Review of animal use requirements in WHO biologics guidelines – database of suggested guideline revisions

Original text

An appropriate in vivo test should be used to evaluate virus seeds and monovalent bulks. Summaries of the MNVT and TgmNVT, including pass and fail criteria, are given in Appendix 2, along with considerations on the choice of assay. The test should be approved by the NRA for the specific product, and may use transgenic mice or nonhuman primates, or both. The test for neurovirulence in nonhuman primates should be carried out as summarized in Appendix 2 and described in the SOP on neurovirulence tests for types 1, 2 or 3 live-attenuated OPV in monkeys, available from WHO.

New text

The potential neurovirulence of a new vaccine strain should be assessed during preclinical development and a risk analysis carried out based on available scientific data and information. If molecular consistency has been demonstrated during characterisation, then assessment of neurovirulence may be omitted for subsequent viral seed lots and/or routine manufacturing. For existing products where animal neurovirulence testing is currently prescribed, this test can be waived when safety and genetic stability of the product are sufficiently assured. This can be established by historical / (pre-) clinical, and pharmacovigilance data, and by data generated with nucleic acid amplification and sequencing techniques, to support the molecular consistency of the virus and for the establishment of a link between genetic sequences and in vivo phenotypes. For all products, a risk-based approach should be performed taking into consideration the genetic features and molecular consistency of the strain (sequence evaluated at different manufacturing steps, determined with traditional or new sequencing technologies) and the nature of the vaccine (attenuated, chimeric, genetically modified), to assess whether a neurovirulence assay is required and what animal model is most suitable. If an in vivo assay is scientifically justified, it should be established at what level (Master Seed Lot, Working Seed Lot, monovalent bulk) the test should be performed to avoid unnecessary duplication.

Original text

Each final lot should be identified by immunological assay on cell culture using specific antibodies, or by a molecular method that has been validated and approved by the NRA. Neutralization tests can distinguish the serotype of polioviruses. Molecular methods, such as sequencing or deep sequencing, can distinguish Sabin virus from wild-type virus. Care should be taken to ensure that the serum samples used are monospecific by titrating them against homotypic and heterotypic viruses of known virus titre. Monoclonal antibodies may be used for this purpose.

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When the vaccine contains more than one poliovirus type, each type should be titrated separately, using appropriate type-specific antiserum to neutralize each of the other types present.

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A sample of the monovalent bulk should be tested for the presence of herpes B virus and other viruses by injection into at least 10 healthy rabbits, each weighing between 1.5 kg and 2.5 kg.
All rabbits that die after the first 24 hours of the test should be examined by necropsy, with the brain and organs removed for detailed examination to establish the cause of death. Animals showing signs of illness should be humanely killed and subjected to a similar necropsy.
A test for the presence of Marburg virus may be carried out in guinea‑pigs.

New text

These tests can be avoided if primary monkey kidney cells are not used for vaccine production. OPV manufacturers are encouraged to avoid the use of primary animal cells for vaccine production. Where animal tests are still required, the use of humane endpoints are encouraged. Manufacturers are also encouraged to adopt contemporary best practice for animal care and welfare.

Original text

At least 10 monkeys should be employeed in each test. Immediately before inoculation, all monkeys should be shown to be serologically negative for varicella. The material under test should be given be injection of 0.5 ml into the thalamic region of each hemisphere. The total amount of varicella virus given to each monkey should be not less than the amount contained in the recommended single human dose od vaccine. The monkey should be observed for 17-21 days.for symptoms of paralysis and other evedence of neurological involvement. Animals that die within 48 hours of injection may be replaced. The test is invalid and should be repeated if more than 20% of the monkeys die from non-specific causes. At the end of the observation period all the monkeys are anaesthetized and killed for autopsy; histopathological examinations of appropriate areas of the brain are made of evidence of central nervous system involvement.

New text

The potential neurovirulence of a new vaccine strain should be assessed during preclinical development and a risk analysis carried out based on available scientific data and information. If molecular consistency has been demonstrated during characterisation, then assessment of neurovirulence may be omitted for subsequent viral seed lots and/or routine manufacturing. For existing products where animal neurovirulence testing is currently prescribed, this test can be waived when safety and genetic stability of the product are sufficiently assured. This can be established by historical / (pre-) clinical, and pharmacovigilance data, and by data generated with nucleic acid amplification and sequencing techniques, to support the molecular consistency of the virus and for the establishment of a link between genetic sequences and in vivo phenotypes. For all products, a risk-based approach should be performed taking into consideration the genetic features and molecular consistency of the strain (sequence evaluated at different manufacturing steps, determined with traditional or new sequencing technologies) and the nature of the vaccine (attenuated, chimeric, genetically modified), to assess whether a neurovirulence assay is required and what animal model is most suitable. If an in vivo assay is scientifically justified, it should be established at what level (Master Seed Lot, Working Seed Lot, monovalent bulk) the test should be performed to avoid unnecessary duplication.

Original text

At the end of the observation period, 25% of the control cell cultures shall be tested for the presence of haemadsorbing viruses, using guinea-pig red cells, and shown to be negative. If the red cells have been stored, the duration of storage shall not have exceeded seven days, and the temperature of storage shall have been in the range of 2–8 °C.

New text

At the end of the observation period a fraction of culture comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using red blood cells from guinea-pig or other suitable red blood cells. It is not necessary to use red blood cells from multiple species. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

Original text

Each final lot shall be tested for the absence of abnormal toxicity in mice and guinea-pigs by appropriate tests approved by the national control authority.

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This is a detailed SOP for the monkey neurovirulence assay.

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This is a detailed SOP for the transgenic mouse neurovirulence assay.

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Each virus master or working seed lot should also be tested in animals that may include guinea-pigs, adult mice, suckling mice and embryonated chicken eggs, as appropriate. For test details, refer to the Requirements for measles vaccines (live) (52). See also section A.4.2.1.1. New molecular methods with broad detection capabilities are being developed for the detection of adventitious agents. These methods include: degenerate NAT for whole virus families with analysis of the amplicons by hybridization, sequencing or mass spectrometry; NAT with random primers followed by analysis of the amplicons on large oligonucleotide microarrays of conserved viral sequencing or digital subtraction of expressed sequences; and high-throughput sequencing. These methods may be used in the future to supplement existing methods, or as alternative methods to both in vivo and in vitro tests, after appropriate validation and approval by the NRA (51).

New text

A strategy for testing adventitious viruses in vaccines must be developed based on a risk assessment. Relevant culture methods and/or specific molecular biology or broad molecular methods should be part of the overall testing package with the agreement of the NRA. In vivo tests may only be used if the risk assessment indicates that this test provides an additional risk mitigation taking into account the overall testing package.