This application claims priority to and benefit of U. S. Provisional Application No. 62/104,252, filed January 16, 2015. The contents of the aforementioned patent application are herein incorporated by reference in their entireties.

Sequence Listing

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 13, 2016, is named LIGO-02701WO_ST25.txt and is 15, 131 bytes in size.

Background of the Invention

Retroviruses are important infectious agents in humans and animals and are responsible for a large number of diseases. A retrovirus may be passed to a subject via contact with a biological sample, e.g., an organ transplant, blood transfusion, and vaccine. Reverse transcriptase (RT) activity is a hallmark of retroviruses and therefore its presence in a biological sample can indicate the presence of a retrovirus particle in the sample.

Cell lines are commonly used for the propagation of recombinant viruses for production of biological products, e.g., vaccines. Such cell lines, which often contain integrated retrovirus- like elements, release RT following cell lysis. Therefore, when a biological product obtained from such a cell line has RT activity, it is unclear whether the RT activity is due to RT released by the cell or due to contamination of the biological product by a viral particle that contains RT; it is import to be able to distinguish between virus particle-contained and free RT activity since the presence of free RT in a biological product is of less significance than the presence of an intact retrovirus particle.

Accordingly, there is an unmet need for methods and kits capable of identifying RT activity in a sample that is due to a virus particle contaminant. Summary of the Invention

Provided herein are methods and kits for detecting in a sample the presence of a virus particle or a virus-like particle that has reverse transcriptase activity and methods for preparing a retroviral contaminant-free substance.

An aspect of the present invention is a method for detecting the presence of a virus particle in a sample of a Virus-like Particle (VLP) drug substance comprising a step of performing PCR-based reverse transcriptase (PBRT) on a sample of the VLP drug substance that has been treated with a protease.

Another aspect of the present invention relates to a method for detecting the presence of virus particle-contained reverse transcriptase activity in a test sample. The method includes the steps of: (1) adding a protease to the test sample and incubating the resultant solution under conditions that allow the protease to digest any soluble reverse transcriptase present in the resultant solution, thereby producing a digested solution; (2) inactivating the protease in the digested solution, thereby producing an inactivated protease solution; (3) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent- containing solution; (4) adding to the detergent-containing solution or to a fraction of the detergent-containing solution an isolated RNA molecule, a first primer that hybridizes to a nucleic acid sequence corresponding to a first part of the isolated RNA molecule, a second primer that hybridizes to the complement of a second part of the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (5) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-contained reverse transcriptase is present in the test sample; and (6) identifying the PCR-amplified product, thereby detecting the presence of the virus particle-contained reverse transcriptase in the test sample. An isolated nucleic acid probe which hybridizes to the nucleic acid sequence corresponding to the isolated RNA molecule may be added during or prior to step (4).

In the above aspect, when the isolated RNA molecule is added in step (4), substantially no DNA template for the isolated RNA molecule is concurrently added. To ensure this, a DNase may be added to the isolated RNA molecule prior to step (4); if added, the DNase is inactivated prior to step (4). Step (4) of the above aspect may further include adding one or more of dNTPs, a reaction buffer, water, Mg ²⁺ , and Mn ²⁺ . The DNA polymerase can be Taq Polymerase, a high- fidelity DNA polymerase, or another polymerase known in the art. In some embodiments, a detergent is added during step (1) of the above aspect in an amount that is insufficient to disrupt an intact virus particle.

Some embodiments of the above aspect include a step of concentrating the inactivated protease solution prior to step (3), e.g., by one or more of centrifugation, dialysis, precipitation, or passing through a column. For example, this step may involve subjecting the inactivated protease solution to high speed centrifugation through a sucrose cushion.

In embodiments of the above aspect, at least one sample is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6). In embodiments, positive control samples comprising a soluble reverse transcriptase, e.g., a recombinant reverse transcriptase {e.g., Avian Myeloblastosis virus αβ holoenzyme (AMV T)), is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6); these positive control samples may include the test sample. In embodiments, positive control samples to which has been added a particle that contains a reverse transcriptase, e.g., a murine leukemia virus (MLV) particle, is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6); these positive control samples may include the test sample. In embodiments, at least one sample is obtained and processed according to steps (1) to (6), (2) to (6), (3) to (6), or (4) to (6) but without adding an isolated RNA molecule in step (4), thereby producing a negative control sample(s); these negative control samples may include the test sample.

In yet another aspect of the present invention is a method for detecting the presence of an RT-containing virus particle in a sample comprising a step of performing PCR-based reverse transcriptase (PBRT) on the sample that has been treated with a protease.

In another aspect of the present invention relates to a method for detecting the presence of an RT-containing virus particle in a Virus-like Particle (VLP) drug substance. The method includes steps of: (1) obtaining a sample of the VLP drug substance; (2) diluting the sample in Proteinase K buffer which is supplemented with Triton X-100 detergent (in an amount that is insufficient to disrupt an intact virus particle) thereby obtaining a diluted sample; (3) adding Proteinase K to the diluted sample and incubating the resultant solution under conditions that allow the Proteinase K to digest any soluble reverse transcriptase present in the resultant solution but not to digest all reverse transcriptase contained in virus particles, thereby producing a digested solution; (4) inactivating the Proteinase K in the digested solution by addition of phenylmethylsulfonyl fluoride (PMSF), thereby producing an inactivated protease solution; (5) centrifuging the inactivated protease solution by high speed centrifugation through a sucrose cushion, thereby producing a concentrated solution; (6) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing concentrated solution; (7) adding to the detergent-containing concentrated solution or to a fraction of the detergent-containing concentrated solution an isolated RNA molecule, a first primer that hybridizes to a 5' end of a nucleic acid sequence corresponding to the isolated RNA molecule, a second primer that hybridizes to the 3' end of the nucleic acid sequence

corresponding to the isolated RNA molecule, an isolated nucleic acid probe which hybridizes to the nucleic acid sequence corresponding to the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (8) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-protected and released reverse transcriptase is present in the PBRT assay solution; and (9) identifying the PCR-amplified product, thereby detecting the presence of the RT-containing virus particle in the VLP drug substance.

Another aspect of the present invention is a retroviral contaminant-free Virus-like

Particle (VLP) drug substance. This substance is identified by a method of an above aspect including its embodiments.

An aspect of the present invention relates to a method for detecting the presence of virus particle-contained reverse transcriptase activity in a test sample. The method includes steps of: (1) adding a protease to the test sample and incubating the resultant solution under conditions that allow the protease to digest any soluble reverse transcriptase present in the resultant solution, thereby producing a digested solution; (2) inactivating the protease in the digested solution, thereby producing an inactivated protease solution; (3) adding a detergent in an amount that is sufficient to disrupt an intact enveloped virus particle, thereby producing a detergent- containing solution; (4) adding to the detergent-containing solution or to a fraction of the detergent-containing solution an isolated RNA molecule, a first primer that hybridizes to a nucleic acid sequence corresponding to a first part of the isolated RNA molecule, a second primer that hybridizes to the complement of a second part of the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution; (5) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-protected and released reverse transcriptase is present in the PBRT assay solution; and (6) identifying the PCR-amplified product, thereby detecting the presence of the virus particle-contained reverse transcriptase in the test sample. An isolated nucleic acid probe which hybridizes to the nucleic acid sequence corresponding to the isolated RNA molecule may be added during or prior to step (4).

In some embodiments, a detergent is added during step (1) in the above aspect of the present invention in an amount that is insufficient to disrupt an intact enveloped virus particle such that the detergent concentration is less than about 0.002%. In some embodiments, a detergent is added during step (3) such that the detergent concentration in the detergent- containing solution or in the PBRT assay solution is between about 0.1% and 0.3%.

Another aspect of the present invention relates to a method for detecting the presence of an enveloped virus particle in a Virus-like Particle (VLP) drug substance. The method includes steps of: (1) obtaining a sample of the VLP drug substance; (2) diluting the sample in Proteinase K buffer which is supplemented with Triton X-100 detergent (in an amount that is insufficient to disrupt an intact enveloped virus particle) thereby obtaining a diluted sample; (3) adding Proteinase K to the diluted sample and incubating the resultant solution under conditions that allow the Proteinase K to digest any soluble reverse transcriptase present in the resultant solution but not to digest all reverse transcriptase contained in enveloped virus particles, thereby producing a digested solution; (4) inactivating the Proteinase K in the digested solution by addition of Phenylmethylsulfonyl fluoride (PMSF), thereby producing an inactivated protease solution; (5) centrifuging the inactivated protease solution by high speed centrifugation through a sucrose cushion, thereby producing a concentrated solution; (6) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing concentrated solution; (7) adding to the detergent-containing concentrated solution or to a fraction of the detergent-containing concentrated solution an isolated RNA molecule, a first primer that hybridizes to a 5' end of a nucleic acid sequence corresponding to the isolated RNA molecule, a second primer that hybridizes to the 3' end of a nucleic acid corresponding to the isolated RNA molecule, an isolated nucleic acid probe which hybridizes to a nucleic acid corresponding to the isolated RNA molecule, and a DNA polymerase, thereby preparing a PCR- based reverse transcriptase (PBRT) assay solution; (8) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if an enveloped virus particle-released reverse transcriptase is present in the PBRT assay solution; and (9) identifying the PCR-amplified product, thereby detecting the presence of the virus particle in the VLP drug substance.

In some embodiments, Triton X-100 is added during step (2) in the above aspect of the present invention such that the detergent concentration is less than about 0.002%. In some embodiments, a detergent is added during step (6) such that the detergent concentration in the detergent-containing concentrated solution or in the PBRT assay solution is between about 0.1% and 0.3%.

Another aspect of the present invention is a method for detecting the presence of a virus particle in a sample comprising a step of performing PCR-based reverse transcriptase (PBRT) on the sample that has been treated with a protease.

Another aspect of the present invention is a method for detecting the presence of an enveloped virus particle containing reverse transcriptase in a sample of a Virus-like Particle (VLP) drug substance comprising a step of performing PCR-based reverse transcriptase (PBRT) on a sample of the VLP drug substance that has been treated with a protease.

The invention includes kits for performing a method of an above aspect including its embodiments. The kits include instructions for use.

Another aspect of the present invention is a retroviral contaminant-free Virus-like Particle (VLP) drug substance. This substance is identified by a method of an above aspect including its embodiments. In some embodiments, an isolated nucleic acid probe, e.g., having a detectable label, is used which hybridizes to a nucleic acid sequence corresponding to the isolated RNA molecule is added to the inactivated protease solution

In some embodiments, a test sample is diluted with a buffer, e.g., about one- to about 20- fold (e.g., about 10-fold).

In some embodiments, the detergent is Triton X-100.

In some embodiments, the protease is Proteinase K.

In some embodiments, the protease inhibitor is PMSF.

In some embodiments, the test sample is a Virus-like Particle (VLP) drug substance, e.g., a norovirus (e.g., Norwalk virus) VLP drug substance.

In some embodiments, the isolated RNA molecule is an in vzYro-transcribed RNA molecule.

In some embodiments along with the DNA polymerase is added one or more of dNTPs, a reaction buffer, water, Mg ²⁺ , and Mn ²⁺ . The DNA polymerase can be Taq Polymerase, a high- fidelity DNA polymerase, or another polymerase known in the art.

In some embodiments, the isolated RNA molecule is at least about 95% identical to a fragment of Dengue virus type 4 genome which has the sequence of SEQ ID NO: 1. The isolated RNA molecule can be at least about 95% identical to the sequence of SEQ ID NO: 5. The first primer may include the sequence of SEQ ID NO: 3 and the second primer may include the sequence of SEQ ID NO: 2. The isolated nucleic acid probe may include the sequence of SEQ ID NO: 4.

Exemplary detergents include Brij-35, Brij-58, CHAPS, CHAPSO, n-Dodecyl-beta-D- Maltoside, NP-40, Octyl glucoside, Octyl thioglucoside, SDS, Sodium Deoxycholate, Triton X- 100, Triton X-l 14, Tween 20, or Tween 80. Preferably, the detergent is Triton X-100.

Examples of a protease includes but is not limited to bromelain, caspase, cathepsins, chymotrypsin, elastase, endoproteinase AspN, endoproteinase GluC, enterokinase

(enteropeptidase), Factor Xa, furin, papain, pepsin, Proteinase K, subtilisin, thrombin, or trypsin. Preferably, the protease is Proteinase K. The step of inactivating the protease includes adding one or more protease inhibitors, for example AEBSF-HCl, Aprotinin, Bestatin, E-64, EDTA, Leupeptin, Pepstatin A, and/or Phenylmethylsulfonyl fluoride (PMSF). Preferably, the protease inhibitor is PMSF.

Examples of the detectable label includes but is not limited to biotin, a colloidal particle, digoxigenin, an electron-dense reagent, an enzyme, a fluorescent dye, hapten, a magnetic bead, a metallic bead, and a radioactive isotope. Preferably, the detectable label is a florescent dye. Examples of the florescent dye include 6-FAM, ABY®, Acridine, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, BioSearch Blue, Coumarin, Cy® 3, Cy® 3.5, Cy® 5, Cy® 5.5, FAM™, FITC®, GPF (and variants thereof), HEX™, JOE™, JUN®, Marina Blue, NED™, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PET®, Pulsar®, Quasar® 570, Quasar® 670 , Quasar® 705, Rhodamine Green, Rhodamine Red, ROX™, SYBR® Green, TAMRA™, TET™, Texas Red®, TRITC, and VIC®.

The invention includes kits for performing a method of an above aspect including its embodiments. The kits include instructions for use.

Any of the above aspects, embodiments, features, or examples can be combined with any other aspect, embodiment, feature, or example.

Other features and advantages of the invention will be apparent from the Detailed

Description, the drawings, and the claims.

Detailed Description of the Invention

The present invention relates to methods and kits including a highly-sensitive PCR-based reverse transcriptase (PBRT) assay for detecting retrovirus particle contaminants in a sample.

The invention relies on the sensitivity of soluble reverse transcriptase (RT) activity to proteinase K digestion and substantially insensitivity of virus particle-contained RT activity; thus, a sample showing RT activity that is sensitive to proteinase K digestion likely does not have a retrovirus contamination whereas a sample showing RT activity that is substantially insensitive to proteinase K digestion likely has retrovirus contamination.

Without being bound by theory, experimental evidence disclosed herein shows that one or more of the following features of the present invention leads to unexpectedly superior results:

1. Dilution of a sample to be tested into a larger volume of buffer, e.g., Proteinase K buffer, in order to dilute out the high concentration of protein-based components in the sample, allows for a more complete proteolytic degradation of RT not contained in virus particles.

2. Addition of a very small amount of detergent, e.g., Triton X-100, during proteolytic digestion, e.g., by Proteinase K, of virus particles helps facilitate complete digestion. Moreover, the amount of detergent used does not diminish virus particle-contained RT activity.

3. Inactivation of the protease, e.g., Proteinase K, by the addition of a protease inhibitor, e.g., PMSF, is performed following the proteolytic digestion step.

4. Collection and concentration, e.g., high speed centrifugation through a sucrose cushion, of viral particles following the inactivation of the protease, is performed in the presence of the protease inhibitor to ensure that protease activity does not persists during the PBRT reaction.

Definitions

As used herein, the terms "virus", "enveloped virus", "virus-particle" are used interchangeably and refer to a structure that in one attribute resembles a virus but which may or may not been demonstrated to be infectious and/or capable of replication. On the other hand, a "virus-like particle (VLP)" refers to a structure that in one attribute resembles a virus but lacks genetic material. Thus, a VLP is not infectious and/or capable of replication; thus, a VLP is not harmful to a subject. However, an immune response is generated when the subject is vaccinated with a VLP as if the immune system has been presented with a virus.

Examples of Human retroviruses include but are not limited to HIV-1, HIV-2, HTLV-I, and HTLV-II; simian retroviruses include but are not limited to SIV-1 and SIV-2; other mammalian retroviruses include but are not limited to MLV, FeLV, BLV, and MMTV; and bird retroviruses include but are not limited to ALV and ASV.

Any virus, e.g., retrovirus, which comprises RT can be detected by the present methods and kits.

As used herein, a sample is any sample (e.g., a test sample) that is suspected of containing a virus particle, e.g., as a contaminant. The sample may be a protein solution, a peptide solution, a salt solution, an intravenous (IV) solution, a drug substance, a culture medium, a cell suspension, a suspension comprising an explanted tissue from a subject (e.g., a tissue biopsy or an organ), a blood product, a biological product, an antibody-containing solution (e.g., a monoclonal antibody), a vaccine (e.g., an inactivated viral vaccine and a live virus vaccine), or VLP preparation.

A "biological product," as used herein, refers to a product or material that is produced by a cell or organism. The biological product may be a natural product of the organism, or may be produced by an organism that has been altered in some way such that it produces the biological product. Examples of biological products include, but are not limited to, vaccines, antibodies (e.g., monoclonal antibodies), therapeutic proteins, viruses (e.g., recombinant viruses for gene therapy such as, for example, adenovirus, vaccinia virus, pox virus, adeno-associated virus, and herpes virus), enzymes, growth factors, polysaccharides, nucleic acids including DNA and RNA, and particles (e.g., virus-like particles). Other non-limiting examples of a biological product include blood (and a blood component), semen, urine, saliva, sputum, and cerebrospinal fluid obtained from a subject. Examples of a blood component include whole blood, serum, plasma, and platelets.

The subject may be from a mammal. The mammal can be a human or a non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, ox, buffalo, sheep, or pig. The subject can also be a bird or fowl. In embodiments, the mammal is a human.

The term "drug substance", as used herein, refers to the material that contains a therapeutic drug and that is used to formulate, along with excipients, a pharmaceutical composition or drug product.

The terms "isolated", "purified", or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state.

"Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. An isolated product can be obtained from a natural source or can be synthetically obtained; for example, a nucleic acid molecule can be obtained from a cell expressing a gene or coding sequence (i.e., in vitro) and/or can be obtained from a machine that synthesizes the molecule. In other words, a nucleic acid used herein can be naturally-produced or synthetically-produced.

As used herein, the term "primer" refers to a string of linked nucleotide residues comprising a sufficient number of residues to be used in a PCR reaction or in a reverse transcription reaction. A primer may be used to amplify, copy, reverse-transcribe, confirm, or reveal the presence of an identical, similar, or complementary DNA or RNA in a sample. As used herein, the term "probe" refers to a nucleic acid sequence used in the detection of identical, similar, or complementary nucleic acid sequences. A primer can be used to bind to RNA, DNA, or cDNA.

As used herein, a sample is "substantially free" of a virus particle means that the sample does not comprise a detectable level of the vims particle as measured by a PGR or RT-PCR assay or the like.

In some embodiments, the methods and kits described herein utilize enzymes to degrade RNA, such as ribonucleases (RNases). RNases are nucleases that degrade RNA. In some embodiments, RNases provide a thorough means to degrade a non-encapsulated RNA that might otherwise be resistant to degradation due to aggregation. RNases can be endoribonucleases such as RNase A, RNase H, RNase III, RNase I, and others; or exoribonucl eases such as RNase II, RNase R, exoribunuclease I, or others. Ribonuclease A (RNase A) is a pancreatic ribonuclease often used in research, and specifically cleaves single-stranded RNA.

The term "DNase" refers to an enzyme that degrades DNA. Any DNase can be used in aspects of the invention. A wide variety of DNases are known and can be categorized including but not limited to DNase I, DNase II, DNase III, DNase IV, DNase V, DNase VI, DNase VII, and DNase VIII.

The term "% identical" as in "95% identical" or "at least about 95% identical" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotides occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The terms "identical" or percent "identity," in the context of two or more nucleic acid sequences, refer to two or more sequences or subsequences that are the same sequences.

Sequences are "substantially identical" if two sequences have a specified percentage of nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, or across the entire sequence where not indicated

Unless specifically stated or obvious from context, as used herein, the term "about", is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about."

Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the methods or processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

Methods for isolating, amplifying, or detecting nucleic acids, including methods for detecting and amplifying target RNA or DNA sequences (for example, by polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), or PCR-based reverse transcriptase (PBRT)) are well known in the art.

One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et ah, Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al. , Molecular Cloning, A Laboratory Manual (3 ^r edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al , The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18 ^th edition (1990). These texts can, of course, also be referred to in making or using an aspect of the invention.

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

In the below examples, the methods of the present invention are used to detect reverse transcriptase (RT) activity in a norovirus vims-like particle (VLP) drug substance that was purified from recombinant baculovirus-infected Sf9 cells, i.e., a cell line used for the propagation of recombinant baculoviruses for production of biologicals and which is known to contain integrated insect retrovirus-like elements and to release RT activity following baculovirus- induced lysis. Trace levels of RT activity present in VLP drug substance was demonstrated to be sensitive to proteinase K digestion and therefore not particle-associated. In contrast, Sf9 cell end-of-production-harvest material was shown to contain significant amounts of RT activity; only a very small fraction of this activity was proteinase K-resistant and therefore, this very small fraction is particle-associated. These data demonstrate that the vast majority of RT activity in harvest material is not particle-associated and that there is no evidence for retroviral particle contamination in purified drug substance. EXAMPLES

Example 1: PCR-based Reverse Transcriptase (PBRT) assay can quantify RT levels in a sample having as few as six RT molecules

In order to measure small quantities of RT activity a highly sensitive PCR-based RT

(PBRT) assay was required. In separate studies involving the development of arbovirus RT-PCR assays, a particular sequence in the Dengue 4 genome was identified that demonstrates reproducible performance in RT-PCR experiments. In vz ^' zro-transcribed RNA containing this sequence as an RNA template was used for the detection of RT activity in a PBRT assay.

Table 1 shows primer and probe sequences used in the employed in the Den4 RT-PCR assay.

Table 1:

A 1 13 nt synthetic DNA sequence representing the above-mentioned portion of the Den4 genome was cloned into plasmid pcDNA 3.1 between the Notl and Kpnl sites. The resulting clone was linearized with Pstl. Linearized DNA was used in an in vitro transcription reaction to generate an approximate a 1.5 kb RNA molecule containing the 113 nt Den4 synthetic sequence which served as the template RNA for the PBRT assay.

For the template RNA to be useful in a PBRT assay it is helpful to eliminate all plasmid DNA template used in the in vitro transcription reaction since residual DNA would effectively yield a PCR signal in the absence of added RT. This was achieved by employing multiple rounds of Turbo™ DNase Treatment (Life Technologies™; Carlsbad, CA, USA) followed by a final purification step employing the RNeasy Mini Kit (Qiagen; Venlo, Limburg, NL). The final purified RNA product showed no evidence of degradation when analyzed by glyoxal agarose gel electrophoresis.

The positive control RT enzyme employed in the PBRT assay was the Avian

Myeloblastosis virus αβ holoenzyme of molecular weight 157,000 daltons supplied by Life Sciences, Advanced Technologies, Inc. (St Petersburg, FL, USA). This is a highly purified enzyme preparation free of nucleases with a specific activity of 62,686 units/mg. A standard curve of this enzyme was established ranging from 0.1 nanounits (nu) to 10,000 nu per μL and 1 μL of each standard curve sample was added to a PBRT standard curve reaction which resulted in a range of 6 molecules to 600,000 molecules of RT in the PBRT standard curve. All PBRT reactions employed 5 ng of Den4 template RNA as well as the Den4 primers and probe and buffer components. Two reactions containing no added RT served as controls for background signals arising from residual DNA in the RNA template preparation. A "no template" control lacking the Den4 RNA was also run. Table 2 shows that when samples are run in triplicate for 40 cycles, a linear RT standard curve is observed with an R ² value of 0.986. The R ² value was slightly less than 0.99 due to a small amount of scatter in the 0.1 nu standard curve triplicate samples (6 molecules of RT per reaction). Importantly, the "No RT" samples exhibited Ct values only slightly less than 40, which demonstrates success in eliminating background signals due to residual DNA.

Table 2:

These data verify the ability of the present method to quantify RT levels down to 0.1 nu or six molecules per sample.

The above-mentioned PBRT assays were run using the TaqMan RNA-to-CT 1-Step Kit (Life Technologies™, catalog numbers 4392653, 4392938, and 4392656) with the exception that the RT enzyme from the kit was not added to the reactions since the assays were being used to detect the presence of contaminating RT or RT associated with the AMV RT and MLV spikes. Cycle times and temperatures were those described by the kit manufacturer.

Example 2: The PBRT assay can distinguish between particle-associated and free RT activity in test samples

Proteinase K (PK) was selected as an exemplary protease for distinguishing between particle-contained and free RT enzymatic activity. It was posited that under appropriate conditions PK would effectively eliminate all non-particle-contained RT activity in a given sample whereas the RT activity in intact, enveloped retroviral particles would be protected from proteolytic digestion. Conditions for this methodology were developed using norovirus VLP drug substance samples as a model. As a control for free (non-virus particle-contained) enzymatic activity samples of the VLP drug substance were supplemented with avian myeloblastosis virus (AMV) RT. As a control for RT contained in intact retrovirus particles, samples of the VLP drug substance were supplemented with murine leukemia virus (MLV) particles. An assay was deemed successful when RT activity due to AMV RT supplementation was eliminated by PK activity whereas RT activity from MLV particle supplementation was protected (i.e., RT activity persisted). It appeared that a crucial part of the assay was the ability to completely inactivate PK activity following PK treatment so that proteolytic activity would not be carried over into the PBRT assay steps and thereby reduce the RT-PCR performances. Also, it was determined that a PK inhibitor must not interfere with the PBRT assay.

It was discovered that a successful method involved one or more of the following steps:

1. Dilution of a sample to be tested into a larger volume of Proteinase K buffer, in order to dilute out the high concentration of virus-like particles in the sample, allowing for a more complete proteolytic degradation of RT not contained in virus particles.

2. Addition of a very small amount of Triton X-100 during Proteinase K digestion of virus particles helps facilitate complete digestion. Moreover, the amount of Triton X-100 used does not diminish virus particle-contained RT activity.

3. Inactivation of the Proteinase K, by the addition of PMSF, is necessary following the proteolytic digestion step. 4. Collection and concentration by high speed centrifugation through a sucrose cushion, of viral particles following the inactivation of Proteinase K should occur in the presence of the PMSF to ensure that Proteinase K activity does not persists during the PBRT reaction.

Example 3: The PBRT assay can distinguish in a test sample activity due to endogenous RT, activity due to supplemented RT, and activity due to supplemented virus particle- contained RT

Three 0.5 mL samples of Norwalk VLP drug substance were analyzed according to the PBRT assay of Example 1. Sample 1 was not supplemented. Samples 2 and 3, which served as positive controls, were supplemented with AMV RT or MLV, respectively. Following supplementation, 25 μΙ_, of each sample was set aside to determine baseline RT activities, i.e., prior to proteolytic digestion. The remainder of each sample was subjected to PK digestion, followed by recovery of intact virus particles by high speed centrifugation. Prior to assaying for RT activity, all samples were diluted 1 :2 with 2x AMV dilution buffer (a source of non-ionic detergent) to lyse (disrupt) any enveloped particles, thereby ensuring that all RT activity was being measured. The PK digested and set aside samples were assayed for RT activity using the PBRT assay described in Example 1. Data from this experiment are shown in Table 3

Table 3:

As shown in Table 3, test sample 1, which was not supplemented, exhibited a small level of baseline RT activity (Ct value = 33.3) prior to PK treatment. However, following PK treatment and recovery, the Ct value increased to 39.7, which is similar to "No RT control" levels. These data indicate that the baseline RT activity is not particle associated, is susceptible to PK digestion, and is not recoverable by high speed centrifugation. Sample 2, which was supplemented with AMV RT, behaved as expected in that it exhibited very strong RT baseline activity (Ct = 17.6) prior to PK digestion but after PK digestion and high speed centrifugation RT activity returned to "No RT Control" levels. This indicates that both the endogenous and supplemented RT activities were degraded by PK. Similar to sample 2, sample 3, which was supplemented with MLV particles, exhibited strong baseline RT activity (Ct value = 18.4).

However, unlike sample 2, in sample 3, PK digestion and high speed recovery of intact particles actually resulted in an increase in RT activity (Ct value = 16.5) due to concentration of the PK- resistant, intact MLV particles by the centrifugation recovery step.

These data support a premise that small amounts of measurable RT activity is present in Norwalk VLP drug substance which is not particle associated and is susceptible to proteolytic degradation. In other words, this endogenous RT activity is not due to intact retrovirus particle contamination.

The experiments described above were replicated with similar results observed.

See, Table 4.

Table 4:

In control experiments for the data presented in Tables 3 and 4, every sample tested in the PBRT reactions was also tested following supplementation with AMV RT in order to test for the possibility of protease carry over into the PBRT reactions. In this case every "spiked" sample showed a similarly strong RT signal demonstrating good performance of the PBRT reaction which was indicative of the absence of protease carryover. Example 4: End of Production harvest material fractions have greater RT activity relative to earlier harvest fractions

It has been shown that particle-associated errantivirus RNA sequences are more prevalent in end of production (EOP) harvest material fractions (i.e., baculovirus-lysed Sf9 cells) relative to medium harvested from healthy, non-baculovirus-infected Sf9 cells; it is believed that EOP harvest material fractions contain greater amounts both free and particle-associated RT than medium harvested from healthy Sf9 cells.

EOP harvest test samples were tested as described in Example 3 samples except that the EOP samples were diluted 10-fold in PK buffer; this was because a preliminary experiment demonstrated exceptionally high initial RT levels in EOP test samples, which would hinder distinguishing RT activity among samples. Table 5 shows the results of this experiment.

Table 5:

As can be seen in Table 5, EOP Sample 1, the un-supplemented sample, had a strong baseline RT activity. Baseline RT activity for EOP Sample 2, the AMV RT-supplemented sample, was only about two-fold greater. EOP Samples 1 and 2 exhibited essentially identical Ct values as each other following PK digestion and their recovery that fell to within the RT standard curve range, which indicates the presence of a PK-resistant, particle-contained fraction of RT activity in EOP harvest material. On the other hand, EOP Sample 3, the MLV-supplemented sample, showed strong baseline RT activity and activity after PK digestion; this is consistent with the PK-resistant-MLV spike which served as a control for the successful recovery of RT from intact retrovirus particles.

Data from the three EOP samples are consistent with the presence of PK-resistant, particle-contained RT in EOP harvest material. However, the particle-contained RT is clearly a small fraction with respect to the total amount of RT activity in the initial harvest sample since the pre- and post-PK signals differ by over three orders of magnitude when compared to the RT standard curve. This observation is consistent with the absence of detectable particle-associated RT activity in purified drug substance, as described in Example 3. The low level of parti cle- associated RT activity in harvest material to begin with, coupled with the use of a detergent step in the VLP purification process, are likely important factors leading to the absence of detectable particle-associated RT activity in the final drug substance.

Example 5: Alternative divalent cation test

Data presented in the prior Examples were generated in experiments employing Mg ²⁺ ions in the RT-PCR buffer. In order to allow for the possibility of detecting novel RT activity from enzymes requiring Mn ²⁺ ions, the drug substance evaluation experiment was repeated with the addition of 3 mM MnCl ₂ in the RT-PCR master mix buffer. Results of these experiments are shown in Table 6.

Table 6:

Table 6 shows that the outcomes are qualitatively similar to the results shown in Tables 3 and 4 with the exception that all of the Ct values were skewed upward Therefore, Mn ²⁺ ions did not stimulate the detection of a novel Mn ²⁺ -dependent RT that might have been present in an as of yet unidentified contaminating retrovirus. RT activity in drug substance and the RT and MLV spikes resulted in the same patterns previously observed. EQUIVALENTS

The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.