DECREASING POTENTIAL IATROGENIC RISKS ASSOCIATED WITH INFLUENZA VACCINES

Abstract

Influenza viruses for use in preparing human vaccines have traditionally been grown on embryonated hen eggs, although more modern techniques grow the virus in mammalian cell culture e.g. on Vero, MDCK or PER.C6 cell lines. The inventor has realised that the conditions used for influenza virus culture can increase the risk that pathogens other than influenza virus may grow in the cell lines and have identified specific contamination risks. Suitable tests can thus be performed during manufacture in order to ensure safety and avoid iatrogenic infections.

Claims

1: A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line, comprising a step in which the vaccine and/or the culture is tested for the presence of an infectious agent that can grow in said cell line but that does not grow in embryonated hen eggs.

2: A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a mammalian cell line, comprising a step in which the vaccine and/or the culture is treated to remove and/or inactivate an infectious agent that can grow in the cell line but does not grow in embryonated hen eggs.

3: The process of claim 1 or claim 2, wherein the mammalian cell line is a MDCK cell line, a Vero cell line, or a PER. C6 cell line.

4: The process of any preceding claim, wherein the infectious agent is selected from the group consisting of: Pneumovirinae; Morbilliviruses of the Paramyxoviridae family; Enteroviruses of the Picornaviridae family; mammalian Reoviridae; and Birnaviridae.

5: The process of claim 4, wherein the infectious agent is selected from the group consisting of: respiratory syncytial virus; measles virus; Coxsackie viruses; echoviruses; enteroviruses; orthoreoviruses; rotaviruses; and infectious bursal disease virus.

6: The process of any preceding claim, wherein the mammalian cell line is a Vero cell line, and wherein the infectious agent is selected from the group consisting of: Metapneumoviruses of the Paramyxoviridae family; Rubulaviruses of the Paramyxoviridae family; Togaviridae; Coronaviridae; Rhinoviruses of the Picornaviridae family; varicella zoster virus; Polyomaviridae; porcine circoviruses; porcine picomaviruses; Chlamydia bacteria; and Parvoviruses.

7: The process of claim 6, wherein the infectious agent is selected from the group consisting of: human metapneumovirus; mumps virus; Rubellavirus; SARS coronavirus; M-strains of Rhinovirus; SV-40 polyomavirus; BK polyomavirus; JC polyomavirus; swine vesicular disease virus; Teschen-Talfan virus; C. trachomatis; C. pneumoniae; C. psittaci; canine parvovirus; and porcine parvoviruses.

8: The process of any preceding claim, further comprising a step in which the vaccine and/or the culture is tested for the presence of a pathogen selected from the group consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; avian circoviruses; and avian Reoviridae.

9: A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a first mammalian cell line, comprising a step in which the vaccine and/or the culture is tested for the presence of an infectious agent that can grow in said first cell line but that does not grow in a second mammalian cell line.

10: A process for preparing an influenza vaccine from influenza virus that has been grown in a culture of a first mammalian cell line, comprising a step in which the vaccine and/or the culture is treated to remove and/or inactivate an infectious agent that can grow in said first cell line but does not grow in a second mammalian cell line.

11: The process of claim 9 or claim 10, wherein (a) the first mammalian cell line is selected from the group consisting of: a MDCK cell line, a Vero cell line, or a PER.C6 cell line; (b) the second mammalian cell line is selected from the group consisting of a MDCK cell line, a Vero cell line, or a PER. C6 cell line; and (c) the first and second mammalian cell lines are different.

12: The process of any one of claims 9 to 11, wherein the first cell line is a Vero cell line, and wherein the infectious agent is selected from the group consisting of: Metapneumoviruses of the Paramyxoviridae family; Rubulaviruses of the Paramyxoviridae family; Togaviridae; Coronaviridae; Rhinoviruses of the Picornaviridae family; varicella zoster v1n1s; Polyomaviridae; porcine circoviruses; porcine picornaviruses; Chlamydia bacteria; Parvoviruses; and Birnaviridae.

13: The process of claim 12, wherein the infectious agent is selected from the group consisting of: human metapneumovirus; mumps virus; Rubellavirus; SARS coronavirus; M-strains of Rhinovirus; SV-40 polyomavirus; BK polyomavirus; JC polyomavirus; swine vesicular disease virus; Teschen-Talfan virus; C. trachomatis; C. pneumoniae; C. psittaci; canine parvovirus; porcine parvoviruses; and infectious bursal disease virus.

14: The process of any one of claims 9 to 13, further comprising a step in which the vaccine and/or the culture is tested for the presence of a pathogen selected from the group consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; avian circoviruses; and avian Reoviridae.

15: The process of claim 2 or claim 10, comprising a further step wherein, after said removal and/or inactivation, the vaccine and/or culture is tested for the presence of said infectious agent.

16: A process for preparing an influenza vaccine from influenza virus that has been grown m a culture of a mammalian cell line or in hens eggs, comprising a step in which the vaccine and/or the culture is tested for the presence of a pathogen selected from the group consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; avian circoviruses; avian Reoviridae; and Birnaviridae.

17: The process of claim 16, wherein the pathogen is selected from the group consisting of: PIV-1; PIV-2; PIV-3; herpes simplex virus 1; herpes simplex virus 2; human adenovirus; simian adenovirus; orthoreoviruses; and infectious bursal disease virus.

18: The process of any preceding claim, where in the culture is tested by immunochemical detection and/or nucleic acid detection.

19: The process of claim 18, wherein detection is by ELISA and/or PCR (including RT-PCR).

20: An influenza vaccine that has been grown in a culture of a mammalian cell line, wherein the vaccine has been confirmed as free from the presence of an infectious agent that can grow in said cell line but that does not grow in embryonated hen eggs.

21: An influenza vaccine that has been grown in a culture of a first mammalian cell line, wherein the vaccine has been confirmed as free from the presence of an infectious agent that can grow in said first cell line but that does not grow in a second mammalian cell line.

22: The vaccine of claim 18 or claim 19, wherein vaccine was grown in a culture of a MDCK cell line, of a Vero cell line, or of a PER.C6 cell line.

23: An influenza vaccine in which mammalian reovirus is undetectable by RT-PCR.

24: The vaccine of claim 23, which is free from ovalbumin and from chicken DNA.

25: An influenza vaccine obtained or obtainable by the process of any one of claims 1 to claim 17.

26: The vaccine of any one of claims 20 to 25, which is a live virus vaccine.

27: The vaccine of any one of claims 20 to 25, which is an inactivated virus vaccine.

28: The vaccine of claim 27, which is a whole virus vaccine, a split virus vaccine, or a viral subunit vaccine.

29: The vaccine of any one of claims 20 to 28, which is a trivalent influenza vaccine.

30: The vaccine of any one of claims 20 to 29, which includes a pandemic influenza virus strain.

31: The vaccine of claim 30, which includes a H5 or H7 influenza virus strain.

32: The vaccine of any one of claims 20 to 31, which is for administration to a patient by injection, by an intranasal route, by an oral route, by an intradermal route, by a transcutaneous route, or by a transdermal route.

33: The vaccine of any one of claims 20 to 32, which is for pediatric immunization.

34: The vaccine of any one of claims 20 to 33, which includes 1-20 g of influenza virus hemagglutinin per strain.

35: The vaccine of any one of claims 20 to 34, which includes an adjuvant.

Description

MODES FOR CARRYING OUT THE INVENTION

MDCK Cells

[0076] The inventor has extensive experience of growing influenza viruses on MDCK cells in serum-free culture for the preparation of vaccines. They have realised that the cells are also suitable hosts for other pathogenic agents, and so the ability of various other pathogens to grow in the same conditions was tested (specifically, culture of MDCK 33016, deposited as DSM ACC2219, in serum-free medium, as disclosed in reference 2).

[0077] When testing for active virus replication or growth in MDCK cells, tests for respiratory syncytial viruses RSV-A2 and RSV-B were negative. Parainfluenzavirus strains PI-3 and SV-5 were detected. Tests for human coronaviruses 229E and SARS were negative, as were tests for poliovirus I, echovirus 6, coxsackievirus A16 and coxsackievirus B3. Type Ib, 37 and NL.9501841 rhinoviruses tested negative. Tests for reovirus Reo3 were positive, as were tests for herpes simplex virus HSV-1. Tests for human adenoviruses 1, 5 and 6 were negative. SV-40 tests were negative, and inoculum titers were stable for 14 days. Canine parvovirus and minute virus of mice tested negative, as did Rous sarcoma virus. Mycoplasma hyorhinis tested negative. Chlamydia trachomatis tested negative, although a very low level of growth could not be excluded during days 3-5 after infection.

[0078] Further investigation revealed that MDCK cells can support growth of vesicular stomatitis (Indiana) virus, vaccinia virus, coxsackievirus B5; reovirus 2; human adenovirus types 4 and 5; vesicular exanthema of swine virus, and infectious canine hepatitis virus [58].

[0079] Of the viruses which could be grown in MDCK cells, parainfluenzaviruses, herpes simplex viruses and adenoviruses can also grow in embryonated hen eggs. In contrast, the human reoviruses (and other mammalian reoviruses) do not readily grow in eggs. If MDCK is used as a cell culture system for influenza virus production, therefore, quality control testing should check for contamination by human reoviruses. The inventor estimates that reovirus levels could increase by 5 logs or more during repeated passages in MDCK suspension cultures, whereas levels of a virus such as adenovirus would decrease by 6 to 10 logs. Herpes simplex virus levels should also be checked, as HSV growth of at least 8 logs is possible. Similarly, PIV-3 growth of 8 logs has been seen after 1 week of culture.

Vero Cells

[0080] Following the testing work on MDCK cells, replication of pathogens in Vero cells was investigated. Vero cells support the growth of pathogens such as: pneumoviruses, such as RSV-A and RSV-B; human metapneumoviruses (HMPV); morbilliviruses, such as measles virus; paramyxoviruses, such as mumps virus and parainfluenza virus; rubellavirus; human and avian coronaviruses; picornaviruses, such as entroviruses, echoviruses and coxsackie viruses, and porcine SVDV and Teschen-Talfan virus; mammalian and avian reoviruses; herpesviruses, such as HSV-1 and HSV-2; simian and human adenoviruses; varicella zoster virus (VZV); polyomaviruses, such as JC, BK and SV-40; birnaviruses, such as gumborovirus; porcine circoviruses; canine parvovirus; and Chlamydia.

[0081] Of these pathogens, the following do not grow in hen eggs, and are thus new risks for contamination of influenza vaccines when Vero cells are used as a substrate: RSV; HMPV; measles virus; rubellavirus; human coronaviruses; enteroviruses; reoviruses; VZV; polyomaviruses; porcine picornaviruses, parvoviruses and circoviruses. Many of these pathogens do not grow in MDCK cells, showing that MDCK is a safer substrate for influenza vaccine production. Emerging viruses such as the SARS coronavirus grow on Vero cells, but not on MDCK cells. Similarly, VZV grows on Vero cells, but not on hen eggs or on MDCK cells. Vaccination with a Vero-derived influenza vaccine that was inadvertently contaminated with this coronavirus or with VZV could lead to an iatrogenic outbreak of SARS and/or chickenpox, which would be disastrous both to the population and to the reputation of vaccines. Having identified these risks, however, appropriate quality control mechanisms can be put in place.

[0082] In addition to Vero cells, PER.C6 cells support growth of adenoviruses [59,60]. Based on known viral characteristics, PER.C6 cells can also be expected to support the growth of at least parainfluenzaviruses and reoviruses.

[0083] It will be understood that the invention is described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention.

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