CELL-BASED METHODS AND REAGENTS
The present invention relates to compositions and methods for isolating cells devoid of unwanted viral contaminants, and to methods for preparing a virus stock substantially devoid of viral contaminants. Virus stocks, cells, and immunogenic reagents produced using such methods are also provided.
1 . A method of isolating a cell that is substantially devoid of a target virus, comprising:
a. contacting a population of cells a portion of which comprises the target virus with an RNA effector molecule that inhibits the growth or replication of the target virus;
b. detecting the presence of the target virus in each cell; and,
c. isolating at least one cell that is substantially devoid of the target virus.
2 . The method of claim 1 , further comprising repeating steps a-c.
3 . The method of claim 2 , further comprising detecting the presence or absence of target viral nucleic acid or protein.
4 . The method of claim 3 , wherein the isolated cell is free of the target virus.
5 . The method of claim 4 , wherein steps a-c are repeated when target viral nucleic acid or protein is detected.
6 . The method of claim 5 , wherein the target viral nucleic acid is detected by a method selected from the group consisting of: polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), dot blot hybridization, Northern hybridization, and Southern hybridization.
7 . The method of claim 5 , wherein the target viral protein is detected by a method selected from the group consisting of: radioimmunoassay, competitive-binding assay, ELISA, Western blot, FACS, immunohistochemistry, immunoprecipitation, proteomics, mass spectrometry, electrophoresis, and immunofluoresence.
8 . The method of claim 1 , wherein the presence of the target virus of step b. is immunologically detected.
9 . The method of claim 8 , wherein the presence of the target virus is detected using an antibody against a capsid protein (Cap).
10 . The method of claim 1 , wherein the cell further comprises a second virus.
11 . The method of claim 10 , further comprising detecting the presence of the second virus.
12 . The method of claim 11 , wherein the presence of the second virus is detected by detection of viral nucleic acid or viral protein of the second virus.
13 . The method of claim 12 , wherein the second viral nucleic acid is detected by a method selected from the group consisting of: polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), dot blot hybridization, Northern hybridization, and Southern hybridization.
14 . The method of claim 12 , wherein the second viral protein is detected by a method selected from the group consisting of: radioimmunoassay, competitive-binding assay, ELISA, Western blot, FACS, immunohistochemistry, immunoprecipitation, proteomics, mass spectrometry, electrophoresis, and immunofluoresence.
15 . The method of claim 10 , wherein the second virus is rotavirus.
16 . The method of claim 1 , wherein the target virus is a non-integrating virus.
17 . The method of claim 16 , wherein the non-integrating virus is porcine circovirus.
18 . The method of claim 1 , wherein the RNA effector molecule is detectably labeled.
19 . The method of claim 1 , further comprising detecting the presence of the RNA effector molecule in the cell.
20 . A cell that is substantially devoid of a target virus, obtained by the process of claim 1 .
21 . A method of producing an immunogenic agent, comprising:
(a) culturing the cell of claim 20 under conditions that allow expression of the immunogenic agent; and,
(b) isolating the immunogenic agent.
22 . A method of preparing a virus stock that is substantially devoid of an unwanted target virus, comprising:
(a) contacting a virus stock that contains unwanted target virus with an agent that binds specifically with the unwanted target virus; and
(b) collecting the unbound virus stock.
23 . The method of claim 22 , further comprising repeating steps (a) and (b).
24 . The method of claim 22 , further comprising detecting the presence or absence of target viral nucleic acid or protein from the virus stock.
25 . The method of claim 22 , wherein the agent is immobilized.
26 . The method of claim 22 , wherein the agent is a receptor, an antibody, or an antigen binding fragment thereof.
27 . The method of claim 26 , wherein the agent is an antibody that binds to the porcine circovirus-1 (PCV-1).
28 . The method of claim 27 , wherein the antibody binds to the porcine circovirus-1 (PCV-1) capsid (Cap) protein.
29 . The method of claim 26 , wherein the agent is a receptor and the receptor is heparin.
30 . The method of claim 22 , wherein the agent is a receptor on a cell surface.
31 . The method of claim 30 , wherein the receptor on a cell surface is heparin.
32 . The method of claim 22 , further comprising contacting the cell with a RNA effector molecule that increases the presence of the receptor on the cell surface.
33 . The method of claim 32 , wherein the RNA effector molecule targets the heparanase gene.
34 . The method of claim 30 , wherein the cell does not have a receptor that binds the desired virus of the virus stock.
35 . The method of claim 34 , wherein the receptor is sialic acid.
36 . The method of claim 30 , wherein the cell is treated to have decreased level of cell receptor to the desired virus stock.
37 . The method of claim 36 , wherein the cell is treated by physical, chemical, or enzymatic means.
38 . The method of claim 34 , wherein the presence of a cell receptor to the desired viral stock has been decreased by contacting the cell with a RNA effector molecule that inhibits expression of the cell receptor.
39 . The method of claim 30 , further comprising
(a) contacting the unbound virus stock with a host cell;
(b) contacting the cell with an RNA effector molecule that inhibits growth or replication of the unwanted target virus; and
(c) isolating the virus stock from the medium.
40 . The method of claim 39 , wherein the host cell does not have a receptor for the unwanted target virus.
41 . A virus stock that is substantially devoid of unwanted target virus prepared by the method of claim 22 .
42 . The virus stock of claim 41 , wherein the virus stock is rotavirus.
43 . The virus stock of claim 42 , wherein the unwanted target virus is PCV1.
44 . A biological product made using the virus stock of claim 41 .
45 . The biological product of claim 44 , wherein the product is rotavirus vaccine.
46 . A method of preparing a virus stock that is substantially devoid of an unwanted target virus, comprising:
(a) propagating a virus stock in a host cell in which expression of a receptor for a suspected viral contaminant has been inhibited; and
(b) collecting the virus stock.
47 . The method of claim 46 , wherein the receptor is heparin.
48 . The method of claim 47 , wherein the heparin is inhibited by contacting the host cell with a RNA effector molecule that targets an epimerase, a xylosyltransferase, a galactosyltransferase, a N-acetylglucosaminyl transferase, a glucuronosyl transferase, or a 2-O sulfotransferase gene.
49 . The method of claim 46 , further comprising contacting the host cell with a RNA effector molecule that inhibits the growth or replication of the unwanted target virus.
50 . The method of claim 46 , wherein the unwanted target virus is PCV1.
51 . A virus stock that is substantially devoid of an unwanted target virus, prepared by the method of claim 46 .
52 . The virus stock of claim 51 , wherein the virus stock is rotavirus.
53 . A biological product made using the virus stock of claim 51 .
54 . The biological product of claim 53 , wherein the product is rotavirus vaccine.
55 . A virus stock that is substantially devoid of an unwanted target virus, prepared by the process comprising:
(a) contacting a virus stock that contains unwanted target virus with an agent that binds specifically with the unwanted target virus; and
(b) collecting the unbound virus stock.
56 . A virus stock that is substantially devoid of an unwanted target virus, prepared by the process comprising:
(a) propagating a virus stock in a host cell in which expression of a receptor for a suspected viral contaminant has been inhibited; and
(b) collecting the stock virus.