# US 2010/0166705 A1 · Culturing circular ssDNA viruses for the production of vaccines

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2010/0166705 A1
- Document ID: US20100166705A1
- Status: Published application
- Kind: A1
- Application number: 12/510,844
- Filed: 2009-07-28
- Published: 2010-07-01
- Earliest claimed: 2005-02-03
- Inventors: Hans Nauwynck; Gerald Misinzo; Sven Arnouts
- Web page: https://patentgrep.com/patent/US20100166705A1

## CPC

- A61K 39/12: Viral antigens
- A61K 38/21: Interferons {[IFN]}
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61P 31/12: Antivirals
- A61P 37/04: Immunostimulants
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/552: {Veterinary vaccine}
- A61K 2039/55522: {Cytokines; Lymphokines; Interferons}
- C12N 2750/10034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to the use of interferon in the in vitro cultivation of animal circular ssDNA virus such as Porcine Circovirus 2 or human TT virus in an animal cell line. Increased titres of animal circular ssDNA virus are obtained by addition of interferons or agents which ensure the production of endogenous interferons by said cell line and/or by the reduction of endosomal-lysosomal system acidification.

## Claims

### Claim 1

A method for the in vitro cultivation of an animal circular ssDNA virus comprising the step of (a) inoculating cells of a continuous animal cell linein a culture medium with said circular ssDNA virus, (b) cultivating said continuous animal cell line in said medium, thereby ensuring that endosomal-lysosomal system acidification is reduced in said continuous animal cell line, and (c) isolating said animal circular ssDNA virus from said medium and/or said infected continuous animal cell line.

### Claim 2 (depends on 1)

The method of claim 1, which comprises, ensuring that said endosomal-lysosomal system acidification is reduced in said continuous animal cell line after said inoculation with said circular ssDNA virus.

### Claim 3 (depends on 1)

The method of claim 1, which comprises cultivating said continuous animal cell line in the presence of an inhibitor of endosomal-lysosomal system acidification.

### Claim 4 (depends on 3)

The method of claim 3, wherein said agent is a lysosomotropic agent.

### Claim 5 (depends on 4)

The method according to claim 4, wherein said lysosomotropic agent is selected from the group consisting of ammonium chloride, chloroquine diphosphate and monensin.

### Claim 6 (depends on 1)

The method according to claim 1, which comprises the steps of:
(a) inoculating cells of a continuous animal cell line in culture medium with a circular ssDNA virus and,
(b) cultivating said continuous animal cell line in the presence of an agent capable of inhibiting endosomal-lysosomal system acidification.

### Claim 7 (depends on 1)

The method according to claim 1, further comprising the step of inoculating cells of a continuous animal cell line in a culture medium with said circular ssDNA virus, thereby ensuring, prior to or after said inoculation, that said medium contains interferon.

### Claim 8 (depends on 6)

The method of claim 6, comprising the steps of:
a) inoculating cells of a continuous animal cell line in culture medium with a circular ssDNA virus,
a′) administering an exogenous interferon or an agent which induces the endogenous production of an interferon by said cells, and
b) cultivating said continuous animal cell line in the presence of an agent capable or inhibiting endosomal-lysosomal system acidification.

### Claim 9 (depends on 6)

The method according to claim 6, which comprises, between steps (a′) and (b) a change of cultivation medium.

### Claim 10 (depends on 8)

The method according to claim 8, wherein said exogenous interferon is added after the inoculation of said cell line with the animal circular ssDNA virus.

### Claim 11 (depends on 8)

The method according to claim 8, wherein said interferon is produced endogenously by said cell line.

### Claim 12 (depends on 11)

The method according to claim 11, wherein said cell line is a transgenic cell line transfected with a polynucleotide encoding said interferon.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said animal circular ssDNA virus belongs to the taxonomic group of circovirus.

### Claim 14 (depends on 13)

The method according to claim 13, wherein said circovirus is Porcine Circovirus 2 (PCV2).

### Claim 15 (depends on 1)

The method according to claim 1, wherein said animal cell line is a porcine cell line.

### Claim 16 (depends on 15)

The method according to claim 15, wherein said porcine cell line is PK-15.

### Claim 17 (depends on 8)

The method according to claim 8, wherein said interferon is interferon-alpha or interferon-gamma.

### Claim 18 (depends on 8)

The method according to claim 8, which comprises ensuring that said medium contains said interferon at a concentration of at least 2 U/ml medium.

### Claim 19

An undiluted cultivation medium of an in vitro culture of a cell-line comprising animal circular ssDNA virus characterised in that it further comprises an agent capable of inhibiting endosomal-lysosomal system acidification.

### Claim 20

An undiluted cultivation medium of an in vitro culture of a cell-line comprising animal circular ssDNA virus characterised in that it further comprises at least 2 U/ml interferon.

### Claim 21

A method for producing a vaccine for protection against an animal circular DNA virus, comprising said animal circular DNA virus or components thereof, said method comprising the steps of:
a) inoculating cells of a continuous animal cell line in a culture medium with said circular ssDNA virus, thereby ensuring, prior to and/or after said inoculation, the reduction of endosomal-lysosomal system acidification of said continuous animal cell line;
b) allowing said ssDNA virus to replicate in said continuous animal cell line; and
c) obtaining said circular ssDNA virus or components thereof from said continuous animal cell line or said medium.

### Claim 22 (depends on 21)

The method according to claim 21, which further comprises ensuring, prior to and/or after said inoculation, that said medium contains interferon.

### Claim 23 (depends on 21)

The method according to claim 21, wherein said step of ensuring, the inhibition of endosomal/lysosomal acidification of said continuous animal cell line is ensured by addition of lysosomotropic endosomal/lysosomal acidification inhibitors to said cultivation medium.

### Claim 24

An in vitro method for determining the infection of a sample by an animal circular ssDNA virus comprising increasing the amount of virus before detection using endosomal-lysosomal system acidification inhibitors.

### Claim 25 (depends on 24)

The method of claim 24, wherein said sample is a cell-containing sample, which method comprises the steps of:
a) adding one or more endosomal-lysosomal system acidification inhibitors to a fraction of said cell-containing sample;
b) allowing the replication of said ssDNA virus; and
c) detecting the presence of said ssDNA virus in said sample.

### Claim 26 (depends on 24)

The method of claim 24, which comprises the steps of:
a) adding a fraction of said sample to a culture of cells susceptible to infection by circular ssDNA virus in a medium;
b) ensuring that endosomal-lysosomal system acidification is reduced in said culture of cells susceptible to infection by circular ssDNA virus; and
c) detecting of the presence of ssDNA virus in said culture of cells or said medium.

### Claim 27 (depends on 25, 26)

The method of any one of claim 25 or 26, which further comprises adding interferon or an agent capable of inducing interferon to said fraction of said cell-containing sample or said culture of cells susceptible to infection by circular ssDNA virus of step (a).

### Claim 28 (depends on 25, 26)

The method according to claim 25 or 26, which further comprises ensuring that the medium of said fraction of said cell-containing sample or of said culture of cells susceptible to infection by circular ssDNA virus comprises interferon.

### Claim 29 (depends on 28)

The method according to claim 28, which comprises adding exogenous interferon to said medium of said culture of cells.

### Claim 30 (depends on 29)

The method according to claim 29, wherein said interferon is IFN-alpha or IFN-gamma.

### Claim 31

A method of improving the immune response of a subject to a vaccine comprising an attenuated animal circular ssDNA virus, said method comprising, administering one or more inhibitors of endosomal-lysosomal system acidification to said subject, simultaneously with or shortly before or after administering said vaccine to said subject.

### Claim 32 (depends on 31)

The method of claim 31, which further comprises administering interferon to said subject, simultaneously with or shortly before or after administering said vaccine.

### Claim 33 (depends on 31)

The method according to claim 31, wherein said animal circular ssDNA virus is a Circovirus.

### Claim 34

A vaccine comprising an attenuated animal circular ssDNA virus and one or more inhibitors of endosomal-lysosomal system acidification.

### Claim 35 (depends on 34)

The vaccine of claim 34, which further comprises an interferon.

### Claim 36

A vaccine comprising an attenuated animal circular ssDNA virus and interferon.

### Claim 37

An undiluted medium of an in vitro culture of a cell-line comprising animal circular ssDNA virus characterized in that it further comprises at least 2 U/ml interferon.

### Claim 38

An undiluted medium of an in vitro culture of a cell-line comprising animal circular ssDNA virus characterized in that it further comprises an agent capable of inhibiting endosomal-lysosomal system acidification.