA number relative to the VP1 protein of AAV-2, however meaning that the insertion may be located directly N- or C-terminal, preferably directly C-terminal of one AA in the sequence of 5 AAs N- or C-terminal of the given AA, preferably 3, more preferably 2, especially 1 AA(s) N- or C-terminal of the given AA. For parvoviruses other than AAV-2 the corresponding insertion sites can be identified by performing an AA alignment or by comparison of the capsid structures, if available. Such alignment has been performed for the parvoviruses AAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV- 10, AAV-1 1, b-AAV, GPV, B19, MVM, FPV and CPV (Figure 3 of WO 2008/145400).

The AA position after which the insertion was introduced and which named the site is underlined. It is also possible likewise to introduce an insertion into the five directly adjacent AAs located next to the underlined AA, because these are likewise located within a loop in the AAV2 capsid. For example the insertion site 1-587 corresponds to an insertion before and/or after one of the following AAs indicated by emphasis: FQSSS_TDPAT (AAV 1 ; SEQ ID NO: 8), LQRGN^RQAAT (AAV2; SEQ ID NO: 9), LQSSN_TAPTT (AAV3b; SEQ ID NO: 10), LQSSS TDPAT (AAV6; SEQ ID NO: 1 1), LQAATAAQT (AAV7; SEQ ID NO: 12), LQQQN.TAPQI (AAV8; SEQ ID NO: 13), LQQAN_TGPIV (AAV10; SEQ ID NO: 14), NQNAJ_TAPIT (AAV1 1 ; SEQ ID NO: 15) and NQSSJ_TAPAT (AAV5; SEQ ID NO: 16).

Further, the insertion site 1-453 corresponds to an insertion directly N- or C-terminal of the following ten AAs each, preferably directly C-terminal of the AA indicated by emphasis

QNQSG_SAQN (AAV1 ; SEQ ID NO: 17), NTPSG^TTTQS (AAV2; SEQ ID NO: 18), GTTSG TTNQS (AAV3b; SEQ ID NO: 19), QNQSG_SAQNK (AAV6; SEQ ID NO: 20), SNPGG.TAGNR (AAV7; SEQ ID NO: 21), GQTTG_TANTQ (AAV8; SEQ ID NO: 22), QSTGGJQGTQ (AAV 10; SEQ ID NO: 23), LSGET.NQGNA (AAV 1 1 ; SEQ ID NO: 24) and FVSTN .NTGGV (AAV5; SEQ ID NO: 25).

In one preferred embodiment the parvovirus mutated structural protein of the invention contains two or more insertions, each containing at least one cross-protective B-cell epitope of an HPV L2 protein and each inserted at a different insertion site of the parvovirus mutated structural protein, preferably wherein one insertion is at 1-587 and one at 1-453, more preferably wherein a B-cell epitope of HPV 16 L2 protein is inserted at 1-587 and a B-cell epitope of HPV31 L2 protein is inserted at 1-453, especially wherein a B-cell epitope derived from amino acids 17-36 of HPV 16 L2 is inserted at 1-587 and a B-cell epitope derived from amino acids 17-36 of HPV31 L2 inserted at 1-453. As described in the examples the L2 peptides 17 to 36 of HPV16 and HPV31 have been successfully inserted into the insertion sites I- 587 (HPV 16) and 1-453 (HPV31) of AAV2. The insertion containing the at least one cross-protective B-cell epitope of the HPV L2 protein preferably contains on its N- and/or C terminus a linker sequence, preferably a linker sequence having 6 to 10 small neutral or polar amino acids (A, G, S, C), which support the inserted epitope to be well accessible to the immune system. C has the advantage that two C on both sides of the linker may be able to form a hydrogen bond. Therefore, it is envisaged that both the N-terminal and C-terminal linker contain at least one C. Generally, it is preferred that the linker sequence(s) is (are) composed of A, G and S. In a preferred embodiment of the invention none of the 5 amino acids directly adjacent to the insertion is R and none of the amino acids of the linker, if present, is R. R in close proximity to the insertion reduces yield of the mutated structural protein/the multimeric structures composed of the mutated structural protein during expression and purification, and therefore is preferably avoided. Accordingly, the Rs at position 585 and 588 for AAV2 have been substituted for example by A. Accordingly, the parvovirus mutated structural protein comprises one or more additional mutations selected from an insertion, a deletion, a N- or C-terminal fusion of a heterologous AA sequence and a substitution, particularly a single-amino-acid exchange, or a combination of these, preferably a mutation of R585 of AAV2 and/or R588 of AAV2, especially a single-amino-acid exchange R585A of AAV2 and/or R58gA of AAV2. 'Heterologous' in this context means heterologous as compared to the virus, from which the parvovirus protein is derived; its should be notes that the protein having an N-terminal fusion is not a VP2 protein or a truncated VP2 protein. The one or more additional mutation of the capsid protein might be adequate to e.g. generate/insert B- cell epitopes of one or more further target protein(s) (multi-target vaccine), T- helper 1 (THi) epitope(s) to further promote the desired THi immune response, peptide sequence(s) to target antigen-presenting cells, or multimeric structures with reduced immunogenicity. The latter might be one possibility to setup an efficient prime/boost regimen.

In a further preferred embodiment the one or more additional mutation might be adequate to introduce at least one cytotoxic T-cell epitope (CTL epitope). For an infectious disease it is most useful to combine both humoral and cellular immune responses to fight these diseases. The multimeric structures according to this invention are in principle capable of pseudo-infecting cells. Accordingly these multimeric structures - like viruses - are able to enter cells, are processed to peptides, the peptides are loaded onto MHC class I and II molecules and finally presented to CD8- or CD4-positive T cells. The T- cells become stimulated after specific recognition of such processed peptide presented by MHC class I or II molecules. As a consequence of such stimulation CD8 cells may differentiate into cytotoxic T cells and then cause a cellular immune response. CD4 cells may develop into T helper cells which stimulate B cells to provide a humoral immune response or CD8-positive T cells to provide a cytotoxic immune response, which may themselves induce lysis of infected cells and other cells carrying and presenting the same peptide. Suitable CTL epitopes are known in the art for various HPV viral antigens, especially for the E6 or E7 protein of the HPV genotype of choice, or they can be predicted from given antigen sequences using for example the peptide prediction program by Parker under http.//www- bimas.cit.nih.gov/molbio/hla_bmd (Parker et al., 1994). Proposed CTL epitopes can be validated according to the methods as exemplified for HPV-epitopes in US 6,838,084, examples 2-8 (herein incorporated by reference). As processing of CTL epitopes occurs within the cell it is not necessary that such CTL epitopes are located on the surface or are present in a specific conformation.

Additionally, the insertion of epitopes at position 1-453 of AAV2 as described in WO 2008/145401 lead to the generation of an R within the linker downstream of the insertion (see example 6.4.3, page 103, lines 12 and 14) due to the generation of useful a endonuclease restriction site. Parvovirus mutated structural proteins where this R was substituted for a small neutral or polar amino acid (in the examples for S - R453S mutant) lead to considerably higher yield of VP3 only AAVLPs during expression and subsequent purification. Therefore, it is preferred, that the linkers, if present, do not contain an R, especially that the linker directly downstream of the inserted epitope at 1-453 does not contain an R.

Preferably the parvoviral mutated structural protein is capable of forming a multimeric structure. Accordingly, another subject of the invention relates to a multimeric structure comprising parvovirus mutated structural proteins of the invention, particularly comprising at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 structural protein. Such multimeric structure can be a capsomer, a virus-like particle (VLP) or a virus. Capsomers are multimeric subunits of a viral capsid, typically consisting of 5-6 capsid proteins (pentamers and hexamers). VLPs are empty viruses, meaning that they do not comprise genetic material such as a viral genome or relevant part thereof. In an especially preferred embodiment, the multimeric structure, preferably a VLP, is composed of essentially only VP3, especially of essentially only VP3 derived from AAV2.

The multimeric structure may also be an aggregate of at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 structural proteins. Compared to capsomers or VLPs aggregates are amorphous structures with no symmetric order.

Preferably the B-cell epitope of a human papillomavirus (HPV) L2 protein is located on the surface of the multimeric structure.

A further embodiment of the present invention is a nucleic acid coding for a parvovirus mutated structural protein of the invention such as DNA, RNA, mRNA etc.. A further embodiment of the present invention is a vector, e.g. a virus, that comprises a nucleic acid encoding the parvovirus mutated structural protein of the invention. Such virus may be infectious or inactive, for example it may have been inactivated through standard techniques such as attenuation or irradiation.

A further embodiment of the present invention is a cell comprising a nucleic acid coding for the parvovirus mutated structural protein. Such cell can be a bacterium, preferably E. coli, a yeast cell, preferably s. cerevisiae, hansenula polymorpha or pichia pastoris, k. lactis, an insect cell, preferably SF- 9, SF+ or High5, or a mammalian cell, preferably HeLa, 293, VERO, PERC6, BHK or CHO.

The parvovirus mutated structural proteins of the invention can be prepared by the method comprising the steps of: a) producing the structural protein by cultivating the cell according to the invention under suitable conditions thereby expressing the nucleic acid of the invention, and b) optionally isolating the expressed parvovirus mutated structural protein produced in step a). In a preferred embodiment, essentially only VP3 is expressed leading to multimeric structures comprising essentially only VP3. Another subject of the invention relates to a composition comprising at least one parvovirus mutated structural protein according to the invention and/or a nucleic acid according to the invention, preferably at least one multimeric structure according to the invention, for use as a medicament.

The medicament is particularly used as a vaccine comprising at least one parvovirus mutated structural protein of the invention and/or a nucleic acid of the invention, preferably at least one multimeric structure of the invention.

Preferably, the medicament is a vaccine. In a preferred embodiment the vaccine is capable of inducing a cross-protective antibody response against at least HPV16, HPV18, HPV31 and HPV45, more preferably HPV16, HPV18, HPV31 , HPV45 and HPV58, more preferably HPV16, HPV18, HPV31, HPV45, HPV52 and HPV58, especially HPV5, HPV6, HPV 1 1 , HPV16, HPV 18, HPV31 and HPV45.

In a preferred embodiment of the invention a vaccine is a mixture of more than one multimeric structures comprising parvovirus mutated structural proteins as further defined herein. Preferably two to three VLPs of a parvovirus displaying different B-cell epitopes as further defined herein are combined for the vaccination of a human subject. Further, it is envisaged that a vaccine according to this invention is combined with some other type of vaccine for convenience of the patient.

In a preferred embodiment the medicament or vaccine encompasses pharmaceutically acceptable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the (poly)peptides herein disclosed. In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e. g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

In a preferred embodiment the medicament further comprises an immunostimulatory substance such as an adjuvant. The adjuvant can be selected based on the method of administration and may include mineral or plant oil-based adjuvants, Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, syntax adjuvant formulation containing muramyl dipeptide, or aluminum salt adjuvants. Preferably, the adjuvant is an oil-based adjuvant, preferably ISA206 (SEPPIC, Paris, France), most preferably ISA51 or ISA720 (SEPPIC, Paris, France). In another preferred embodiment the parvovirus mutated structural protein is co-formulated with at least one suitable adjuvant such as CpG, Imidazoquinolines, MPL, MDP, MALP, flagellin, LPS, LTA, or cholera toxin or derivative thereof, HSP60, HSP70, HSP90, saponins, QS21 , ISCOMs, CFA, SAF, MF59, adamantanes, aluminum hydroxide, aluminum phosphate or a cytokine.

In a more preferred embodiment the immunostimulatory substance is selected from the group comprising polycationic polymers, especially polycationic peptides such as polyarginine, immunostimulatory deoxynucleotides (ODNs), peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK (SEQ ID NO: 92), neuroactive compounds, especially human growth hormone, alumn, adjuvants or combinations thereof. Preferably, the combination is either a polycationic polymer and immunostimulatory deoxynucleotides or of a peptide containing at least two LysLeuLys motifs and immunostimulatory deoxynucleotides. In a still more preferred embodiment the polycationic polymer is a polycationic peptide. In an even more preferred embodiment of the invention the immunostimulatory substance is at least one immunostimulatory nucleic acid. Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acids derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine dinucleotides (CpG) in a defined base context (e.g. as described in WO 96/02555). Alternatively, also nucleic acids based on inosine and cytidine as e.g. described in WO 01/93903, or deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 and WO 02/095027) may preferably be used as immunostimulatory nucleic acids in the present invention. Preferably, mixtures of different immunostimulatory nucleic acids are used in the present invention. Additionally, the aforementioned polycationic compounds may be combined with any of the immunostimulatory nucleic acids as aforementioned. Preferably, such combinations are according to the ones described in WO 01/93905, WO 02/32451 , WO 01/54720, WO 01/93903, WO 02/13857 and WO 02/095027 and the AU application A 1924/2001.

Preferably the medicament or vaccine is lyophilized or formulated to be stable at room temperature for at least one year and/or at stress conditions (6 hours at 50°C). Stable in this context means that during the given time at the given temperature, degradation (or other form of inactivation of the vaccine) is below < 20%, preferably < 5%, especially < 1% (measurable as loss of cross-protective titer for e.g. HPVl 6). This is especially important as there is a high medical need in the developing world where transport of vaccines with required cooling is a large problem. According to this invention, AAVLPs, especially VP3 AAVLPs are preferably stable at room temperature for at least one year and/or at stress conditions.

Also encompassed by the present inventions are methods for vaccination and/or for treating or preventing the diseases specified herein by administering to a patient an effective amount of a parvovirus mutated structural protein of the invention and or nucleic acid coding for a parvovirus mutated structural protein of the invention. Accordingly the composition according to the invention can be used in a method of preventing or treating an HPV infection, preferably an HPV infection caused by a malignant HPV genotype, preferably selected from HPV 16, HPVl 8, HPV31, HPV33, HPV35, HPV 39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73 and HPV82, more preferably selected from HPV 16, HPV 18, HPV31, HPV45 and HPV58, even more preferably from HPV 16, HPV 18, HPV31, HPV45, HPV52 and HPV58, especially HPV 16 and HPV31 , and/or from a benign genotype, preferably selected from HPVl, HPV2, HPV5, HPV6, HPV8, HPVl 1 and HPV63, especially selected from HPV5, HPV6 and HPVl 1.

Above mentioned problems are solved by the invention as claimed herein.

Figures

Figure 1: VP3 particle production in insect cells

A) Schematic representation of constructs used for AAV production in insect cells. B) Western blot analysis of expressed VP proteins was performed using antibody SA7885 (1 : 10000 dilution) a polyclonal rabbit serum that detects all three capsid proteins and subsequent the secondary antibody anti rabbit IgG-HRP 1 :2500 (Dianova, Hamburg, Germany). C) Capsid formation was quantified by an ELISA based on monoclonal antibody A20. Means +/- standard deviations of 2 (VP2 construct) or 4 (VP3 and VPl_Mod4) independent experiments are shown.

Figure 2: Comparison of parvovirus AAP sequences.

Alignment of predicted AAP protein sequences derived from ORF2 of the cap gene of different parvoviruses. Conserved amino acids that are 100% identical in at least 60% of aligned sequences are represented as lines in the lower row. Position of the predicted AAV2 AAP translation start is highlighted by a frame. Non-translated sequences upstream of the potential translation initiation codons are included as well. NCBI entree numbers of the corresponding DNA sequences are listed in Table 3.

Figure 3: Western blot analysis of purified, modified HPV L2 candidates

Purified HPV L2 vaccine candidates consisting of VP3 only from constructs AAVLP-L2-Bi_R588A, AAVLP-L2-Bi_R585A_R588A or AAVLP-L2-Bi_R585A_R588A_R453S (50 ng each) were separated through a 10% Bis-Tris polyacrylamide gel. Proteins were transferred onto a positively charged nylon membrane and incubated with a polyclonal anti-AAV2 antibody (SA7885). Western blotting was performed according to the standard procedures. Marker: SeeBlue Plus2 ladder.

Figure 4: Interaction of AAVLPs with anti-HPV L2 polyclonal antibodies

0.5 to 2 μg AAVLP particles of a bivalent HPV vaccine (AAVLP-L2-Bi) targeting HPV 16 and HPV31 L2 were dotted onto a nitrocellulose membrane. As negative control equal amounts of wtAAV2 VP3 AAVLPs used (lower lanes). As a positive control 5 and 10 μg of the inserted L2-peptide of HPV 16 and HPV31 were dotted (right lanes). The membrane was incubated with (A) anti-HPV 16 L2 polyclonal antibody and (B) with anti-HPV31 L2 polyclonal antibody. Binding of the antibody to the AAVLPs /peptides was detected using a secondary HRP-labeled antibody.

Figure 5: Induction of HPV L2 specific antibodies by AAVLP-L2-Bi vaccine

Rabbits (n=3) were immunized with an AAVLP-based bivalent HPV vaccine (AAVLP-L2-Bi) targeting HPV 16 and HPV31 L2. Immune sera of the vaccinated animals were analyzed after the 3rd boost immunization measuring the induction of HPV 16 L2 (white bars) and HPV31 L2 (black bars) epitope specific antibodies.

AMINO ACID SEQUENCES OF AAP

AAV1 (SEQ ID NO: 26) SSRHKSQTPP RASARQASSP LKRDSILVRL ATQSQSPIHN LSENLQQPPL LWDLLQWLQA VAHQWQTITK

APTEWVMPQE IGIAIPHGWA TESSPPAPAP GPCPPTITTS TSKSPVLQRG PATTTTTSAT APPGGILIST

DSTATFHHVT GSDSSTTIGD SGPRDSTSNS STSKSRRSRR MMASQPSLIT LPARFKSSRT RSTSFRTSSA

LRTRAASLRS RRTCS AAV2 (SEQ ID NO: 27)

ILVRLETQTQ YLTPSLSDSH QQPPLVWELI RWLQAVAHQW QTITRAPTEW VIPREIGIAI PHGWATESSP PAPEPGPCPP TTTTSTNKFP ANQEPRTTIT TLATAPLGGI LTSTDSTATF HHVTGKDSST TTGDSDPRDS TSSSLTFKSK RSRRMTVRRR LPITLPARFR CLLTRSTSSR TSSARRIKDA SRRSQQTSSW CHSMDTSP

AAV3b (SEQ ID NO: 28)

ISVRLATQSQ SQTLNLSENH QQPPQVWDLI QWLQAVAHQW QTITRVPMEW VIPQEIGIAI PNGWATESSP PAPEPGPCPL TTTISTSKSP ANQELQTTTT TLATAPLGGI LTLTDSTATS HHVTGSDSLT TTGDSGPRNS ASSSSTSKLK RSRRTMARRL LPITLPARFK CLRTRSISSR TCSGRRTKAV SRRFQRTSSW SLSMDTSP

AAV4 (SEQ ID NO: 29)

LNPPSSPTPP RVSAKKASSR LKRSSFSKTK LEQATDPLRD QLPEPCLMTV RCVQQLAELQ SRADKVPMEW VMPRVIGIAI PPGLRATSRP PAPEPGSCPP TTTTSTSDSE RACSPTPTTD SPPPGDTLTS TASTATSHHV TGSDSSTTTG ACDPKPCGSK SSTSRSRRSR RRTARQRWLI TLPARFRSLR TRRTNCRT

AAV5 (SEQ ID NO: 30)

TTTFQKERRL GPKRTPSLPP RQTPKLDPAD PSSCKSQPNQ PQVWELIQCL REVAAHWATI TKVPMEWAMP

REIGIAIPRG WGTESSPSPP EPGCCPATTT TSTERSKAAP STEATPTPTL DTAPPGGTLT LTASTATGAP ETGKDSSTTT GASDPGPSES KSSTFKSKRS RCRTPPPPSP TTSPPPSKCL RTTTTSCPTS SATGPRDACR

PSLRRSLRCR STVTRR

AAV6 (SEQ ID NO: 3 1)

SSRHKSQTPP RALARQASSP LKRDSILVRL ATQSQSPTHN LSENLQQPPL LWDLLQWLQA VAHQWQTITK APTEWVMPQE IGIAIPHGWA TESSPPAPEH GPCPPITTTS TSKSPVLQRG PATTTTTSAT APPGGILIST DSTAISHHVT GSDSSTTIGD SGPRDSTSSS STSKSRRSRR MMASRPSLIT LPARFKSSRT RSTSCRTSSA LRTRAASLRS RRTCS

AAV7 (SEQ ID NO: 32)

SRHLSVPPTP PRASARKASS PPERDSISVR LATQSQSPTL NLSENLQQRP LVWDLVQWLQ AVAHQWQTIT KVPTEWVMPQ EIGIAIPHGW ATESLPPAPE PGPCPPTTTT STSKSPVKLQ VVPTTTPTSA TAPPGGILTL TDSTATSHHV TGSDSSTTTG DSGPRSCGSS SSTSRSRRSR RMTALRPSLI TLPARFRYSR TRNTSCRTSS ALRTRAACLR SRRTSS

AAV8 (SEQ ID NO: 33)

SHHPSVLQTP LRASARKANS PPEKDSILVR LATQSQFQTL NLSENLQQRP LVWDLIQWLQ AVAHQWQTIT

KAPTEWVVPR EIGIAIPHGW ATESSPPAPE PGPCPPTTTT STSKSPTGHR EEPPTTTPTS ATAPPGGILT LTDSTATFHH VTGSDSSTTT GDSGPRDSAS SSSTSRSRRS RRMKAPRPSP ITSPAPSRCL RTRSTSCRTF

SALPTRAACL RSRRTCS

AAV9 (SEQ ID NO: 34) SSLLRNRTPP RVLANRVHSP LKRDSISVRL ATQSQSQTLN QSENLPQPPQ VWDLLQWLQV VAHQWQTITK

VPMEWVVPRE IGIAIPNGWG TESSPPAPEP GPCPPTTITS TSKSPTAHLE DLQMTTPTSA TAPPGGILTS

TDSTATSHHV TGSDSSTTTG DSGLSDSTSS SSTFRSKRLR TTMESRPSPI TLPARSRSSR TQTISSRTCS GRLTRAASRR SQRTFS AAV10 (SEQ ID O: 35)

TLGRLASQSQ SPTLNQSENH QQAPLVWDLV QWLQAVALQW QTITKAPTEW VVPQEIGIAI PHGWATESSP PAPEPGPCPP TTTTSTSKSP TGHREEAPTT TPTSATAPPG GILTSTDSTA TSHHVTGSDS STTTGDSGQK DSASSSSTSR SRRSRRMKAP RPSPITLPAR FRYLRTRNTS CRTSSAPRTR AACLRSRRMS S

AAV 1 1 (SEQ ID NO: 36)

SHHKSPTPPR ASAKKANNQP ERGSTLKRTL EPETDPLKDQ IPAPCLQTLK CVQHRAEMLS MRDKVPMEWV MPRVIGIAIP PGLRARSQQP RPEPGSCPPT TTTCTCVSEQ HQAATPTTDS PPPGDILTST DSTVTSHHVT GKDSSTTTGD YDQKPCALKS SISKLRRSQR RTARLRSLIT LPARFRYLRT RRMSSRT

AAV 12 (SEQ ID NO: 37)

KRLQIGRPTR TLGRPRPRKS KKTANQPTLL EGHSTLKTLE QETDPLRDHL PEKCLMMLRC VRRQAEMLSR RDKVPMEWVM PPVIGIAIPP GQRAESPPPA PEPGSYPRTT TTCTCESEQR PTATPTTDSP PPGDTLTLTA STATFPHATG SDSSTTTGDS GRNRCVLKSS TYRSRRSRRQ TARLRSLITL PARFRSLRIR RMNSHT

AAV 13 (SEQ ID NO: 38)

ILVRLATQSQ SQTLNHSDNL PQPPLVWDLL QWLQAVAHQW QTITRVPMEW VIPQEIGIAI PNGWATESSP PAPAPGPCPP TTITSTSKSP ANQEPPTTTT TLATAPPGGI LTSTDSTATF HHVTGKDSST TTGDSDPRDS TSSSLTFKSK RSRRMTVRRR LPITLPARFR CLLTPSTSSR TSSARRIRDA SRRSQQTSSW SHSMDTSP

b-AAV (bovine) (SEQ ID NO: 39)

SRVLKSQTPR AELARKANSL PERDSTLTTN LEPETGLPQK DHLPELCLLR LKCVQQLAEM VAMRDKVPRE WVMPPVIGIA IPLGQRATSP PPQPAPGSCR PTTTTCTCGS ARATPATPST DSPPPGDTLT LTASTATSRQ ETGKGSSTTT GDCAPKACKS ASSTSKLRRS RRLTGRRPYP TTSPARSRSL RTARTSSRT

Avian AAV ATCC VR-865 (SEQ ID NO:40 )

VKPSSRPKRG FSNPLVWWKT QRRLRPETSG KAKTNLVCPT LLHRLPRKTR SLARKDLPAG QKIRAKAPLP TLEQQHPPLV WDHLSWLKEV AAQWAMQARV PMEWAIPPEI GIAIPNGWKT ESSLEPPEPG SCPATTTTCT NESKDPAEAT TTTNSLDSAP PGDTLTTIDS TATFPRETGN DSSTTTGASV PKRCALDSLT SRLKRSRSKT STPPSATTSP VRSRSLRTRT TNCRTSSDRL PKAPSRRSQR ISTRSRSTGT AR

Mouse AAV 1 (SEQ ID NO: 41 )

TRRTVSSLPL QRRPKLEALP PPAIWDLVRW LEAVARQSTT ARMVPMEWAM PREIGIAIPH GWTTVSSPEP LGPGICQPTT TTSTNDSTER PPETKATSDS APPGDTLTST ASTVISPLET GKDSSTITGD SDQRAYGSKS LTFKLKKSRR KTQRRSSPIT LPARFRYLRT RSTSSRT

Avian AAV strain DA-1 (SEQ ID NO: 42)

LNNPTTRPGP GRSVPNASTT FSRKRRRPRP SKAKPLLKRA KTPEKEPLPT LDQAPPLVWD HLSWLKEVAV

QWAMQAKVPT EWAIPREIGI AIPNGWTTES LPEPLEPGSC PATTTTCTSG SKDREEPTPT INSLDSAPPG

GTLTTTDSTA TSPPETGNDS STTTGASDPK RCALDSLTSR LKKSLSKTPT PPSPTTSPAR SKSLRTRTTS

CRTSSDRLQR APSRRSQRIS TRSRSMVTAR Caprine AAV l isolate AAV-Go. l (SEQ ID NO: 43)

TTTFQKERRL GPKRTPSLPP RQTPKLDPAD PSSCKSQHNQ PQVWELIQCL REVAAHWATI TKVPMEWAMP REIGIAIPRG WGTESSPSPP APGCCPATTT TSTERSKAAP STEATPTPTL DTAPPGGTLT LTASTATGAP ETGKDSSTTI GASDPGLSES KSSTSKSKRS RCRTPPPPSP TTSPPPSKCL RTTTTNSRTS SATGPRDACR PSPRRSLRCR STATRR

Rat AAVl (SEQ ID NO: 44)

ASRSRSWLLQ SSVHTRPRKP QRTRRVSRDR IPGRRPRRGS SSPISLDLQQ TYLHPHNSPS LPQGFPVWFL VRCLQEEALQ WTMLNKVPTE WAMPREIGIA IPNGWATEFS PDPPGPGCCP ATTTTCTSRS QTPPACTASP GADTLATAPP GGTSTSIAST ATSRPETGSA SSITTGASDP RDCESNSSTS RSRRSRLLIR RPRSPTTSRA RSRSSQTTST SCRTSAATPP RDACRRSPRT SSRCRSTATR R

Goose parvovirus strain DB3 (SEQ ID NO: 45)

KTEEPPRRAP NLWQHLKWQR EEAELWATLQ GVPMEWVMPR EIGIAIPNGW ETQSSQRPPE PGSCQATTTT STKQLPVEPL KMQMSSMQDT VPPGGTLIST ASTATSPLET GRDLSTTIGE SDPNLLNSRS SMSKSKKSQR RIKQRPLQTI SPQRFKSLRM MSINSRMSWA RLRKAPCRRS RRMSMPCRST GTAQCTPTRM EHGSMTVVHS TA

Duck parvovirus strain 90-0219 (SEQ ID NO: 46)

KSLNYLKKTL LHPVIVEEKQ VQLPPKAPNL WQHLTWQREE AELWATLQGV PMEWVMPQEI GIAIPNGWET QSLPRLQEPG SCQATTTTST KPSQAEQTQT QIPNMLDTAP PGGTLISTDS TAISLQETGR DSSTTIGGLD RKHSNSRYSM CKLKKSRRKT RQRLLLTTLP LQSRYSRIMN TSCPMFWARP RRGRCHRSPQ MCMPCPSTAT AQCTPTRVEL DSMTEVPSIA

Snake parvovirus 1 (SEQ ID NO: 47)

TNTILKLKRP NKACRYQLHL KAEKKKLHRH NLEGAQQVPI LAAHLSWLQE EAVRWQTITR APREWVIPQV

IGIAIPSGWE TTSLQSQPEL GCSPLTGIIS TGLSTLTAPQ VRVL QPMQD TRLPGGTLTS IDSIATSPPE

TGKDSSTTTQ ASGRKDSKSK SLTSKSKKLQ HKIQRKQLPT ISPAPYRSLR TRTTTYHMY

NUCLEIC ACID SEQUENCES

AAV l (SEQ ID NO: 48)

AGCAGTCGCC ACAAGAGCCA GACTCCTCCT CGGGCATCGG CAAGACAGGC CAGCAGCCCG C AAAAAGAG

ACTCAATTTT GGTCAGACTG GCGACTCAGA GTCAGTCCCC GATCCACAAC CTCTCGGAGA ACCTCCAGCA ACCCCCGCTG CTGTGGGACC TACTACAATG GCTTCAGGCG GTGGCGCACC AATGGCAGAC AATAACGAAG

GCGCCGACGG AGTGGGTAAT GCCTCAGGAA ATTGGCATTG CGATTCCACA TGGCTGGGCG ACAGAGTCAT

CACCACCAGC ACCCGCACCT GGGCCTTGCC CACCTACAAT AACCACCTCT ACAAGCAAAT CTCCAGTGCT

TCAACGGGGG CCAGCAACGA CAACCACTAC TTCGGCTACA GCACCCCCTG GGGGTATTTT GATTTCAACA

GATTCCACTG CCACTTTTCA CCACGTGACT GGCAGCGACT CATCAACAAC AATTGGGGAT TCCGGCCCAA GAGACTCAAC TTCAAACTCT TCAACATCCA AGTCAAGGAG GTCACGACGA ATGATGGCGT CACAACCATC

GCTAATAACC TTACCAGCAC GGTTCAAGTC TTCTCGGACT CGGAGTACCA GCTTCCGTAC GTCCTCGGCT

CTGCGCACCA GGGCTGCCTC CCTCCGTTCC CGGCGGACGT GTTCATGA

AAV2 (SEQ ID NO: 49) ATTTTGGTCA GACTGGAGAC GCAGACTCAG TACCTGACCC CCAGCCTCTC GGACAGCCAC CAGCAGCCCC CTCTGGTCTG GGAACTAATA CGATGGCTAC AGGCAGTGGC GCACCAATGG CAGACAATAA CGAGGGCGCC GACGGAGTGG GTAATTCCTC GGGAAATTGG CATTGCGATT CCACATGGAT GGGCGACAGA GTCATCACCA CCAGCACCCG AACCTGGGCC CTGCCCACCT ACAACAACCA CCTCTACAAA CAAATTTCCA GCCAATCAGG AGCCTCGAAC GACAATCACT ACTTTGGCTA CAGCACCCCT TGGGGGTATT TTGACTTCAA CAGATTCCAC TGCCACTTTT CACCACGTGA CTGGCAAAGA CTCATCAACA ACAACTGGGG ATTCCGACCC AAGAGACTCA ACTTCAAGCT CTTTAACATT CAAGTCAAAG AGGTCACGCA GAATGACGGT ACGACGACGA TTGCCAATAA CCTTACCAGC ACGGTTCAGG TGTTTACTGA CTCGGAGTAC CAGCTCCCGT ACGTCCTCGG CTCGGCGCAT CAAGGATGCC TCCCGCCGTT CCCAGCAGAC GTCTTCATGG TGCCACAGTA TGGATACCTC ACCCTGA AAV3b (SEQ ID NO: 50)

ATTTCGGTCA GACTGGCGAC TCAGAGTCAG TCCCAGACCC TCAACCTCTC GGAGAACCAC CAGCAGCCCC CACAAGTTTG GGATCTAATA CAATGGCTTC AGGCGGTGGC GCACCAATGG CAGACAATAA CGAGGGTGCC GATGGAGTGG GTAATTCCTC AGGAAATTGG CATTGCGATT CCCAATGGCT GGGCGACAGA GTCATCACCA CCAGCACCAG AACCTGGGCC CTGCCCACTT ACAACAACCA TCTCTACAAG CAAATCTCCA GCCAATCAGG AGCTTCAAAC GACAACCACT ACTTTGGCTA CAGCACCCCT TGGGGGTATT TTGACTTTAA CAGATTCCAC TGCCACTTCT CACCACGTGA CTGGCAGCGA CTCATTAACA ACAACTGGGG ATTCCGGCCC AAGAAACTCA GCTTCAAGCT CTTCAACATC CAAGTTAAAG AGGTCACGCA GAACGATGGC ACGACGACTA TTGCCAATAA CCTTACCAGC ACGGTTCAAG TGTTTACGGA CTCGGAGTAT CAGCTCCCGT ACGTGCTCGG GTCGGCGCAC CAAGGCTGTC TCCCGCCGTT TCCAGCGGAC GTCTTCATGG TCCCTCAGTA TGGATACCTC ACCCTGA AAV4 (SEQ ID NO: 51)

TTGAATCCCC CCAGCAGCCC GACTCCTCCA CGGGTATCGG CAAAAAAGGC AAGCAGCCGG CTAAAAAGAA

GCTCGTTTTC G AAG AC G AAA CTGGAGCAGG CGACGGACCC CCTGAGGGAT CAACTTCCGG AGCCATGTCT

GATGACAGTG AGATGCGTGC AGCAGCTGGC GGAGCTGCAG TCGAGGGCGG ACAAGGTGCC GATGGAGTGG

GTAATGCCTC GGGTGATTGG CATTGCGATT CCACCTGGTC TGAGGGCCAC GTCACGACCA CCAGCACCAG

AACCTGGGTC TTGCCCACCT ACAACAACCA CCTCTACAAG CGACTCGGAG AGAGCCTGCA GTCCAACACC

TACAACGGAT TCTCCACCCC CTGGGGATAC TTTGACTTCA ACCGCTTCCA CTGCCACTTC TCACCACGTG

ACTGGCAGCG ACTCATCAAC AACAACTGGG GCATGCGACC CAAAGCCATG CGGGTCAAAA TCTTCAACAT

CCAGGTCAAG GAGGTCACGA CGTCGAACGG CGAGACAACG GTGGCTAATA ACCTTACCAG CACGGTTCAG

ATCTTTGCGG ACTCGTCGTA CGAACTGCCG TACGTGA

AAV 5 (SEQ ID NO: 52)

ACGACCACTT TCCAAAAAGA AAGAAGGCTC GGACCGAAGA GGACTCCAAG CCTTCCACCT CGTCAGACGC

CGAAGCTGGA CCCAGCGGAT CCCAGCAGCT GCAAATCCCA GCCCAACCAG CCTCAAGTTT GGGAGCTGAT

ACAATGTCTG CGGGAGGTGG CGGCCCATTG GGCGACAATA ACCAAGGTGC CGATGGAGTG GGCAATGCCT

CGGGAGATTG GCATTGCGAT TCCACGTGGA TGGGGGACAG AGTCGTCACC AAGTCCACCC GAACCTGGGT

GCTGCCCAGC TACAACAACC ACCAGTACCG AG AG AT C AAA AGCGGCTCCG TCGACGGAAG CAACGCCAAC

GCCTACTTTG GATACAGCAC CCCCTGGGGG TACTTTGACT TTAACCGCTT CCACAGCCAC TGGAGCCCCC

GAGACTGGCA AAGACTCATC AACAACTACT GGGGCTTCAG ACCCCGGTCC CTCAGAGTCA AAATCTTCAA

CATTCAAGTC AAAGAGGTCA CGGTGCAGGA CTCCACCACC ACCATCGCCA ACAACCTCAC CTCCACCGTC

CAAGTGTTTA CGGACGACGA CTACCAGCTG CCCTACGTCG TCGGCAACGG GACCGAGGGA TGCCTGCCGG

CCTTCCCTCC GCAGGTCTTT ACGCTGCCGC AGTACGGTTA CGCGACGCTG A AAV6 (SEQ ID NO: 53)

AGCAGTCGCC AC AAG AG CCA GACTCCTCCT CGGGCATTGG CAAGACAGGC CAGCAGCCCG CTAAAAAGAG

ACTCAATTTT GGTCAGACTG GCGACTCAGA GTCAGTCCCC GACCCACAAC CTCTCGGAGA ACCTCCAGCA

ACCCCCGCTG CTGTGGGACC TACTACAATG GCTTCAGGCG GTGGCGCACC AATGGCAGAC AATAACGAAG

GCGCCGACGG AGTGGGTAAT GCCTCAGGAA ATTGGCATTG CGATTCCACA TGGCTGGGCG ACAGAGTCAT

CACCACCAGC ACCCGAACAT GGGCCTTGCC CACCTATAAC AACCACCTCT ACAAGCAAAT CTCCAGTGCT

TCAACGGGGG CCAGCAACGA CAACCACTAC TTCGGCTACA GCACCCCCTG GGGGTATTTT GATTTCAACA

GATTCCACTG CCATTTCTCA CCACGTGACT GGCAGCGACT CATCAACAAC AATTGGGGAT TCCGGCCCAA

GAGACTCAAC TTCAAGCTCT TCAACATCCA AG TC AAG GAG GTCACGACGA ATGATGGCGT CACGACCATC

GCTAATAACC TTACCAGCAC GGTTCAAGTC TTCTCGGACT CGGAGTACCA GTTGCCGTAC GTCCTCGGCT

CTGCGCACCA GGGCTGCCTC CCTCCGTTCC CGGCGGACGT GTTCATGA

AAV7 (SEQ ID NO: 54)

AGCCGTCACC TCAGCGTTCC CCCGACTCCT CCACGGGCAT C G G C AAG AAA GGCCAGCAGC CCGCCAGAAA

GAGACTCAAT TTCGGTCAGA CTGGCGACTC AGAGTCAGTC CCCGACCCTC AACCTCTCGG AGAACCTCCA

GCAGCGCCCT CTAGTGTGGG ATCTGGTACA GTGGCTGCAG GCGGTGGCGC ACCAATGGCA GACAATAACG

AAGGTGCCGA CGGAGTGGGT AATGCCTCAG GAAATTGGCA TTGCGATTCC ACATGGCTGG GCGACAGAGT

CATTACCACC AGCACCCGAA CCTGGGCCCT GCCCACCTAC AACAACCACC TCTACAAGCA AATCTCCAGT

GAAACTGCAG GTAGTACCAA CGACAACACC TACTTCGGCT ACAGCACCCC CTGGGGGTAT TTTGACTTTA

ACAGATTCCA CTGCCACTTC TCACCACGTG ACTGGCAGCG ACTCATCAAC AACAACTGGG GATTCCGGCC

CAAGAAGCTG CGGTTCAAGC TCTTCAACAT CCAGGTCAAG GAGGTCACGA CGAATGACGG CGTTACGACC

ATCGCTAATA ACCTTACCAG CACGATTCAG GTATTCTCGG ACTCGGAATA CCAGCTGCCG TACGTCCTCG

GCTCTGCGCA CCAGGGCTGC CTGCCTCCGT TCCCGGCGGA CGTCTTCATG A

AAV8 (SEQ ID NO: 55)

AGCCATCACC CCAGCGTTCT CCAGACTCCT CTACGGGCAT C G G C AAG AAA GGCCAACAGC CCGCCAGAAA

AAGACTCAAT TTTGGTCAGA CTGGCGACTC AGAG CAGTT CCAGACCCTC AACCTCTCGG AGAACCTCCA

GCAGCGCCCT CTGGTGTGGG ACCTAATACA ATGGCTGCAG GCGGTGGCGC ACCAATGGCA GACAATAACG

AAGGCGCCGA CGGAGTGGGT AGTTCCTCGG GAAATTGGCA TTGCGATTCC ACATGGCTGG GCGACAGAGT

CATCACCACC AGCACCCGAA CCTGGGCCCT GCCCACCTAC AACAACCACC TCTACAAGCA AATCTCCAAC

GGGACATCGG GAG GAG CC AC CAACGACAAC ACCTACTTCG GCTACAGCAC CCCCTGGGGG TATTTTGACT

TTAACAGATT CCACTGCCAC TTTTCACCAC GTGACTGGCA GCGACTCATC AACAACAACT GGGGATTCCG

GCCCAAGAGA CTCAGCTTCA AGCTCTTCAA CATCCAGGTC AAGGAGGTCA CGCAGAATGA AGGCACCAAG

ACCATCGCCA ATAACCTCAC CAGCACCATC CAGGTGTTTA CGGACTCGGA GTACCAGCTG CCGTACGTTC

TCGGCTCTGC CCACCAGGGC TGCCTGCCTC CGTTCCCGGC GGACGTGTTC ATGA

AAV9 (SEQ ID NO: 56)

AGCAGTCTCC TCAGGAACCG GACTCCTCCG CGGGTATTGG CAAATCGGGT GCACAGCCCG CTAAAAAGAG

ACTCAATTTC GGTCAGACTG GCGACACAGA GTCAGTCCCA GACCCTCAAC CAATCGGAGA ACCTCCCGCA

GCCCCCTCAG GTGTGGGATC TCTTACAATG GCTTCAGGTG GTGGCGCACC AGTGGCAGAC AATAACGAAG

GTGCCGATGG AGTGGGTAGT TCCTCGGGAA ATTGGCATTG CGATTCCCAA TGGCTGGGGG ACAGAGTCAT

CACCACCAGC ACCCGAACCT GGGCCCTGCC CACCTACAAC AATCACCTCT ACAAGCAAAT CTCCAACAGC

ACATCTGGAG GATCTTCAAA TGACAACGCC TACTTCGGCT ACAGCACCCC CTGGGGGTAT TTTGACTTCA ACAGATTCCA CTGCCACTTC TCACCACGTG ACTGGCAGCG ACTCATCAAC AACAACTGGG GATTCCGGCC

TAAGCGACTC AACTTCAAGC TCTTCAACAT TCAGGTCAAA GAGGTTACGG ACAACAATGG AGTCAAGACC

ATCGCCAATA ACCTTACCAG CACGGTCCAG GTCTTCACGG ACTCAGACTA TCAGCTCCCG TACGTGCTCG

GGTCGGCTCA CGAGGGCTGC CTCCCGCCGT TCCCAGCGGA CGTTTTCATG A AAV10 (SEQ ID O: 57)

ACTTTGGGCA GACTGGCGAG TCAGAGTCAG TCCCCGACCC TCAACCAATC GGAGAACCAC CAGCAGGCCC

CTCTGGTCTG GGATCTGGTA CAATGGCTGC AGGCGGTGGC GCTCCAATGG CAGACAATAA CGAAGGCGCC

GACGGAGTGG GTAGTTCCTC AGGAAATTGG CATTGCGATT CCACATGGCT GGGCGACAGA GTCATCACCA

CCAGCACCCG AACCTGGGCC CTGCCCACCT ACAACAACCA CCTCTACAAG CAAATCTCCA ACGGGACATC GGGAGGAAGC ACCAACGACA ACACCTACTT CGGCTACAGC ACCCCCTGGG GGTATTTTGA CTTCAACAGA

TTCCACTGCC ACTTCTCACC ACGTGACTGG CAGCGACTCA TCAACAACAA CTGGGGATTC CGGCCAAAAA

GACTCAGCTT CAAGCTCTTC AACATCCAGG TCAAGGAGGT CACGCAGAAT GAAGGCACCA AGACCATCGC

CAATAACCTT ACCAGCACGA TTCAGGTATT TACGGACTCG GAATACCAGC TGCCGTACGT CCTCGGCTCC

GCGCACCAGG GCTGCCTGCC TCCGTTCCCG GCGGATGTCT TCATGA AAV 1 1 (SEQ ID NO: 58)

AGTCACCACA AGAGCCCGAC TCCTCCTCGG GCATCGGCAA AAAAGGCAAA CAACCAGCCA GAAAGAGGCT

CAACTTTGAA GAGGACACTG GAGCCGGAGA CGGACCCCCT GAAGGATCAG ATACCAGCGC CATGTCTTCA

GACATTGAAA TGCGTGCAGC ACCGGGCGGA AATGCTGTCG ATGCGGGACA AGGTTCCGAT GGAGTGGGTA

ATGCCTCGGG TGATTGGCAT TGCGATTCCA CCTGGTCTGA GGGCAAGGTC ACAACAACCT CGACCAGAAC CTGGGTCTTG CCCACCTACA ACAACCACTT GTACCTGCGT CTCGGAACAA CATCAAGCAG CAACACCTAC

AACGGATTCT CCACCCCCTG GGGATATTTT GACTTCAACA GATTCCACTG TCACTTCTCA CCACGTGACT

GGCAAAGACT CATCAACAAC AACTGGGGAC TACGACCAAA AGCCATGCGC GTTAAAATCT TCAATATCCA

AGTTAAGGAG GTCACAACGT CGAACGGCGA GACTACGGTC GCTAATAACC TTACCAGCAC GGTTCAGATA

TTTGCGGACT CGTCGTATGA GCTCCCGTAC GTGA AAV 12 (SEQ ID NO: 59)

AAAAGACTCC AAATCGGCCG ACCAACCCGG ACTCTGGGAA GGCCCCGGCC AAGAAAAAGC AAAAAGACGG

CGAACCAGCC GACTCTGCTA GAAGGACACT CGACTTTGAA GACTCTGGAG CAGGAGACGG ACCCCCTGAG

GGATCATCTT CCGGAGAAAT GTCTCATGAT GCTGAGATGC GTGCGGCGCC AGGCGGAAAT GCTGTCGAGG

CGGGACAAGG TGCCGATGGA GTGGGTAATG CCTCCGGTGA TTGGCATTGC GATTCCACCT GGTCAGAGGG CCGAGTCACC ACCACCAGCA CCCGAACCTG GGTCCTACCC ACGTACAACA ACCACCTGTA CCTGCGAATC

GGAACAACGG CCAACAGCAA CACCTACAAC GGATTCTCCA CCCCCTGGGG ATACTTTGAC TTTAACCGCT

TCCACTGCCA CTTTTCCCCA CGCGACTGGC AGCGACTCAT CAACAACAAC TGGGGACTCA GGCCGAAATC

GATGCGTGTT AAAATCTTCA ACATACAGGT CAAGGAGGTC ACGACGTCAA ACGGCGAGAC TACGGTCGCT

AATAACCTTA CCAGCACGGT TCAGATCTTT GCGGATTCGA CGTATGAACT CCCATACGTG A AAV 13 (SEQ ID NO: 60)

ATTTTGGTCA GACTGGCGAC AC AG AG TC AG TCCCAGACCC TCAACCACTC GGACAACCTC CCGCAGCCCC

CTCTGGTGTG GGATCTACTA CAATGGCTTC AGGCGGTGGC GCACCAATGG CAGACAATAA CGAGGGTGCC

GATGGAGTGG GTAATTCCTC AGGAAATTGG CATTGCGATT CCCAATGGCT GGGCGACAGA GTCATCACCA

CCAGCACCCG CACCTGGGCC CTGCCCACCT ACAACAATCA CCTCTACAAG CAAATCTCCA GCCAATCAGG AGCCACCAAC GACAACCACT ACTTTGGCTA CAGCACCCCC TGGGGGTATT TTGACTTCAA CAGATTCCAC TGCCACTTTT CACCACGTGA CTGGCAAAGA CTCATCAACA ACAACTGGGG ATTCCGACCC AAGAGACTCA ACTTCAAGCT CTTTAACATT CAAGTCAAAG AGGTCACGCA GAATGACGGT ACGACGACGA TTGCCAATAA CCTTACCAGC ACGGTTCAGG TGTTTACTGA CTCCGAGTAC CAGCTCCCGT ACGTCCTCGG CTCGGCGCAT CAGGGATGCC TCCCGCCGTT CCCAGCAGAC GTCTTCATGG TCCCACAGTA TGGATACCTC ACCCTGA

b-AAV (bovine) (SEQ ID NO: 61 )

AGCAGAGTCC TCAAGAGCCA GACTCCTCGA GCGGAGTTGG CAAGAAAGGC AAACAGCCTG CCAGAAAGAG

ACTCAACTTT GACGACGAAC CTGGAGCCGG AGACGGGCCT CCCCCAGAAG GACCATCTTC CGGAGCTATG

TCTACTGAGA CTGAAATGCG TGCAGCAGCT GGCGGAAATG GTGGCGATGC GGGACAAGGT GCCGAGGGAG

TGGGTAATGC CTCCGGTGAT TGGCATTGCG ATTCCACTTG GTCAGAGAGC CACGTCACCA CCACCTCAAC

CCGCACCTGG GTCCTGCCGA CCTACAACAA CCACCTGTAC CTGCGGCTCG GCTCGAGCAA CGCCAGCGAC

ACCTTCAACG GATTCTCCAC CCCCTGGGGA TACTTTGACT TTAACCGCTT CCACTGCCAC TTCTCGCCAA

GAGACTGGCA AAGGCTCATC AACAACCACT GGGGACTGCG CCCCAAAAGC ATGCAAGTCC GCATCTTCAA

CATCCAAGTT AAGGAGGTCA CGACGTCTAA CGGGGAGACG ACCGTATCCA ACAACCTCAC CAGCACGGTC

CAGATCTTTG CGGACAGCAC GTACGAGCTC CCGTACGTGA

Avian AAV ATCC VR-865 (SEQ ID NO: 62)

GTAAAGCCAT CTTCCAGGCC AAAAAGAGGG TTCTCGAACC CTTTGGTCTG GTGGAAGACT CAAAGACGGC

TCCGACCGGA GACAAGCGGA AAGGCGAAGA CGAACCTCGT TTGCCCGACA CTTCTTCACA GACTCCCAAG

AAAAACAAGA AGCCTCGCAA GGAAAGACCT TCCGGCGGGG CAGAAGATCC GGGCGAAGGC ACCTCTTCCA

ACGCTGGAGC AGCAGCACCC GCCTCTAGTG TGGGATCATC TATCATGGCT GAAGGAGGTG GCGGCCCAGT

GGGCGATGCA GGCCAGGGTG CCGATGGAGT GGGCAATTCC TCCGGAAATT GGCATTGCGA TTCCCAATGG

CTGGAAAACG GAGTCGTCAC TCGAACCACC CGAACCTGGG TCTTGCCCAG CTACAACAAC CACCTGTACA

AACGAATCCA AGGACCCAGC GGAGGCGACA ACAACAACAA ATTCTTTGGA TTCAGCACCC CCTGGGGATA

CTTTGACTAC AATCGATTCC ACTGCCACTT TTCCCCGCGA GACTGGCAAC GACTCATCAA CAACAACTGG

GGCATCCGTC CCAAAGCGAT GCGCTTTAGA CTCTTTAACA TCCAGGTTAA AGAGGTCACG GTCCAAGACT

TCAACACCAC CATCGGCAAC AACCTCACCA GTACGGTCCA GGTCTTTGCG GACAAGGACT ACCAACTGCC

GTACGTCCTC GGATCGGCTA CCGAAGGCAC CTTCCCGCCG TTCCCAGCGG ATATCTACAC GATCCCGCAG

TACGGGTACT GCACGCTAA

Mouse AAV 1 (SEQ ID NO: 63)

ACGAGGAGGA CCGTGAGTTC GCTGCCGCTG CAGCGGAGAC CGAAACTGGA AGCGCTCCCC CCACCGGCAA

TTTGGGACCT GGTACGATGG CTGGAGGCGG TAGCGCGCCA ATCGACGACG GCTCGTATGG TGCCGATGGA

GTGGGCAATG CCTCGGGAGA TTGGCATTGC GATTCCACAT GGCTGGACAA CTGTGTCATC ACCCGAACCA

CTCGGACCTG GAATCTGCCA ACCTACAACA ACCACATCTA CAAACGACTC AACGGAACGA CCTCCGGAGA

CCAAAGCTAC TTCGGATTCA GCACCCCCTG GGGATACTTT GACTTCAACC GCTTCCACTG TCATTTCTCC

CCTCGAGACT GGCAAAGACT CATCAACAAT AACTGGGGAC TCCGACCAAA GAGCCTACGG TTCAAAATCT

TTAACATTCA AGTTAAAGAA GTCACGACGC AAGACTCAAC GAAGATCATC TCCAATAACC TTACCAGCAC

GGTTCAGGTA TTTGCGGACA CGGAGTACCA GCTCCCGTAC GTGA

Avian AAV strain DA-1 (SEQ ID NO: 64)

TTGAACAACC CGACAACACG GCCGGGACCG GGGAGAAGCG TCCCGAACGC GTCGACGACT TTTTCCCGAA

AAAGAAGAAG GCCAAGACCG AGCAAGGCAA AGCCCCTGCT CAAACGGGCG AAGACCCCGG AGAAGGAACC

TCTTCCAACG CTGGATCAAG CGCCCCCTCT AGTGTGGGAT CATCTGTCAT GGCTGAAGGA GGTGGCGGTC CAATGGGCGA TGCAGGCCAA GGTGCCGACG GAGTGGGCAA TTCCTCGGGA AATTGGCATT GCGATTCCCA

ATGGCTGGAC AACGGAGTCG TTACCCGAAC CACTCGAACC TGGGTCCTGC CCAGCTACAA CAACCACTTG

TACAAGCGGA TCCAAGGACC GGGAGGAACC GACCCCAACA ATAAATTCTT TGGATTCAGC ACCCCCTGGG

GGTACTTTGA CTACAACCGA TTCCACTGCC ACTTCTCCCC CCGAGACTGG CAACGACTCA TCAACAACAA

CTGGGGCATC CGACCCAAAG CGATGCGCTT TAGACTCTTT AACATCCAGG TTAAAGAAGT CACTGTCCAA

GACTCCAACA CCACCATCGC CAACAACCTC ACCAGCACGG TCCAAGTCTT TGCGGACAAG GACTACCAGC

TGCCGTACGT CCTCGGATCG GC TACAGAGG GCACCTTCCC GCCGTTCCCA GCGGATATCT ACACGATCCC

GCAGTATGGT TACTGCACGC TAA

Caprine AAVl isolate AAV-Go. l (SEQ ID NO: 65)

ACGACCACTT TCCAAAAAGA AAGAAGGCTC GGACCGAAGA GGACTCCAAG CCTTCCACCT CGTCAGACGC

CGAAGCTGGA CCCAGCGGAT CCCAGCAGCT GCAAATCCCA GCACAACCAG CCTCAAGTTT GGGAGCTGAT

ACAATGTCTG CGGGAGGTGG CGGCCCATTG GGCGACAATA ACCAAGGTGC CGATGGAGTG GGCAATGCCT

CGGGAGATTG GCATTGCGAT TCCACGTGGA TGGGGGACAG AGTCGTCACC AAGTCCACCC GCACCTGGGT

GCTGCCCAGC TACAACAACC ACCAGTACCG AG AG AT C AAA AGCGGCTCCG TCGACGGAAG CAACGCCAAC

GCCTACTTTG GATACAGCAC CCCCTGGGGG TACTTTGACT TTAACCGCTT CCACAGCCAC TGGAGCCCCC

GAGACTGGCA AAGACTCATC AACAACTATT GGGGCTTCAG ACCCCGGTCT CTCAGAGTCA AAATCTTCAA

CATCCAAGTC AAAGAGGTCA CGGTGCAGGA CTCCACCACC ACCATCGCCA ACAACCTCAC CTCCACCGTC

CAAGTGTTTA CGGACGACGA C

Rat AAVl (SEQ ID NO: 66)

GCGTCGAGGA GCCGGAGCTG GCTCCTCCAG TCAAGCGTCC ACACTCGCCC GAGAAAACCC CAGAGAACCA

GAAGGGTCAG CCGCGACCGG ATCCCCGGAC GCCGGCCAAG AAGAGGCTCG AGTTCTCCGA TCAGCCTGGA

TCTTCAGCAG ACTTACCTGC ATCCTCACAA CAGTCCCAGC CTCCCGCAGG GGTTCCCGGT GTGGTTCCTG

GTACGATGTC TGCAGGAGGA GGCGCTCCAG TGGACGATGC TCAACAAGGT GCCGACGGAG TGGGCAATGC

CTCGGGAGAT TGGCATTGCG ATTCCAAATG GCTGGGCAAC CGAGTTCTCA CCCGATCCAC CCGGACCTGG

GTGCTGCCCA GCTACAACAA CCACCTGTAC AAGCAGATCT CAGACGCCTC CGGCGTGCAC AGCCTCCCCG

GGAGCCGATA CTTTGGCTAC AGCACCCCCT GGGGGTACTT CGACTTCAAT CGCTTCCACT GCCACTTCTC

GCCCAGAGAC TGGCAGCGCC TCGTCAATAA CCACTGGGGC TTCCGACCCA AGAGACTGCG AGTCAAACTC

TTCAACATCC AGGTCAAGGA GGTCACGACT ACTGATTCGA CGACCACGGT CTCCAACAAC CTCACGAGCA

CGGTCCAGGT CTTCACAGAC GACGAGTACC AGCTGCCGTA CGTCTGCGGC AACGCCACCG AGGGATGCCT

GCCGCCGTTC CCCCCGGACG TCTTCACGCT GCCGCAGTAC GGCTACGCGA CGCTGA

Goose parvovirus strain DB3 (SEQ ID NO: 67)

AAGACGGAGG AGCCACCGCG GAGGGCACCG AACCTGTGGC AGCATCTGAA ATGGCAGAGG GAGGAGGCGG

AGCTATGGGC GACTCTTCAG GGGGTGCCGA TGGAGTGGGT AATGCCTCGG GAAATTGGCA TTGCGATTCC

CAATGGATGG GAAACACAGT CATCACAAAG ACCACCAGAA CCTGGGTCCT GCCAAGCTAC AACAACCACA

TCTACAAAGC AATTACCAGT GGAACCTCTC AAGATGCAAA TGTCCAGTAT GCAGGATACA GTACCCCCTG

GGGGTACTTT GATTTCAACC GCTTCCACTG CCACTTCTCC CCTAGAGACT GGCAGAGACT TATCAACAAC

CATTGGGGAA TCCGACCCAA ATCTCTTAAA TTCAAGATCT TCAATGTCCA AG T C AAAGAA GTCACAACGC

AGGATCAAAC AAAGACCATT GCAAACAATC TCACCTCAAC GATTCAAGTC TTTACGGATG ATGAGCATCA

ACTCCCGTAT GTCCTGGGCT CGGCTACGGA AGGCACCATG CCGCCGTTCC CGTCGGATGT CTATGCCCTG CCGCAGTACG GGTACTGCAC AATGCACACC AACCAGAATG GAGCACGGTT CAATGACCGT AGTGCATTCT

ACTGCTTAG

Duck parvovirus strain 90-0219 (SEQ ID NO: 68)

AAAAGCCTAA ATTATCTGAA GAAAACTCTC CTTCACCCAG TAATAGTGGA GGAGAAGCAA GTGCAGCTGC

CACCGAAGGC TCCGAACCTG TGGCAGCACC TAACATGGCA GAGGGAGGAA GCGGAGCTAT GGGCGACTCT

GCAGGGGGTG CCGATGGAGT GGGTAATGCC TCAGGAAATT GGCATTGCGA TTCCCAATGG CTGGGAGACA

CAGTCATTAC CAAGACTACA AGAACCTGGG TCCTGCCAAG CTACAACAAC CACATCTACA AAGCCATCAC

AAGCGGAACA AACCCAGACA CAAATACCCA ATATGCTGGA TACAGCACCC CCTGGGGGTA CTTTGATTTC

AACAGATTCC ACTGCCATTT CTCTCCAAGA GACTGGCAGA GACTCATCAA CAACCATTGG GGGATTAGAC

CGAAAGCACT CAAATTCAAG ATATTCAATG TGCAAGTTAA AGAAGTCACG ACGCAAGACC AGACAAAGAC

TATTGCTAAC AACCTTACCT CTACAATCCA GATATTCACG GATAATGAAC ACCAGCTGCC CTATGTTCTG

GGCTCGGCCA CGGAGGGGAC GATGCCACCG TTCCCCTCAG ATGTGTATGC CTTGCCCCAG TACGGCTACT

GCACAATGCA CACCAACCAG AGTGGAGCTA GATTCAATGA CAGAAGTGCC TTCTATTGCT 1 TAG

Snake parvovirus (SEQ ID NO: 69)

ACGAATACTA TCCTAAAGCT AAAAAGGCCA AACAAGGCTT GCAGATACCA GCTCCACCTA AAGGCGGAGA

AGAAGAAGCT ACATCGTCAC AATCTGGAGG GAGCCCAGCA GGTTCCGATA CTAGCGGCAC ATCTGTCATG

GCTACAGGAG GAGGCGGTCC GATGGCAGAC GATAACCAGG GCGCCGAGGG AGTGGGTAAT TCCTCAGGTG

ATTGGCATTG CGATACCAAG TGGATGGGAG ACCACGTCAT TACAAAGTCA ACCAGAACTT GGGTGCTCCC

CACTTACGGG AATCATCTCT ACGGGCCTAT CAACTTTGAC GGCACCACAG GTTCGGGTGC TAATGCAGCC

TATGCAGGAT ACAAGACTCC CTGGGGGTAC TTTGACTTCA ATCGATTCCA TTGCCACTTC TCCCCCCGAG

ACTGGCAAAG ACTCA CAAC AACCACACAG GCATCAGGCC GAAAGGACTC AAAATCAAAG TCTTTAACGT

CCAAGTCAAA GAAGTTACAA CACAAGATTC AACGAAAACA ATTGCCAACA ATCTCACCAG CACCGTACAG

ATCTTTGCGG ACGAGAACTA CGACTTACCA TATGTATTAG

Examples

The following examples exemplify the invention for AAV, especially for AAV2. Due to the general similarities within the structures of the adeno-associated viruses and other parvoviruses the invention can be easily transferred to other parvoviruses encoding three viral capsid proteins.

1. Manufacture and Purification

Production and purification of AAV variants was described elsewhere (see WO 2008/145401 , especially example 4).

1.1. Production of AAV (like particles) in mammalian cells 1.1.1. Plasmids

Ad helper plasmid: An Ad helper plasmid encoding adenoviral proteins E2, E4 and VAI-VAII was used for AAV manufacturing in 293 or 293-T cells. The helper plasmid pUCAdE2/E4-VAI-VAII was constructed by subcloning the BamHI restriction fragment encoding the adenovirus (Ad) E2 and E4- ORF6 from pAdEasy-1 (Stratagene, La Jolla, USA) into the BamHI site of pUC19 (Fermentas, St. Leon-Rot, Germany). The resulting plasmid is referred to as pUCAdE2/E4. The VAI-VAII fragment from pAdVAntage™ (Promega, Mannheim, Germany) was amplified by PCR using the primers

5 XbaI-VAI-780-3': 5 ' -TCT AGA GGG CAC TCT TCC GTG GTC TGG TGG-3 1

(SEQ ID NO: 70), and

XbaI-VAII-1200-5': 5 ' -TCT AGA GCA AAA AAG GGG CTC GTC CCT GTT TCC- 3 '

(SEQ ID NO: 71)

cloned into pTOPO (Invitrogen, Carlsbad, USA) and then subcloned into the Xbal site of pUCAdE2/E4. 10 This plasmid was named pUCAdV.

AAV encoding plasmids: The construction of pUCAV2 is described in detail in US 6,846,665. Plasmid pTAV2.0 is described in Heilbronn (1990), pVP3 is described in Warrington (2004). Further AAV viral protein encoding plasmids are described within the respective examples.

1.1.2. Transfection for large scale virus production

15 293-T cells (ATCC, Manassas, USA) (7.5 x 106/dish) were seeded in 15 cm dishes (i.e. dish with a diameter of 15 cm) 24 h prior to transfection (cultivated in DMEM/10% FCS). Cells were transfected by calcium phosphate precipitation as described in US 2004/0053410.

In case of AAV promoter p40 dependent transcription a co-transfection with an adenoviral helper plasmid was performed. For co-transfection of the AAV encoding plasmid and pUCAdV a molar ratio

20 of the plasmids of 1 : 1 was chosen. For transfection of one culture plate with 293-T cells the calcium phosphate transfection protocol was used as described above, 12 μg AAV Cap encoding plasmid (pUCAV2, pTAV2.0, and pVP3, respectively) and 24 μg pUCAdV were used. In case of p40 independent transcription cells were transfected with the respective AAV VP1 , VP2 and/or VP3 encoding plasmid. For transfection of one culture plate of 293-T cells the calcium phosphate

25 transfection protocol was used as disclosed in US 2004/0053410, 36 μg total DNA were mixed in 875 μΐ 270 mM CaCl2. In brief, 875 μΐ 2x BBS (50 mM BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethane sulfonic acid) (pH 6.95), 280 mM NaCI and 1.5 mM Na2HP04) was added to the mixture and the resulting solution was carefully mixed by pipetting. The solution was incubated for 20 min at room temperature (RT) and then added drop-wise to the cell culture plate. After 18 h incubation of cells in a

30 humidified atmosphere at 35°C and 3% C02, medium was changed into a serum free DMEM (Invitrogen Carlsbad, USA) and cells were cultivated for an additional 2 d at 37°C, 5% C02 in a humidified atmosphere. 293-T cells were harvested with a cell lifter, transferred into 50 ml plastic tubes (Falcon) and centrifuged at 3000g, 4°C for 10 min. The cell pellet was resuspended in 0.5 ml lysis buffer (150 mM NaCI, 50 mM Tris, pH 8.5) per 15 cm dish and objected to three rounds of freeze and 5 thaw cycles (liquid nitrogen/37°C). The cell lysate was cleared by two centrifugation steps (3700g, 4°C, 20 min) and the AAV-containing supernatant was used for further purification. Alternatively the whole dishes were objected to freeze and thaw cycles (-50°C/RT). The remaining supernatant was collected and further purified as described in 1.2.

1.1.3. Small scale transfection and preparation of virus supernatants

Cells (5 x 1 OVdish) were seeded in 6 cm dishes 24 h prior to transfection. 293-T cells were transfected by calcium phosphate precipitation as described in US 2004/0053410. For HeLa and COS-1 cells transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer's manual. In case of promoter p40 dependent transcription of the cap gene (pTAV2.0, derivates thereof, and pVP3) cells were infected with adenovirus type 5 (Ad5) (MOI=10). After additional incubation for 24-48 h, cells were harvested in the medium and lysed by three freeze-thaw cycles (-80°C and 37°C). Lysates were incubated at 56°C for 30 min to inactivate Ad5. Cell debris was removed by centrifugation at l O.OOOg for 5 min.

1.1.4. Cell culture

HeLa and 293-T cells were maintained at 37°C and 5% C02 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat- inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine.

1.2. Purification

1.2.1. Tangential Cross Flow Filtration (TFF)

The binding of modified AAVLP to Fractogel EMD S03 " (M) resin beads (cation exchange chromatography) was intensively investigated. In general, AAVLPs which bind only at a pH <5.0 and a sodium chloride concentration of <30 mM could not be purified effectively by cation exchange chromatography. At this pH, host cell protein binding was very strong. Therefore, crude lysate was purified by an alternatively step. Crude lysate obtained after three freeze/thaw cycles was further processed by Tangential Cross Flow Filtration (TFF). First, lysate was cleared by centrifugation at 3.700g, 4°C for 20 min and cleared supernatant was further concentrated using a Tangential Cross Flow Filtration Unit (Sartoflow Slice 200 Benchtop Crossflow System, Sartorius Biotech GmbH, Gottingen, Germany) using a 100 kDa or a 300 kDa cut off membrane (SARTOCON Slice 200). The resulting TFF concentrate («45 ml) was then further processed by iodixanol density ultracentrifugation (ii) or ion exchange chromatography (iii). (i) Sucrose density gradient analysis: 1.5 x 106 cells were seeded in 10 cm dishes 24 h prior to transfection. They were harvested 48 h post transfection and lysed in 300 μΐ PBS-MK (phosphate- buffered saline: 18.4 mM Na2HP04, 10.9 mM KH2P04, 125 mM NaCl supplemented with 1 mM MgCl2, 2.5 mM KC1) by three freeze-thaw cycles (-80°C and 37°C). After treatment with 50 U/ml Benzonase (Sigma, Deisenhofen, Germany) for 30 min at 37°C and centrifugation at 3,700g for 20 min the supernatant was loaded onto a 1 1 ml 5-50% sucrose gradient (sucrose in PBS-MK, 10 mM EDTA, containing one tablet of complete mini EDTA free protease inhibitor (Roche, Mannheim, Germany)) in polyallomer centrifuge tubes (14 by 89 mm; Beckman Coulter, Marseille, France). After centrifugation at 160,000 g for 1 h at 16°C (SW41 rotor; Beckman), 500 μΐ fractions were collected from the bottom of the tubes. As reference empty AAV2 capsids (60 S) were analyzed in a separate gradient. For immuno dot blot assay 50 μΐ of heat denatured (99°C for 10 min) or non denatured aliquots of the fractions were transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using a vacuum blotter. Membranes were blocked for 1 h in PBS containing 10% skim milk powder and then incubated for 1 h with monoclonal antibodies B l (Progen, Heidelberg, Germany, Cat. No: 65158) to detect denatured capsid proteins or A20 to detect non denatured capsids. Antibodies Bl and A20 were applied in 1 : 10 dilutions. Membranes were washed several times with PBS and incubated for 1 h with a peroxidase-coupled goat anti-mouse antibody (1 :5000 dilution) (Dianova, Hamburg, Germany). Then, membranes were washed again and the antibody reaction was visualized using an enhanced chemiluminescence detection kit (Amersham, Braunschweig, Germany). For Western blot analysis 15 μΙ per fraction were processed for SDS-PAGE and then probed with monoclonal antibodies A69 (Progen, Heidelberg, Germany, Cat. No: 65157) or Bl .

(ii) Purification of AAV particles by density gradient centrifugation using iodixanol: 20 ml of the resulting TFF concentrate were transferred to Qickseal ultracentrifugation tubes (26x77 mm, Beckman Coulter, Marseille, France). Iodixanol solutions (purchased from Sigma, Deisenhofen, Germany) of different concentrations were layered beneath the virus containing lysate. By this an Iodixanol gradient was created composed of 4 ml 60% on the bottom, 5 ml 40%, 6 ml 25% and 7 ml 15% Iodixanol with the virus solution on top (20 ml). The gradient was spun in an ultracentrifuge at 416,000 g for 1 h at 18°C. The 40% phase containing the AAV particles was then extracted with a canula by puncturing the tube underneath the 40% phase and allowing the solution to drip into a collecting tube until the 25% phase was reached.

(iii) Purification of AAV particles by chromatography - empty wtVP3 and modified VP3 AAVLPs

Cation exchange chromatography (AKTA explorer system): Total lysate containing empty wtVP3 and modified AAVLPs was obtained by performing three freeze thaw cycles (-54°C/37°C). Total lysate was cleared by two filter capsules (Sartopure PP2; 065 μηι and Sartopore 2 (0,2/0, 1 μπι) obtained from Sartorius, Gottingen, Germany. The pH of the resulting cleared lysate was adjusted to 5.5 - 7.5 depending on the modified AAVLPs. In addition, the conductivity of salt was reduced to approximately 3.5 or 6.5 mS/cm (-30 or 60 mM NaCl) by adding sterile water. A Fractogel EMD S03 " (M) chromatography column (120 mm in height; 15 mm in diameter, XK16, GE Healthcare, Munich, Germany) was packed and equilibrated using 5 CV running buffer consisting of 30 or 60 mM NaCl, 50 mM HEPES (pH 6.0), 2.5 mM MgCl2. After equilibration, cleared lysate was separated through the Fractogel EMD S03~ (M) packed chromatography column (flow rate 15 ml/min). After separation, column was washed using 5 CV running buffer mentioned above. Bound particles (peak 1 ; wtVP3 or modified AAVLPs) were effectively eluted at a sodium chloride concentration of 600 mM (peak 1«45 ml).

Buffer exchange (AKTA explorer system): To adjust the pH and the salt concentration of the eluted proteins (peak 1) for successive anion exchange chromatography, buffer exchange was performed using a Sephadex G25 packed chromatography column (500 mm in height; 15 mm in diameter, XK26, GE Healthcare, Munich, Germany) (flow rate 15 ml/min). After column equilibration using 3 CV CaptoQ running buffer consisting of 25 mM Tris (pH 8.2), 100 - 150 mM, 2.5 mM MgCl2 peak 1 was separated through the column. Protein fraction («120 ml) was collected.

Anion exchange chromatography (AKTA explorer system): A CaptoQ chromatography column (120 mm in height; 15 mm in diameter, XK16, GE Healthcare, Munich, Germany) was equilibrated using 5 CV CaptoQ running buffer consisting of 25 mM Tris (pH 8.2), 100 - 150 mM NaCl , 2.5 mM MgCl2. After equilibration, the protein fraction obtained after buffer exchange (appr. 120 ml) was loaded and separated through the chromatography column (flow rate 10 ml/min). Flow-through containing 90% of the particles (appr. 120 ml) was collected.

Alternatively, column was equilibrated using CaptoQ buffer consisting of 25 mM Tris (pH 8.2), 20 mM NaCl, 2.5 mM MgCl2. Bound particles (wtVP3/modified AAVLPS) were eluted by 100 - 150 mM NaCl (peak 1 fraction).

Particle concentration using centrifugal filter devices: Flow-through containing wtVP3 or modified AAVLPs were concentrated using Centricon Plus-70 (cut off 100 kDa) centrifugal filter devices (Millipore). Concentration was carried out using a swinging-bucket rotor (MULIFUGE L-R; Heraeus, Hanau, Germany) at 3500 g, 20°C for 15 min. Resulting concentrate (appr. 45 ml) was immediately separated through a size exclusion chromatography.

Size exclusion chromatography (AKTA explorer system): A Superdex 200 (prep grade) chromatography column (500 mm in height; 50 mm in diameter, XK50, GE Healthcare, Munich, Germany) was packed and equilibrated using 2 CV running buffer consisting of 200 mM NaCl, 50 mM HEPES (pH 6.0), 2.5 mM MgCl2. The particle concentrate or the eluted peak 1 fraction mentioned above was separated through the column (flow-rate 15 ml/min). Particles eluted first (SEC fraction no. 1-13; each 5 ml). SEC fractions with a particle purity of greater than 95% were pooled, sterile filtered (0.2 μηι) (Minisart; Sartoriusstedim) and stored at -84°C.

1.3. Analysis of protein expression by Western blot

Identical portions of harvested cells or identical amounts of purified particles were processed for SDS- PAGE. Protein expression was analyzed by Western blot assay using monoclonal antibodies A69, Bl (Progen, Heidelberg, Germany), anti-AU l (Covance, Emeryville, USA), anti-GFP (clone B-2; Santa Cruz Biotechnology, Santa Cruz, USA) as described previously (Wistuba et al., 1995). Variations of the protocols are indicated within the description of the respective examples.

1.4. Titer analysis

Capsid titers were determined using a commercially available AAV2 titration ELISA kit (Progen, Heidelberg, Germany Cat. No: PRATV) or the respective AAV1 titration ELISA kit (Progen, Heidelberg, Germany Cat. No: PRAAV1) according to the manufacturer's manual.

VP3 capsid manufacture was also described in Sonntag et al. (2010).

1.5. General results

Electron microscopic images of AAV VLPs confirmed that the morphology of virus-like particles assembled of VP1 , VP2 and VP3 (VP 1 ,2,3 VLP) is comparable to that of VLPs assembled only of VP3 (data in PCT/EP2010/001343).

Capsid assembly of VP3 cloned from AAV l, AAV2 and AAV5, respectively, was compared after co- transfection of pVP2N-gfp cloned from AAV2 and AAV l, respectively, or Bluescript vector (pBS). Expression of VP3 in the absence of any other viral protein (pBS control) showed no detectable capsid formation, irrespective of its origin. In contrast, expression of AAP (expressed from the respective pVP2N-gfp construct) from serotype AAV l completely restored AAV2 VP3 assembly (compared to assembly mediated by AAP from AAV2). Also vice e versa, AAP from AAV2 completely restored AAVl VP3 assembly (compared to assembly mediated by AAP from AAVl). AAP from AAV5 was only partially able to complement AAV2 VP3 assembly and failed to complement AAVl VP3 assembly. Further, AAV2 and AAVl AAP failed to complement AAV5 VP3 assembly. The failure of trans-complementation with respect to AAV5 constructs may be due to the fact that AAPs in these experiments were fused to GFP leading to a short C-terminal deletion of AAP which might interfere with the complementation of more distant parvoviruses while activity is sufficient for closely related serotypes. A further likely explanation is that more distant AAV serotypes are only partially able to complement each other with respect to VP3 assembly. Whereas AAP from AAV l and AAV2 have a 71.5% identity and 81.0% similarity (Smith- Waterman Alignment), AAV2 and AAV5 only have a 56.2% identity and 60.8% similarity. These numbers are even lower with respect to AAVl compared to AAV5 (53.8% identity and 58.1% similarity). Accordingly, the skilled artisan will be able to select functionally active AAPs from different serotypes and/or other functionally active variants by looking at identities / similarities of AAP (data in PCT/EP2010/001343).

Still, these results confirm that parvoviruses other than AAV2 encode functional AAP and make use of the same mechanism for capsid assembly enabling the manufacture of VLPs made from VP3 only. Further, AAP and VP3 are in principal interchangeable between different parvoviruses, especially between closely related viruses.

2. VP3 capsid assembly can be achieved in insect cells 2.1. Cloning of the VPl mutant "Modification 4"

The construct pVL_VPl_MOD4 was generated to produce viral particles consisting essentially of the capsid protein VP3 in the absence of any Rep expression. In detail, pUC19AV2 (described in detail in US 6,846,665) was used as template to amplify VPl according to standard PCR conditions in the presence of the following primers:

Insect_mod_4_s: 5 ' -CAC CCG CGG GGA TCC GCC GCT GCC GAC GGT TAT CTA CCC GAT TGG

CTC- 3 ' (SEQ ID NO: 72), and

E_VP2_rev: 5 ' -CGC GAA TTC CTA TTA CAG ATT ACG AGT CAG G- 3 * (SEQ ID NO: 73) Thereby, the wild type translation start codon ATG (coding for Methionin) of VPl was changed into GCC (Alanin) and inactivated. The resulting EcoRI/BamHI fragment was cloned into pBSIIKS (Stratagene, La Jolla, CA, USA). This vector was used to inactivate the translation start codon of VP2 by site directed mutagenesis according to the instructions of the QuickChange II Site directed mutagenesis kit (Stratagene) using the following primers:

Insect-muta_4_s: 5 ' -ACC TGT TAA GAC AGC TCC GGG AAA AAA G- 3 ' (SEQ ID NO: 74)

Insect-muta_4_as: 5 ' -CTT τττ TCC CGG AGC TGT CTT AAC AGG T- 3 ' (SEQ ID NO: 75) Thereby, the wildtype translation start codon ACG of VP2 was changed into ACA (both coding for Threonin). The resulting construct was digested with restriction enzymes BamHI and EcoRI and cloned into the baculo transfer vector pVL1393. As a result, the construct contained the complete AAV cap gene with mutations of the VPl and VP2 start codons but no rep cds. (Figure 1)

2.2. cloning of pVL_VP2

AAV2 VP2 was amplified using the primers E_VP2_for and E_VP2_rev listed below. Thereby, the wildtype VP2 translation start codon ACG (coding for Threonine) was changed into ATG (Methionine). Primers:

E_VP2_for: 5~-cac ccg egg gga tec act atggct ccg gga aaa aag agg- 3 "

(SEQ ID NO: 76)

E_VP2_rev: 5v-cgc gaa ttc eta tta cag att acg agt cag g-3 (SEQ ID NO: 77) The resulting construct was cloned into the baculo transfer vector pVL1393.

2.3. cloning of pVL_VP3

AAV2 VP3 was amplified using the primers E_VP3_for and E_VP3_rev listed below. Primers: E_VP3_for: 5~-CAC CCG CGG GGA TCC ACT ATG get ACA GGC AGT GGC GCA C-3~ (SEQ ID NO: 78)

E_VP2_rev: 5"-cgc gaa ttc eta tta cag att acg agt cag g-3 v (SEQ ID NO: 79).

The resulting construct was cloned into the baculo transfer vector pVL1393. 2.4. Analysis of particle production

AAV particles were produced as described in 1.1. Cell lysates were investigated by Western blot analysis for protein expression. pVL_VPl_MOD4 showed only VP3 expression, pVL_VP2 VP2 expression, while pVL_VP3 showed in addition to VP3 smaller degradation signals (Fig. 12 B) . Titers were obtained by an A20 ELISA. A titer of lxl 012 particles/ml was observed for the modification 4 construct while VP2 pVL_VP2 showed a titer of 9xl08 particles/ml and pVL_VP3 only a titer of lxlO8 particles/ml (Fig. 12 C).

Conclusion

This result shows that AAV VLPs can be produced in insect cells as efficiently as in mammalian cells. The data show that in insect cells the N-terminal sequence of VP3 also seems to be required and sufficient for efficient VP3 capsid assembly. Further a change of the VP2 start codon from ACG into ATG comes along with loss of efficiency in capsid assembly (Figure 1C). We speculate that particle assembly from pVL_VP2 goes along with minor VP3 expression initiated from a VP3 ATG which was left intact in the construct.

3. Trans-complementation of AAP and VP3 cloned from different serotypes

Capsid assembly of VP3 cloned from AAV1, AAV2 and AAV5, respectively, was compared (data not shown). As expected, expression of VP3 in the absence of any other viral protein showed no detectable capsid formation, irrespective of its origin. In contrast, expression of AAP from serotype AAV1 completely restored AAV2 VP3 assembly (compared to assembly mediated by AAP from AAV2). Also vice e versa, AAP from AAV2 completely restored AAV1 VP3 assembly (compared to assembly mediated by AAP from AAV1 ). AAP from AAV5 was only partially able to complement AAV2 VP3 assembly and failed to complement AAVl VP3 assembly. Further, AAV2 and AAVl AAP failed to complement AAV5 VP3 assembly. The failure of trans-complementation with respect to AAV5 constructs may be due to the fact that AAPs in these experiments were fused to GFP leading to a short C-terminal deletion of AAP which might interfere with the complementation of more distant parvoviruses while activity is sufficient for closely related serotypes. A further likely explanation is that more distant AAV serotypes are only partially able to complement each other with respect to VP3 assembly. Whereas AAP from AAVl and AAV2 have a 71.5% identity and 81.0% similarity (Smith- Waterman Alignment), AAV2 and AAV5 only have a 56.2% identity and 60.8% similarity. These numbers are even lower with respect to AAVl compared to AAV5 (53.8% identity and 58.1% similarity). Accordingly, the skilled artisan will be able to select functionally active AAPs from different serotypes and/or other functionally active variants by looking at identities / similarities of AAP.

4. Cloning of an expression vector for generation of an monovalent AAVLP-based HPV16 vaccine

For generation of empty VLPs composed of VP3 proteins containing the cross-reacting HPV16 L2 peptide QLYKTCKQAGTCPPDIIPKV ("HPV16 L2 epitope"; SEQ ID NO: 3) at position 1-587 (amino acid number relative to the VP 1 protein of AAV2) the L2 epitope sequence was cloned into the vector pCIVP2mutACG_NotI/BspEI_ANot3. The vector contains the overlapping AAV2 VP2 and VP3 coding sequences cloned into the Xhol/Notl site of pCI (Promega). In pCFVP2mutACG_NotI/BspEI_ANot3 the ACG start-codon of VP2 is destroyed and replaced by a GAG codon. The vector was further modified by site directed-mutagenesis to introduce a Notl and BspEI restriction site at position 1-587. Simultaneously, the Arg residue at position 1-588 of AAV -2 VP (amino acid number relative to the VP1 protein of AAV-2) was substituted by an Ala residue (substitution R588A). In addition, the Notl site of the multiple cloning site of the vector pCI was destroyed by site directed mutagenesis.

The HPV16 L2 epitope was cloned into the Notl/BspEI site of pCIVP2mutACG_NotI/BspEI_ANot3 using annealed oligonucleotides encoding the epitope sequence flanked by an adaptor sequence and 5 '- site extensions. The 5 '-site extensions of the oligonucleotides were designed so that annealing of the sense and anti-sense oligonucleotides results in a dsDNA with 5 '-site and 3 '-site overhangs compatible with overhangs generated by Notl and BspEI restriction of pCIVP2mutACG_NotI/BspEI_ANot3. The sequences of the oligonucleotides and the respective epitope sequence are shown in Table 3.

To anneal the oligonucleotides 25 μg of the sense oligonucleotide and 25 μg of the antisense oligonucleotide were mixed in a total volume of 100 μΐ lx PCR-Buffer (Qiagen) and incubated for 3 min at 95°C in a thermomixer. After 3 min at 95°C the thermomixer was switched off and the tubes were left in the incubator for an additional 2 h to allow annealing of the oligonucleotides during the cooling down of the incubator. To clone the annealed oligonucleotides into pCrVP2mutACG_NotI/BspEI_ANot3 the vector was linearized by restriction with Notl and Mrol (BspEI isoenzyme) and the cloning reaction was performed using the Rapid DNA Ligation Kit (Roche). Briefly, the annealed oligonucleotides were diluted 10-fold in lx DNA Dilution Buffer and incubated for 5 min at 50°C. 100 ng of the annealed oligonucleotides and 50 ng of the linearized vector were used in the ligation reaction, which was performed according to the instructions of the manufacturer of the Rapid DNA Ligation Kit (Roche). E. coli DH5a were transformed with an aliquot of the ligation reaction and plated on LB-Amp agar plates. Plasmids were prepared according to standard procedures and were analyzed by sequencing. The resulting expression vector was named pCIVP2mutACGJL2. 5. Cloning of a vector for generation of a bivalent AAVLP-based HPV16/HPV31 vaccine

For cloning of expression vectors encoding AAVLPs composed of VP3 capsid proteins containing a L2 epitope derived from HPV31 and HPV16 at position 1-453 and 1-587 (amino acid number relative to the VP1 protein of AAV2), respectively, the HPV16 L2 epitope was cloned into pCrVP2mutACG_NotI/BspEI_DNot3 at the site corresponding to 1-587 as described above.

The cross-reacting HPV31 L2 peptide QLYQTCKAAGTCPSDVEPKI ("HPV31 L2 eptiope"; SEQ ID NO: 4) was initially cloned into the Notl/AscI restriction site of the vector pCIVP2-I453-NotI-AscI (described in WO 2008/145400). Briefly, the vector pCI-VP2-I453-Not-AscI was created by PCR amplification of the AAV2 VP2 gene and cloning of the respective PCR product into the Xhol/Notl site of vector pCI (Promega). The resulting vector pCIVP2 was modified by destruction of the Notl restriction site of the cloning site by site-directed mutagenesis. The vector was further modified by introduction of a novel singular Notl and Ascl restriction site allowing the insertion of epitope sequences at position 1-453 of the AAV2 capsid. In addition, an Fsel site located between 1-453 and I- 587 was introduced in-frame into the VP coding sequence of pCIVP2-I453 -Notl- Ascl by site directed mutagenesis.

For cloning of the HPV-31 L2 epitope sequences into the Notl/AscI site of the vector pCIVP2-I453- Notl-Ascl sense- and anti-sense oligonucleotides were designed that encode the respective epitope flanked by an adaptor sequence and 5 '-site extensions. The 5 '-site extension of the oligonucleotides was designed so that annealing of the sense and anti-sense oligonucleotides results in a dsDNA with 5 '-site and 3 '-site overhangs compatible with overhangs generated by Notl and Ascl restriction of pClVP2- 1453-Not-AscI. Cloning of the annealed oligonucleotides was performed as described above. The sequences of the oligonucleotides and the respective epitope sequences are shown in Table 3.

Table 3: Sequences of the oligonucleotides

Name/ Forward Reverse

Type

Peptide Seq. Oligonucleotide Oligonucleotide

HPV16 L2 5 ' - 5 ' -

GGCCGCAGGTGGCGGACAGCTGTAC CCGGACCCGCCCCCCACCTTGGGGA

QLYKTCKQAGTCPP AAGACCTGCAAGCAGGCCGGCACCT TGATGTCGGGGGGGCAGGTGCCGGC

DI I PKV Epitope

GCCCCCCCGACATCATCCCCAAGGT CTGCTTGCAGGTCTTGTACAGCTGT

(SEQ ID NO: 3)

GGGGGGCGGGT -3 ' CCGCCACCTGC- 3 '

(SEQ ID NO: 80) (SEQ ID NO: 81) Name/ Forward Reverse

Type

Peptide Seq. Oligonucleotide Oligonucleotide

5 ' - 5 ' -

HPV31 L2 GGCCGGCGGTGGAGGCGGTCAGCTG CGCGCCCTCCACCGCCTCCGATCTT

QLYQTCKAAGTC PS TACCAGACCTGCAAGGCCGCCGGCA GGGGATCACGTCGCTGGGGCAGGTG

Epitope

DVI PKI CCTGCCCCAGCGACGTGATCCCCAA CCGGCGGCCTTGCAGGTCTGGTACA

(SEQ ID NO: 4) GATCGGAGGCGGTGGAGGG- 3 ' GCTGACCGCCTCCACCGCC-3 '

(SEQ ID NO: 82) (SEQ ID NO: 83)

For generation of bivalent AAVLPs displaying both HVP16 and HPV31 L2 epitopes at 1-587 and 1-453 the BsiWI/Fsel fragment of pCIVP2-I453-NotI-AscI containing the HPV31 L2 epitope inserted at 1-453 was subcloned into the vector pCIVP2mutACG_L2, which contains the HPV16 L2 epitope inserted into 1-587 (described above). To allow subcloning of the fragment an Fsel site located between 1-453 and I- 587 was introduced in-frame into the VP coding sequence of pCrVP2mutACG_L2 by site directed mutagenesis. Subcloning was performed according to standard procedures.

The resulting vector (pAAVLP_L2_Bi) already containing the RsgsA substitution (Arg residue at position of 588 substituted by Ala - amino acid number relative to the VPl protein of AAV2) was used for production of bivalent AAVLP displaying the HPV16 and HPV31 L2 epitope at position 1-587 and I- 453, respectively.

6. Site directed mutagenesis of an expression vector encoding a bivalent AAVLP-based HPV-16/HPV-31 vaccine

The vector pAAVLP_L2_Bi (described above) was modified by the following additional mutation: The Arg residue at position 585 (amino acid number relative to the VPl protein of AAV2) was substituted by an Ala residue (substitution Ρν585Α). The substitution was performed by site-directed mutagenesis using the oligonucleotides

L2-Pv585A-uni: 5'-ctaccaacctccag GCC ggcaacgcggccgcag-3' (SEQ ID NO: 84) and

L2-R585A-rev: 5'-ctgcggccgcgttgcc GGC ctggaggttggtag-3' (SEQ ID NO: 85) and the Quick Change site-directed mutagenesis kit (Stratagene). Mutagenesis was performed according to the instructions of the manufacturer. The resulting plasmid was named pAAVLP_L2_Bi_R585A. The plasmid pAAVLP_L2_Bi_R585A was further modified by the substitution of the adjacent Arg and Ala residues within the C-terminal adaptor sequence of the HPV31 L2 epitope at 1-453 by Ser and Gly, respectively. The substitutions were performed by site-directed mutagenesis using the oligonucleotides R453S-uni: 5'-ggcggtggaggg TCC GGA gctaccaccacgcag-3' (SEQ ID NO: 86) and R453S-rev: 5'-ctgcgtggtggtagc TCC GGA ccctccaccgcc-3' (SEQ ID NO: 87) and the Quick Change site-directed mutagenesis kit (Stratagene). The resulting plasmid was named pAAVLP_L2_Bi_R5g5A_R453S. The individual substitutions within the different expression vectors are summarized in Table 4.

Table 4: Sequence insertions

Figure imgf000039_0001

Epitope sequences are underlined. Adaptor sequences are shown in italic letters. Residues of the AAV2 VP protein are shown in lower-case letters. Substitutions are shown in bold/underlined letters (R585A, R588A and RA->SG in adaptor sequence at 1-453).

7. Manufacture of AAVLPs with anti-HPV L2 polyclonal antibodies

Bivalent AAVLPs with different Arg substitutions (AAVLP-L2-Bi_R588A, AAVLP-L2-Bi_R585A_R588A and AAVLP-L2-Bi_R585A_R588A_R453S) displaying the HPV 16 L2 epitope at position 1-587 and the HPV-31 L2 epitope at position 1-453 of the capsid were produced as described in 1.1 and 1.2 from 20 15 cm dishes each. The amount of AAVLPs was quantified prior to and after purification using the AAV2 Elisa obtained from Progen (Heidelbert). Table 5: Yields AAVLP-L2-B. vaccine candidates

Figure imgf000040_0001

Whereas both vaccine candidates AAVLP-L2-Bi_R58gA and AAVLP-L2-Bi_R585A_R588A have a relatively low yield during expression (amount prior to purification), the starting amount produced for the AAVLP-L2-Bi_R.585A_R.58gA R453S vaccine candidate is higher by a factor of about 3.5 although all constructs were expressed under identical conditions (see Table 5). Although AAVLP-L2-Bi_R588A and AAVLP-L2-Bi_R585A_R588A were expressed almost with an equal efficiency, the mutant with the double Arginin substitution at positions 585 and 588 had an about 3 fold increased yield after purification compared to the single Arginin substitution at position 588. The triple Arginin substitution mutant has a 52% recovery of AAVLPs equaling a 3.5 fold increase in yield after purification compared to the double Arginin substitution.

Purified AAVLPs were analyzed by western blotting. Results show that all three AAVLP HPV L2 vaccine candidates are stable (see Figure 3). Apparently, differences in yield are not due to degradation. We conclude that the Arginin substitutions have an a pronounced effect on yield prior to purification and during purification.

8. Interaction of AAVLPs with anti-HPV L2 polyclonal antibodies

The AAVLPs (AAVLP-L2-B _R585A_R588A_R453S) were analyzed by dot blot experiments. 0.5 to 2 μg AAVLP particles of a bivalent HPV vaccine targeting HPV 16 and HPV31 L2 were spotted onto a nitrocellulose membrane using a vacuum device. As negative control equal amounts of wtAAV2 VP3 AAVLPs used (lower lanes). As a positive control 5 and 10 μg of the inserted L2-peptide of HPV 16 and HPV31 were dotted (right lanes). After blocking of the membrane with blocking buffer (5% milk powder in PBS containing 0.1% Tween-20), the membrane was incubated with anti-HPV 16 L2 polyclonal antibody or anti-HPV31 L2 polyclonal antibody. After washing of the membrane with PBS/0.05% Tween-20, binding of the antibody to the AAVLPs /peptides was detected using a secondary HRP-labeled antibody (Figure 4). The data demonstrate that the bivalent HPV L2 AAVLP are recognized very well (compared to the isolated peptide spotted at much higher amounts) by both the anti-HPV16 L2 polyclonal and the anti- HPV31 L2 polyclonal antibody in the native state, whereas wild type VP3 AAVLP are not recognized at all. Cross-reactivity of the anti-L2 polyclonal antibodies can be seen as the anti-HPV16 L2 antibody to a minor extent also recognizes the HPV31 L2 peptide (A) and vice e versa (B). Accordingly, it is difficult to conclude whether both L2 insertions into the AAVLP are functional. However, from the observed effect that recognition by the anti-HPV31 L2 antibody can be largely reduced by heat inactivation, whereas recognition by the anti-HPV 16 L2 antibody not, we assume that both insertions are functional, but the insertion of HPV31 L2 17-36 at position 1-453 is heat-sensitive.

9. Immunization of rabbits with a bivalent AAVLP-based HPV vaccine

Three rabbits were immunized by four intramuscular administrations of the bivalent AAVLPs. Each dose of the AAVLP vaccine (250 μΐ; 13.5 μg) was formulated with 500 μΐ Montanide ISA 720 (Seppic) as adjuvant. The first boost immunization of rabbits was performed 2 weeks after an initial prime immunization. Rabbits were boosted another two times with the vaccine at intervals of 3 weeks. Serum of the immunized animals was prepared two weeks after each boost immunization.

Induction of HPV specific-antibodies in vaccinated animals was determined by ELISA using the synthetic HPV 16 L2 peptide or the HPV31 L2 peptide (see Table 2) as antigen. Briefly, a 96-well Maxisorp plate (Nunc) was coated with the peptides (1.0 μg/well) for 18h at 4°C. After coating wells were washed with wash buffer (PBS / 0.1% Tween-20) and subsequently incubated with blocking buffer (5% skim milk in wash buffer) for lh at 37°C. After blocking of the wells, immobilized peptides were incubated with serial dilutions of the immune sera in dilution buffer (wash buffer with 1% skim milk and 1% BSA) for lh at 37°C. Rabbit pre-immune sera served as negative control. After washing binding of rabbit IgG to the immobilized peptides was detected using a HRP-labeled anti-rabbit IgG antibody (DAKO). Signals were detected using TMB (KemEnTec) as substrate.

Antibody titers were determined by end point dilution. The titer of the immune serum corresponds to the intersection point of the titration curve of the immune sera with the limit of detection of the assay. The limit of detection (LOD) of the assay was calculated as follows:

Mean OD (pre-immune sera) + 3.3 x standard deviation OD (pre-immune sera)

Results are depicted in

Figure 5. The data demonstrate that the tested bivalent AAVLP-based HPV vaccine (AAVLP-L2-Bi) induces high titers of antibodies specific for the HPV16 and HPV31 L2 derived epitopes that are in the range of 40,000 - 220,000. 10. Immunization of mice with bivalent AAVLP-based HPV vaccine and cross-neutralization analysis

C57BL/6 (H-2b) and Balb/c female mice at 6-8 weeks of age were purchased from Charles River Wiga 5 (Sulzfeld, Germany) and kept in an isolator at the animal facilities of the DKFZ were housed in accordance to the institutional guidelines.

For immunization, female mice were kept under deep ketamine 10% / rompun 2% anesthesia by intraperitoneal injection. Low doses (LD, lE+1 1 particles / dose) or high doses (HD, 5E+12 particles / 10 dose) of AAVLP-L2-Bi_R585A_R588A R453S comprising HPV 16 and 31 epitopes (which generation is described afore) were delivered intramusculary into the tibia anterior muscle of the right leg with or without adjuvant. One tenth of the human dose of Cervarix® or Gardasil® (50 μΐ) was delivered in the same way. Two weeks (schedule 1) or two and four weeks (schedule 2) later a boost was administrated. Samples of blood were collected 45 days after the first immunization. All samples were stored at -20°C.

15

As adjuvant Montanide ISA 51 (Seppic GmbH, Germany) was applied in a mixture of 1 : 1 with the antigen per dose.

Antibody responses were measured as follows: The presence of HPV 16 L2-specific IgG antibodies in 20 sera of immunized mice was determined by GST L2-ELISA. Briefly, 96-well plastic plates were coated overnight at 4°C with 50 μΐ of coating buffer containing glutathione-casein, followed by the addition of 50 μΐ of GST-L2 for 1 h at 37 °C. After washing with PBS-T, plates were blocked with 0.2% casein in PBS for 1 hr at 37°C. Prediluted sera (in 2-fold dilutions starting from 1 :50 to 1 :500,000) were added, and plates were incubated for 1 hr at 37°C. After washing, plates were incubated for 1 hr at 37°C with 25 1 :5000 diluted HRP-coupled antimouse IgG secondary antibody (Southern Biotechnology, Birmingham, MA) in 0.2% casein, TMB (3,3',5,5'-tetramethylbenzidine) substrate solution (Sigma) was used as substrate. OD was measured in an ELISA reader at 450 nm after 10 min and 30 min incubation at room temperature. Nonspecific binding was determined by incubating mice sera in wells containing GST only.

30

The pseudovirions of HPV16, 18, 31, 45, 52 and 58 were prepared as described by Rubio et al. 2009.

The neutralizing activity of the various AAVLP-L2-Bi antisera were measured by neutralization assays in titration experiments carried out against the HPV 16, 18, 31, 45, 52 and 58 pseudovirions (Rubio et al. 35 2009). The results of the neutralization assays for the antisera derived from the above described immunization experiments are depicted in Tables 6 to 8. Table 6. In vitro pseudovirion neutralization titers of antisera from C57BL/6 (H-2b) mice vaccinated twice with AAVLP-L2-Bi

Neutralization titers of pseudovirions

homologous heterologous

Group Mouse HP VI 6 HPV31 HP VI 8 HPV45 HPV52 HPV58

Schedule

1 1280 0 160 320 40 80 1, LD

2 640 0 640 160 40 160

3 2756 40 574 0 0 0

4 2560 40 0 40 0 40

5 777 0 0 0 0 0

Schedule

1 1251 60 40 0 0 40 1 , HD

2 2031 603 908 763 40 1630

3 320 80 0 0 160 160

Table 7. In vitro pseudovirion neutralization titers of antisera from Balb/c mice vaccinated twice or three times with AAVLP-L2-Bi

Neutralization titers of pseudovirions

homologous heterologous

Group Mouse HP VI 6 HPV31 HPV18 HPV45 HPV52 HPV58

Schedule

1 686 319 565 40 61 150 1, LD

2 41 15 90 1 125 72 501 5258

3 60 0 0 0 0 0

4 141 3668 0 106 70 1010

5 40 533 40 40 127 221

6 6354 40 2159 0 0 0

7 1737 699 416 74 60 582

8 2153 40 2844 0 0 0

9 1 174 1 14 0 0 102 104

10 228 239 185 40 0 0

Schedule

1 98 88 92 102 60 0 2, LD

2 569 3348 439 395 153 1854

3 89 0 0 0 0 0 4 561 40 615 0 0 0

5 15000 1018 1817 724 40 1012

6 2198 120 40 74 0 0

7 5397 0 1767 0 0 0

8 1202 0 209 198 0 0

9 1007 3671 454 124 0 0

10 0 382 0 0 101 0

Table 8. In vitro pseudovirion neutralization titers of antisera from Balb/c mice vaccinated three times Cerverix® or Gardasil®

Neutralization titers of pseudovirions

homologous heterologous

Group Mouse HPV16 HPV18 HPV31 HPV45 HPV52 HPV58

Cervarix® 1 15000 15000 0 0 0 0

2 15000 15000 0 0 0 0

3 15000 15000 0 897 0 0

4 15000 15000 0 98 0 0

5 15000 15000 0 0 0 0

Gardasil® 1 15000 15000 0 0 0 0

2 15000 15000 0 0 0 0

3 15000 15000 0 0 0 0

4 15000 15000 0 0 0 0

5 15000 5446 0 0 0 0

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Claims

What is claimed is:
1. Parvovirus mutated structural protein which comprises an insertion containing at least one cross-protective B-cell epitope of a human papillomavirus (HPV) L2 protein,
wherein the parvovirus structural protein is capable of forming a multimeric structure , and wherein the B-cell epitope is located at the surface of the multimeric structure.
2. Parvovirus mutated structural protein of claim 46, characterized in that the parvovirus is derived from adeno-associated virus (AAV), Goose parvovirus, Duck parvovirus, Snake parvovirus, feline panleukopenia virus, canine parvovirus, B 19 or minute virus of mice (MVM), preferably from an AAV selected from the group consisting of bovine AAV (b- AAV), canine AAV (CAAV), mouse AAV1 , caprine AAV, rat AAV, avian AAV (AAAV), AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1 1 , AAV 12, and AAV 13, especially AAV2.
3. Parvovirus mutated structural protein of any of the claims 1 to 2, wherein the mutated
structural protein is VP3.
4. Parvovirus mutated structural protein of any of the claims 1 to 3, wherein the cross-protective B-cell epitope is derived from a HPV L2 protein of a malignant HPV genotype, preferably selected from HPV 16, HPV 18, HPV31 , HPV33, HPV35, HPV 39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73 and HPV82, more preferably selected from
HPV 16, HPV 18, HPV31, HPV45, HPV 52 and HPV58, especially HPV 16 and HPV31 , and/or of a benign genotype, preferably selected from HPV1 , HPV2, HPV5, HPV6, HPV8, HPV1 1 and HPV63, especially selected from HPV5, HPV6 and HPV1 1.
5. Parvovirus mutated structural protein of any of the claims 1 to 4, wherein the cross-protective B-cell epitope is derived from the N-terminal 200 amino acids of HPV L2 protein, preferably from the N-terminal 100 amino acids of HPV L2 protein, more preferably from amino acids 10 to 40 of HPV L2 protein, especially from amino acids 17-36 of HPV L2 protein.
6. Parvovirus mutated structural protein of any of the claims 1 to 4, wherein the insertion
contains at least 9, preferably at least 15, especially at least 20 amino acids from the HPV L2 protein.
7. Parvovirus mutated structural protein of any of the claims 1 to 4, wherein two or more insertions are inserted, each containing at least one cross-protective B-cell epitope of an HPV L2 protein and each inserted at a different insertion site of the parvovirus mutated structural protein, preferably wherein one insertion is at 1-587 and one at 1-453, more preferably wherein a B-cell epitope of HPV 16 L2 protein is inserted at 1-587 and a B- cell epitope of HPV31 L2 protein is inserted at 1-453, especially wherein a B-cell epitope derived from amino acids 17-36 of HPV16 L2 is inserted at 1-587 and a B-cell epitope derived from amino acids 17-36 of HPV31 L2 inserted at 1-453.
8. Parvovirus mutated structural protein of any of the claims 1 to 7, wherein the insertion
containing at least one cross-protective B-cell epitope of the HPV L2 protein contains on its N- and/or C terminus a linker sequence, preferably a linker sequence having 6 to 10 small neutral or polar amino acids, preferably selected from A, G and S.
9. Parvovirus mutated structural protein of any of the claims 1 to 7, wherein none of the 5 amino acids directly adjacent to the insertion is R and none of the amino acids of the linker of claim 8, if present, is R.
10. Parvovirus mutated structural protein of any of the claims 1 to 9, wherein the parvovirus mutated structural protein comprises one or more additional mutations selected from an insertion, a deletion, a N- or C-terminal fusion and a substitution, particularly a single-amino- acid exchange, or a combination of these, preferably a mutation of R585 of AAV2 and/or R588 of AAV2, especially a single-amino-acid exchange 585A of AAV2 and/or RsssA of AAV2.
1 1. Multimeric structure comprising parvovirus mutated structural proteins of any of the claims 1 to 10, particularly comprising at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 structural protein and/or particularly being a capsomere, a viruslike particle (VLP) or a virus, especially a VLP, preferably a VLP composed only of VP3, especially of AAV2 VP3.
12. Nucleic acid coding for a parvovirus mutated structural protein according to any of the claims 1 to 10.
13. Cell comprising a nucleic acid according to claim 12, preferably wherein the cell is a bacterium, a yeast cell, an insect cell or a mammalian cell.
14. Method of preparing a structural protein according to any of the claims 1 to 10, the method comprising the steps of:
a) producing the structural protein by cultivating a cell according to claim 13 under suitable conditions thereby expressing the nucleic acid of claim 12, and
b) optionally isolating the parvovirus mutated structural protein produced in step a).
15. A composition comprising at least one parvovirus mutated structural protein according to any of claims 1 to 10 and/or a nucleic acid according to claim 12, preferably at least one multimeric structure according to claim 1 1 , for use as a medicament.
16. The composition for use of claim 15, wherein the medicament is a vaccine, preferably capable of inducing a cross-protective antibody response against HPV 16, HPV 18, HPV3 1 , HPV45 and HPV58, preferably against HPV 16, HPV 18, HPV31 , HPV45 and HPV58, especially against HPV5, HPV6, HPVl 1 , HPV l 6, HPV 16, HPV31 and HPV45.
17. The composition for use according to any of the claims 15 or 16, wherein the medicament is lyophilized or formulated to be stable at room temperature for at least one year.
18. The composition for use according to claim 15 or 16 for use in a method of preventing or treating an HPV infection, preferably an HPV infection caused by a malignant HPV genotype, preferably selected from HPV 16, HPV 18, HPV31 , HPV33, HPV35, HPV 39, HPV45, HPV51 , HPV52, HPV 56, HPV58, HPV59, HPV68, HPV73 and HPV82, more preferably selected from HPV 16, HPVl 8, HPV31, HPV45, and HPV58, especially HPV 16 and HPV31 , and/or from a benign genotype, preferably selected from HPVl, HPV2, HPV5, HPV6, HPV8, HPVl 1 and HPV63, especially selected from H V5, HPV6 and HPVl 1.
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