Patent Application
20130129683
The present invention relates to a rodent parvovirus variant capable of propagating and spreading through human tumor cells which is obtainable by serially passaging a rodent parvovirus as starting strain in semi-permissive human tumor cells. It also relates to a parvovirus variant capable of propagating and spreading through human tumor cells characterized by particular amino acid deletions and/or substitutions, e.g. a deletion of several amino acids in the C-terminus of NS1/middle exon of NS2. The present invention also relates to a pharmaceutical composition containing such parvovirus as well as its use for the treatment of cancer, preferably a glioblastoma.
Oncolytic viruses such as rodent parvoviuses represent novel tools for cancer treatment. Besides specifically killing cancer cells (oncolysis), these agents also provide danger signals prompting the immune system to eliminate virus-infected tumours. As a consequence of oncolytic events, the innate and adaptive immune systems gain access to tumour antigens, which results in cross-priming and vaccination effects. Rodent parvoviruses are single-stranded DNA viruses possessing intrinsic oncolytic activity, i.e. they preferentially replicate in and kill cancer cells of both murine and human origin.
Glioblastoma is a devastating disease with only limited treatment possibilities. Evidently, the prognosis for patients requires new therapies. The replication competent oncolytic viruses discussed above are considered promising since they are able to spread through malignant tissues and induce anti-tumor immune-responses. Among them, rodent parvoviruses appear to be excellent candidates, due to their natural oncotropism, their capacity to infect human cells, the specific toxicity for neoplastic cells, and the low pathogenicity for humans. In a rat model, H1-PV was able to cause a complete regression of established gliomas without any relaps and they proved able to target various human gliomas, thereby efficiently killing these cells irrespective of their acquired resistance towards known death inducers. Although, H1-PV proved able to infect and efficiently kill most of the human glioblastoma cell lines tested so far, the majority of these cells resisted efficient virus replication, progeny particle production and spreading. This failure to propagate and spread through human tumor tissue could have major impact for the treatment efficacy of human gliomas in vivo, not only because cell killing is limited to a single hit event reaching only a limited proportion of the tumor cells, but also due to the lack of an active intracellular transport of progeny virions to the cell surface.
Therefore, it is the object of the present invention to overcame this draw back of the prior art strains, i.e., to provide parvoviruses that are capable to propagate and spread through human tumor tissue.
According to the invention this is achieved by the subject matters defined in the claims. Initial experiments of the inventors have shown that progeny PV virions are shuttled from the nucleus to the cell periphery through vesicles prior to cell lysis where they are released into the medium supernatant. Moreover, this vesicular egress is associated with multiple intracellular proteins (including potential tumor antigens) which are carried as “co-cargo” to the cell surface and might contribute to the host anti-tumor immune stimulation. To mend the defects of the original H1-PV isolate (pSR19) in virus propagation and spreading in human glioblastoma, the inventors aimed to obtain propagation competent H1-PV variants through serial passaging in semi-permissive patient-derived human glioblastoma cell lines, i.e., to circumvent potential bottle-necks during the infection process (uncoating, delivery of the viral genome to the nucleus, double-strand conversion), infectious clone DNA of H1-PV (pSR-19) was transfected into different human glioblastoma cell lines (e.g. NCH149, NCH82, NCH89) and this replication competent DNA was passaged until cytopathic effects became visible. Additional viral passages (>15) were made combining cell associated progeny virions obtained through repeated freezing and thawing in vTE pH 8.7 with medium released viruses through infection. A pool of propagation competent viruses was then analyzed after 25 passages on NCH149 cells presenting efficient viral DNA amplification, generation of progeny virions and spreading through a variety of human glioblastoma cultures. From this original pool, individual virus clones were isolated through plaque-purification on NB 324K cells, amplified again on NCH149 cells, cloned and sequenced.
The genetic analysis of all H1-PV variants has revealed a deletion of 84 nts affecting 28 amino acids of the coding region of NS1 (C-terminus) and NS2 and a single cytosine to thymidine transition at position 3913 changing His374 to Tyr in VP2. Two additional guanidine to adenosine substitutions in the VP2 region (3964 and 4108) leading to a change from Asp391 to Asn and Asp439 to Ser have only been assigned to two isolates. A few changes in the 3′ non-coding regions remained of various prevalence. Apparently, the region approximately between nts 2000 to 2200 comprises a hot-spot of variability allowing H1-PV through modulation of NS1/NS2 function to adapt to the host environment. Therefore, it is possible to modify actively the host range of H1-PV, but also the other closely related rat PV strains, H3 and TVX through insertion/deletions within this region.
The newly generated H1-PV variants are able to propagate and spread through human glioblastoma cell(line)s. This allows an increased infection/destruction of already established human tumors and in consequence it is expected to give rise to a better anti-tumor immune stimulation. In agreement with the inventors' previous findings of parvovirus vesicular egress, it is reasonable to assume that the co-transport of intracellular proteins to the surface might serve to unmask tumor cells for a host immune response. This co-cargo effect is not present in absence of progeny virion formation using the original H1-PV isolate (pSR-19).
With the adaptation process (passaging) in semi-permissive cells as outlined in the examples an H1-PV variant proficient for propagation in many other human glioblastoma cell lines could be generated. The outlined procedure can be expanded to other human tumor cell(line)s including tumor stem cells.
Adaptation of the rat H1-PV on human glioblastoma cells led to a deletion in the C-terminus of H1 NS1/middle exon of NS2. This region represents a hot spot area for variation determining H1-PV host range and with additional adapatations/analyses of other so far uncharacterized rodent PV isolates additional variations can be found in this area.
Besides generating an H1-PV variant that is potentially able to spread from cell to cell in human glioblastoma tumor tissue and therefore has a higher potential for tumor cell killing (as indicated by the fitness assays), progeny particles of this isolate are actively transported to the cell periphery through vesicles. Besides newly synthesized virions, multiple cellular proteins being associated with such vesicles were found after infection which were not present in non-infected human glioblastoma cells. This co-cargo could serve to unmask tumor cells to make them available for the host immune system, thereby contributing for a virus-induced anti-tumor immune response.
(To.) Schematical Presentation of the Adaptation Approach
Rat parvovirus H1-PV readily propagates in transformed rat cells such as rat glioma RG-2 (left), but is limited to produce and spread in human glioblastoma cell lines. To overcome these limitations, serial passages were performed in semi-permissive human glioblastoma allowing mutations and amplification of permissive H1-PV variants to occur. Propagation competent variants were obtained/analyzed after 20 consecutive passages in NCH149 cells.
(B, C) Determination of Viral DNA Amplification in NCH149
NCH149 cells were infected with 10 pfu/cell H1-PV and the presence of replication intermediates was measured by Southern blotting after indicated times post infection.
(B) Infection with rat H1-PV (pSR19).
(C) Primary and secondary DNA amplification with human glioma adapted H1-PV, determination of gelsolin functioning for vesicular egress and spreading. Inf 1, primary infection, Inf 2, supernatants from Inf 1 were used to infect naive NCH149 cells. The generation of viral replication intermediates was measured by Southern blotting. αCon, control serum; αGln, neutralizing antiserum raised against gelsolin; dRF, dimeric replication intermediate; mRF, monomeric replication intermediate; ssDNA, progeny virus particles=single-stranded virus genomes.
(A) NCH149 cells were infected with 10 pfu/cell human glioma adapted H1-PV and fractionated for nuclei (Nuc), heavy mitochondrial fraction (HMF), and light mitochondrial fraction (LMF), containing cellular vesicles. To determine the presence of progeny virions in the vesicular fraction, LMF components were further separated by iodexanol gradients and the presence of virion DNA was measured by Southern blotting as compared to the presence of vesicular markers (determined by Western blotting).
(B) Determination of vesicular proteins in hgH1-PV and mock infected NCH149 cells. Vesicles were purified as described under (A) and analyzed for their protein content by SDS-PAGE and silver staining. Proteins overrepresented in hgH1-PV infected cellular vesicles are indicated by arrows.
(A) Determination of Viral Protein Expression After Infection of NCH149 Cells with Rat (pSR19) Versus Human Glioma Adapted H1-PV
NCH149 cells were infected with 10 pfu/cell and harvested at indicated times post infection. Accumulation of viral proteins were determined by Western blotting. Encircles (red) the large heterogeneity of NS1 produced after hgH1-PV infection and the larger accumulation of NS2 and NS3 (blue).
(B) RG-2 and NCH149 Cells were Infected with the Indicated Virus Preparations at a Multiplicity of 10, and Metabolically Labelled with 32P-Orthophosphate at 24 h p.i. for Four Hours
Cells were harvested, NS1 purified by immunoprecipitations and SDS-PAGE and the tryptic phosphopeptide pattern was determined by two-dimensional electrophoresis/chromatography. The phosphopeptide pattern of NS1 produced by rat H1-PV (pSR-19) in rat glioma (left), NCH149 (middle), as well as hgH1-PV in NCH149 cells (right) are shown. Encircled: phosphorylations known to drive viral DNA amplification.
(A) Multiplication of Input Virus After Infection of Various Target Cell Lines
Indicated cell lines were infected at a multiplicity 10, washed and harvested 4 (Input virus) 24 and 48 h post infection. Cell and medium associated virus was determined by plaque assay. The average amplification 24 and 48 h post infection is expressed over input (4 h) virus titer.
(B) Virus Fitness Assay
Target cell lines were seeded in 24 well dishes, infected at indicated multiplicities with the appropriate virus preparations, and living cells were fixed and stained with crystal violet 6 days post infection.
Top: Genome organization of H1-PV with indication of variation found after adaptation in human glioma
Middle section: PCR-fragments generated from plaque purified virus preparations
Bottom: Genetic determination of plaque purified hgH1-PV variants
Cloned PCR fragments are indicated by black lines, sequenced material is indicated by dotted squares and the presence of the individual variations are indicated in green.
b: H1-PV variant plaque #13:
After the initial characterization of five plaques (1,2,3,5, and 8) an additional plaque derived from the same adaptation experiment was cloned and sequenced (plaque #13). This additional variant was recognized by a different deletion in the C-terminal part of NS1/middle exon of NS2 together with mutations 2 and 6 previously found in the first round of characterizations (plaques 1 through 8).
b: Detailed characterization of the additional deletion in the coding region of NS1/NS2 found by sequencing of plaque #13.
Protein sequences corresponding to the H1-PV nucleotide sequence published under NC—001358. The three exons of NS2p as deduced from the transcription pattern of Qiu et al., (2006) are indicated in italics (common Nterminus with NS1=exon1)−regular (middle exon)−italics(C-terminal exon [unique for each NS2 isoform). Amended sequencing errors are indicated in light gray (these differences to the published sequence were found in all our isolates as well). Variations created through passaging in NCH149 cells are indicated in bold and marked with light-grey. VP1-specific sequences are denoted in italics.
b: Changes in the H1-PV protein sequences in plaque #13 as deduced from nucleotide changes obtained by DNA-sequencing and summarized in
Protein sequences corresponding to the H1-PV nucleotide sequence published under NC 001358. The three exons of NS2p as deduced from the transcription pattern of Qiu et al., (2006) are indicated in italics (common Nterminus with NS1=exon1)−regular (middle exon)−italics(C-terminal exon [unique for each NS2 isoform).Amended sequencing errors are indicated in light gray (these differences to the published sequence were found in all our isolates as well). Variations created through passaging in NCH149 cells are indicated in bold. VP1-specific sequences are denoted in italics. Coding changes of the deletion D1b and VP1/2 mut4 of Plaque #13 are given in bold and marked with light-gray.
The present invention provides in a first aspect a parvovirus variant capable of propagating and spreading through human tumor cells which is obtainable by serially passaging a rodent parvovirus as starting strain in semi-permissive human tumor cells.
In addition, the present invention provides in a second aspect a parvovirus variant capable of propagating and spreading through human tumor cells characterized in that it comprises a deletion of 28 amino acids from position 619 to 646 (Rhode and Paradiso, 1983) in the C-terminus of NS1/position 126 to 153 in the middle exon of NS2 (Rhode and Paradiso, 1983; Qiu et al., 2006).
The present invention also provides a parvovirus variant capable of propagating and spreading through human tumor cells characterized in that it comprises the following amino acid substitution(s) in VP1/2:
(a) H374Y;
(b) D391N; and/or
(c) D439S.
The term “propagating” as used herein means that the progeny virions of the parvovirus variant show amplification significantly over input levels (i.e. >5 fold).
The term “spreading through” as used herein means that the progeny virions of the parvovirus variant are capable not only of killing primarily infected cell populations, but are able to cause secondary infections of naive cells with particles produced during the first round of infection.
The term “semi-permissive” as used herein for characterizing the host cells means that the infection with the virus results in incomplete viral replication within the particular type of host cells but are characterized by viral DNA amplification and eventually low yields of progeny virions. The latter do not necessarily have to be infectious in the beginning of the procedure.
The expression “parvovirus” comprises any parvovirus, particularly a rodent parvovirus, such as minute virus of mice (MVM) and H-1 virus as well as related viruses or vectors based on such viruses or derivatives. The person skilled in the art can arrive at the parvovirus variant according to the present invention by subjecting a starting strain to serial passaging as outlined in the Examples, below, or by introducing a deletion resulting in the desired modification of the biological properties, i.e., capability of the parvovirus variant to propagate and spread through, thereby killing, human tumor cells starting from the known nucleic acid sequence and amino acid sequences of the non-structural proteins of parvoviruses, e.g., from parvovirus H-1 (Rhode and Paradiso, 1983; Qiu et al., 2006). The person skilled in the art can also easily assay whether a particular variant exhibits the desired biological properties recited by using the assays explained in the examples, below.
Preferably, the parvovirus variant of the invention is derived from parvovirus H-1 (H-1PV) or a related rodent parvovirus. Examples of preferred related rodent parvovirus are LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV) or Rat virus (RV).
In principle, any tumor can be cured using a parvovirus variant of the present invention with gliomas being preferred.
A particular preferred glioma is a glioblastoma.
A preferred human semi-permissive tumor cell line is NCH149.
As regards the procedure of serial passaging, the person skilled in the art can easily determine how many passages are sufficient for arriving at a parvovirus of the present invention. In general, however, this method of the invention comprises at least 10 passages, preferably at least 20 to 25 passages, or more. Preferably, passaging is on NCH149 cells.
In order to ensure the purity and stability of the virus variants the individuals clones can be plaque-purified after passaging, preferably on NB 324K cells.
The present invention also provides a method of generating a parvovirus variant capable of propagating and spreading through human tumor cells said method comprising serially passaging a rodent parvovirus as starting strain in semi-permissive human tumor cells as described above and in the examples, below.
A further subject matter of the present invention relates to a nucleic acid, particularly a DNA, which codes for an above parvovirus variant.
A DNA according to the invention can be present in a vector and expression vector, respectively. A person skilled in the art is familiar with examples thereof. In the case of an expression vector for E. coli these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b, T7 based expression vectors and pQE-8. For the expression in yeast, e.g. pY100 and Ycpad1 have to be mentioned while e.g. pKCR, pEFBOS, cDM8, pMSCND, and pCEV4 have to be indicated for the expression in animal cells. The baculovirus expression vector pAcSGHisNT-A is especially suitable for the expression in insect cells.
In a preferred embodiment, the vector containing the DNA according to the invention is a virus, e.g. an adenovirus, vaccinia virus, an AAV virus or a parvovirus, such as MVM or H-1, a parvovirus being preferred. The vector may also be a retrovirus, such as MoMULV, MoMuLV, HaMuSV, MuMTV, RSV or GaLV.
For constructing expression vectors which contain the DNA according to the invention, it is possible to use general methods known in the art. These methods include e.g. in vitro recombination techniques, synthetic methods and in vivo recombination methods.
Furthermore, the present invention relates to host cells which contain the above described variants or vectors. These host cells include human 293(T), NBK, rat RG2 cells, NCH149, NCH82, or U87 cells. Methods of transforming these host cells, of phenotypically selecting transformants and of expressing the DNA according to the invention by using the above described vectors are known in the art.
Thus, the present invention also provides a method of producing the parvovirus variant of the invention, comprising the culturing of a host cell of the invention under suitable conditions and harvesting the parvovirus variant from the cells or the medium.
Moreover, the present invention relates to antibodies which specifically recognize an above describe parvovirus variant, i.e. the polypeptide region of the parvovirus variant where the deletion is located characterizing the rodent parvovirus variant in that it is capable of propagating and spreading through human gliomas. The antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof, e.g. Fab, Fv or scFV fragments. Preferably monoclonal antibodies are concerned. For the production it is favorable to immunize animals—particularly rabbits or chickens for a polyclonal antibody and mice for a monoclonal antibody—with an above parvovirus variant or with fragments thereof. Further boosters of the animals can be affected with the same parvovirus variant or with fragments thereof. The polyclonal antibody can then be obtained from the animal serum and egg yolk, respectively. The monoclonal antibody can be obtained according to standard methods, reference being made particularly to the method by Köhler and Milstein (Nature 256 (1975), 495) and Galfre (Meth. Enzymol. 73 (1981), 3). In this case, mouse myeloma cells are fused with spleen cells originating from the immunized animals. Antibodies according to the invention can be used in many ways, e.g. for the immunoprecipitation of the above described parvovirus variants or for the isolation thereof. The antibodies can be bound in immunoassays in liquid phase or to a solid carrier. In this connection, the antibodies can be labeled in various ways. The person skilled in the art is familiar with suitable markers and labeling methods. Examples of immunoassays are ELISA and RIA.
A kit is also provided for the application of the present invention. This kit comprises the following:
Of component (a) to (d) one or more representatives can be present each.
The present invention also provides a pharmaceutical composition containing a parvovirus variant of the invention, vector or a cell producing said parvovirus variant (“parvotherapeutic agent” or “parvotherapeutic composition”), e.g. human 293(T), NBK or rat RG2 cells.
For administration, the parvotherapeutic agent can be combined with suitable pharmaceutical carriers. Suitable pharmaceutical carriers of a type well known in the art and readily commercially available, include phosphate buffered saline (PBS) solutions, water, emulsions such as oil/water emulsions, wetting agents of various types, sterile solutions, etc. Such carriers can be formulated with the parvotherapeutic agent(s) by conventional formulating methods for administration to the subject at a suitable dose.
Additional pharmaceutically compatible carriers can include gels, biosorbable matrix materials, implantation elements containing the therapeutic agent, or any other suitable vehicle, delivery or dispensing means or material(s).
Administration of the parvotherapeutic pharmaceutical compositions to a patient, e.g. a brain tumor patient, may be effected in any of numerous suitable ways, e.g., by intravenous, intraperetoneal, subcutaneous, intramuscular, topical, intradermal, intracranial, and intratumoral administration. The route of administration, of course, depends on the nature of the disease and the specific therapeutic agent(s) contained in the pharmaceutical composition.
If such parvotherapeutic agent(s) comprise infectious virus particles with the ability to penetrate through the blood-brain barrier, treatment can be performed or at least initiated by intravenous injection of the viral therapeutic agent, e.g., a H1-PV variant.
Since long-term intravenous treatment is susceptible to becoming inefficient as a result of the formation of neutralizing antibodies to the viral therapeutic agent, different modes of administration can be adopted after an initial regimen of intravenous viral administration, or such different administration techniques, e.g., intracranial or intratumoral virus administration, can be alternatively used throughout the entire course of parvoviral treatment.
As another specific administration technique, the parvotherapeutic agent (virus, vector and/or cell agent) can be administered to the patient from a source implanted in the patient. For example, a catheter, e.g., of silicone or other biocompatible material, can be connected to a small subcutaneous reservoir (Rickham reservoir) installed in the patient during tumor removal or by a separate procedure, to permit the parvotherapeutic composition to be injected locally at various times without further surgical intervention. The parvovirus variant or derived vectors can also be injected, e.g., into a tumor, by stereotactic surgical techniques or by neuronavigation targeting techniques.
Administration of the parvoviral agents or compositions can also be performed by continuous infusion of viral particles or fluids containing viral particles through implanted catheters at low flow rates using suitable pump systems, e.g., peristaltic infusion pumps or convection enhanced delivery (CED) pumps.
A yet another method of administration of the parvotherapeutic composition is from an implanted device constructed and arranged to dispense the parvotherapeutic agent to the desired locus, e.g., tumor. For example, wafers can be employed that have been impregnated with the parvotherapeutic composition, e.g., a parvovirus H-1 variant, wherein the wafer is attached to the edges of the resection cavity at the conclusion of surgical tumor removal. Multiple wafers can be employed in such therapeutic intervention.
Cells that actively produce the parvotherapeutic agent, e.g., a parvovirus H-1 variant, or H-1 variant vectors, can be injected into the desired tumor, or into a tumoral cavity after tumor removal.
Combinations of two or more of the above-described administration modes can be employed in any suitable manner, e.g., concurrently, contemporaneously, or sequentially.
The dosage regimen of the parvotherapeutic agent is readily determinable within the skill of the art, by the attending physician based on patient data, observations and other clinical factors, including for example the patient's size, body surface area, age, sex, the particular virus, vector, cell, etc. to be administered, the time and route of administration, the tumor type and characteristics, general health of the patient, and other drugs or therapies to which the patient is being subjected.
Accordingly, the present invention also relates to the use of a parvovirus variant according to the present invention, a cell producing said parvovirus variant, a DNA, an expression vector or antibody of the present invention for the preparation of a pharmaceutical composition for the treatment of cancer. Brain tumors (preferably a glioma, medulloblastoma, meningioma or glioblastoma) are expected to be particularly amenable to treatment with an agent of the present invention.
The below examples explain the invention in more detail.
Infection of many human glioblastoma cell lines with H1-PV derived from pSR19 isolate (Accession No: NC—001358) led to the expression of viral proteins and in consequence cell killing irrespective of their resistance towards drug-induced apoptosis. However, monitoring viral DNA amplification and progeny particle production, with exception of NCH89, the majority of the tested human glioblastoma cell lines only allowed little DNA amplification and virtually no increase in progeny production (Herrero y Calle et al., 2004; DiPiazza et al., 2007). To mend this defect, it was aimed to obtain (a) propagation competent H1-PV variant(s) through serial passaging of pSR19 in human glioblastoma cells, which were competent for DNA amplification although progeny virion production was hampered. The infectious clone DNA of H1-PV, pSR19 was transfected into NCH149 cells, a cell line that was shown to allow low levels of DNA amplification by Southern blotting (
After more than 20 consecutive passages the inventors obtained a virus stock competent for viral DNA amplification, progeny virion production and spreading in NCH149 cultures. The properties of this virus pool were analyzed. As shown in
As shown in
Then, the nature of the virus modification(s) causing the permissive phenotype in NCH149 cells was identified. Original H1-PV (pSR19) was produced in rat RG-2 glioma, whereas the adapted H1-PV variant (hgH1-PV) was generated in NCH149. Both virus stocks were titrated by plaque assays in permissive NB324K cells. Infections were performed at a multiplicity of 10 in NCH149 cells and the production of viral proteins was determined in a time-course experiment by Western blotting. As shown in
Finally, the adapted hgH1-PV stock was tested for its fitness to propagate and spread through a variety of (human) glioblastoma cultures in comparison to the pSR19 isolate. As shown in
To determine genetic variations hgH1-PV acquired during passaging in NCH149 as compared to the original inoculum (pSR19), endpoint titrations were performed to plaque purify individual virus variants and virion DNA was isolated. As shown in
To further characterize the properties of individual clones, virus stock of individual plaque isolates was generated and their fitness to spread and kill human glioblastoma cell cultures in comparison to pSR19 generated in rat RG-2 cells was tested. As shown in
U87 glioblastoma cells were infected or not ex vivo with a MOI of 1.5 pfu/cell. (WT:H-1PV wildtype; Plaque #3: adapted H-1PV derived from plaque #3, mock: uninfected U87 cells). Cells were washed and dissolved in PBS 4 h p.i. and 2×106 mock or H-1PV infected cells in 100 μl PBS were implanted subcutaneously into 8 mice per group. Tumour size (length, width, height) was measured mechanically with a calliper twice a week, starting 12 days after tumour cell implantation. Volume was calculated as follows: ½×length×width2. Animals were sacrificed at a tumour size of maximum 2500 mm3, in case of appearance of peritoneal carcinosis or malignant ascites. All surviving animals were sacrificed at day 61 after cell implantation. (1×WT, 2×Plaque #3) as indicated by “survivors”. It was shown in
Rhode, S. L. III and Paradiso, P. R. (1983. Parvovirus genome: nucleotide sequence of H-1 and mapping of its genes by hybrid-arrested translation. J. Virol. 45, 173-184.
Qiu, J., Yoto, Y., Tullis, G., and Pintel, D. J. (2006). Parvovirus processing strategies. pp. 253-274. In “Parvoviruses” Eds. Kerr, J. R., Cotmore, S. F., Bloom, M. E., Linden, R. M., and Parrish, C. R. Hodder Arnold, London.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10004897.4 | May 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/002306 | 5/9/2011 | WO | 00 | 12/12/2012 |
