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WHO/INFLUENZA/DRAFT/NOVEMBER 2017 2
ENGLISH ONLY 3
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WHO biosafety risk assessment and guidelines for the production 5
and quality control of novel human influenza candidate vaccine 6
viruses and pandemic vaccines 7
(Proposed revision of WHO TRS No. 941, Annex 5; H1N1 specific update 2009; and H7N9 8
update 2013) 9
10
NOTE: 11
This document has been prepared for the purpose of inviting comments and suggestions on the 12
proposals contained therein, which will then be considered by the Expert Committee on 13
Biological Standardization (ECBS). Publication of this early draft is to provide information 14
about the proposed WHO biosafety risk assessment and guidelines for the production and 15
quality control of novel human influenza candidate vaccine viruses and pandemic vaccines to a 16
broad audience and to improve transparency of the consultation process. 17
The text in its present form does not necessarily represent an agreed formulation of the 18
Expert Committee. Written comments proposing modifications to this text MUST be 19
received by 12 February 2018 in the Comment Form available separately and should be 20
addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: 21
Department of Essential Medicines and Health Products (EMP). Comments may be submitted 22
electronically to the Responsible Officer: Dr TieQun Zhou at email: [email protected]. 23
The outcome of the deliberations of the Expert Committee will be published in the WHO 24
Technical Report Series. The final agreed formulation of the document will be edited to be in 25
conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). 26
© World Health Organization 2017 27
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health 28
Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: 29
[email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for 30
non-commercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-31
mail: [email protected]). 32
The designations employed and the presentation of the material in this publication do not imply the expression of 33
any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, 34
territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on 35
maps represent approximate border lines for which there may not yet be full agreement. 36
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The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 1
recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. 2
Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 3
All reasonable precautions have been taken by the World Health Organization to verify the information contained in 4
this publication. However, the published material is being distributed without warranty of any kind, either expressed 5
or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the 6
World Health Organization be liable for damages arising from its use. 7
The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. 8
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Contents 1
2
Summary 3
1. Introduction 4
2. Scope 5
3. Terminology 6
4. Hazard identification 7
5. Safety Testing of candidate vaccine viruses 8
6. Risk assessment and management 9
Authors and acknowledgements 10
References 11
Appendix 1. Testing for attenuation of influenza vaccine viruses in ferrets 12
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Abbreviations 1
A/Len/17 Influenza A/Leningrad/134/17/57 virus
BSL Biosafety Level
CVV Candidate vaccine virus
EID Egg infectious dose
GAP WHO Global Action Plan for Influenza Vaccines
GISRS WHO's Global Influenza Surveillance and Response System
GMP Good manufacturing practice
HA Haemagglutinin
H&E haemotoxylin and eosin (staining)
HEPA High-efficiency particulate air filtration
HP High pathogenicity
HPAI Highly pathogenic avian influenza virus
IVPI (Chicken) Intravenous pathogenicity index. Any virus with an index greater
than 1.2 is considered an HPAI
LP Low Pathogenicity
LPAI Low-pathogenic avian influenza virus
MDCK Madin-Darbey Canine Kidney (cells)
MVA Modified Vaccine Ankara
NA Neuraminidase
OIE World Organization for Animal Health
PAPR Powered air-purifying respirators
PFU Plaque forming unit
PPE Personal protection equipment
PR8 Influenza A/Puerto Rico/8/34 virus (A/PR8/34)
PTC Pass through cabinets
RG Reverse Genetics
TCID Tissue culture infective dose
VCM WHO Vaccine Composition meeting for Influenza Vaccines
VSV Vesicular stomatitis virus
WHO CC WHO Collaborating Centre for Influenza
WHO ERL WHO Essential Regulatory Laboratory for Influenza
wt Wild-type
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Summary 1
International biosafety expectations for both the pilot-scale and large-scale production of 2
human vaccines in response to a pandemic influenza virus, and the quality control of these 3
vaccines, are described in detail in these WHO Guidelines. Tests required to evaluate the safety 4
of candidate influenza vaccine viruses (CVVs) prior to release to vaccine manufacturers are 5
also specified in this document, which is thus relevant to both development and production 6
activities, and also to vaccine and biosafety regulators. A detailed risk assessment is presented 7
that concludes that the likelihood of direct harm to human health would be high if reassorted 8
H5 or H7 viruses or other subtypes that have high in vivo pathogenicity are used for vaccine 9
production; moreover, low pathogenic avian influenza viruses that are highly virulent for 10
humans may also present a high risk and all such viruses could also pose a significant risk to 11
animal health. Stringent vaccine biosafety control measures, defined as Biosafety Level (BSL)3 12
enhanced, are defined to manage the risk from vaccine production and quality control using 13
such viruses in the pre-pandemic period. For all other vaccine viruses, for example reassortants 14
containing a highly attenuated backbone (e.g. from PR8) or the handling of low pathogenic 15
avian influenza viruses that are not highly virulent for humans, the direct risk to human health 16
is considered to be low. Nevertheless, there is an indirect risk to human health due to a 17
theoretical risk of secondary reassortment with circulating human influenza viruses, resulting in 18
a novel virus capable of replicating in humans. Although extremely unlikely, such a secondary 19
reassortant could become adapted to human infection and transmission and result in serious 20
public health consequences. The biosafety control measures that are proposed, defined as BSL2 21
enhanced (pandemic influenza vaccine), take this and also potential risks to animal health, into 22
account. Specifications for personal protection are provided for both BSL2 enhanced and BSL3 23
enhanced biosafety levels, and guidance is provided on biosafety management and 24
implementation within a vaccine production facility. Tests to be performed on CVVs prior to 25
release to vaccine manufacturers depend on the type of virus but include, at a minimum, in vivo 26
tests in ferrets and, where appropriate, chickens and embryonated eggs embryos, plaque assays 27
and sequencing. 28
29
30
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1. Introduction 1
Due to the inherent risks in handling pandemic influenza virus(es) for the production of 2
vaccines where an uncontrolled release could have a significant public health impact, risk 3
assessment, biosafety and biosecurity precautions are needed in laboratory and manufacturing 4
environments. WHO developed a biosafety risk assessment and guidelines for the production 5
and quality control of human influenza pandemic vaccines in 2005, published as Annex 5 to 6
TRS 941, in response to the threat of a pandemic posed by the highly pathogenic avian 7
influenza (HPAI) viruses A(H5N1) and the need to begin development of experimental 8
vaccines (2). This threat still persists and several countries have produced and stockpiled H5N1 9
vaccine. Moreover, other threats have arisen since the appearance of H5N1, such as the 10
H1N1pdm09 subtype virus causing the pandemic in 2009 and the emergence of low pathogenic 11
avian influenza (LPAI) viruses A(H7N9) that were able to replicate in humans causing severe 12
disease with a high fatality rate (mostly in adults and the elderly). The document was updated 13
to include pandemic influenza A(H1N1) virus in 2009 and A(H7N9) in 2013 (3-5). However, it 14
has been over a decade since the original publication of the WHO guideline and a revision was 15
requested by industry, regulators and GISRS laboratories during several WHO informal 16
consultations, such as the WHO Vaccine Composition Meetings (VCM), Global Action Plan 17
for Influenza Vaccines (GAP) meetings and ‘Switch’ meetings (Meetings on Influenza vaccine 18
response at the start of a pandemic), in order to review and reduce testing timelines for CVVs, 19
which have been identified as one of the bottlenecks to rapid vaccine responses (6-10). 20
Moreover, since the initial publication of TRS 941 Annex 5 guidance experience with viruses 21
of pandemic potential and with pandemic viruses has increased globally and is reflected in this 22
update. Further experience gained to date from the development and testing of CVVs derived 23
by reverse genetics (RG) from HPAI viruses has also been incorporated. WHO convened a 24
Working Group meeting during 9-10 May 2017 attended by experts and representatives of 25
WHO Collaborating Centres (CC), Essential Regulatory Laboratories (ERL) and other national 26
regulatory authorities for vaccine and biosafety regulation, manufacturers, and the World 27
Organisation for Animal Health (OIE) to review the up-to-date experience, discuss the revision 28
of TRS 941, Annex 5 and reach consensus on the outline and key issues for the revision (11). 29
This document follows the risk-assessment scheme used in the WHO biosafety guidance for 30
pilot-lot vaccine production, but is extended to include considerations relating to the greater 31
production-scale needed to supply large quantities of vaccines (12, 13). The risks associated 32
with large-scale production are likely to be different from pilot lots, e.g. the “open” aspect of 33
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some production processes and quantity of virus-containing waste. It also takes into account the 1
considerable experience gained from handling HPAI viruses and those classed as low 2
pathogenic in avian species but highly virulent in humans, as well as the hazards associated 3
with such viruses. 4
Furthermore, the range of options for vaccine development is broader than originally 5
considered in the WHO risk assessment for pilot lot production and the present document has 6
been expanded to encompass current vaccine development pathways. 7
2. Scope 8
Efforts have continued into the development and manufacture of H5N1 vaccines and the 9
guidance presented in this document reflects experience gained with this virus and our greater 10
knowledge of H5 subtype viruses in general. Moreover, a great deal has also been learned from 11
our experience with the A/H1N1pdm09 viruses, and from the production of vaccines to this 12
pandemic virus. It is, nevertheless, intended that the guidance will also be applicable to threats 13
from other potential pandemic viruses (e.g., H2, H9, H7 subtype viruses, etc.), as well as low 14
pathogenic avian and mammalian influenza viruses of various subtypes, which are potentially 15
virulent in humans. Manufacturers and laboratories handling HPAI should consult their 16
national regulatory authorities to determine whether additional biosecurity measures are 17
required to handle these viruses. 18
Transmission and pathogenicity of influenza viruses are multi-factorial traits that are currently 19
not completely understood (14). There is therefore a significant range of diversity in the 20
pathogenicity of viruses used to make CVVs which are then used in vaccine production, not 21
only for humans but also for other mammals and avian species. The haemagglutinin (HA) 22
protein has been identified as a major determinant for virulence of avian influenza viruses (15). 23
For example, H5N1 HPAI viruses that can also cause fatal disease in humans have been used to 24
produce reassortant viruses containing an HA that has been genetically modified to generate 25
viruses of low pathogenicity for chickens. In case of viruses that are inherently less pathogenic 26
for humans, wild-type (wt) virus might be used directly for vaccine production (16). Thus, 27
reassortants derived by classical means, RG, or synthetic means, which may or may not be 28
genetically modified, as well as wt viruses are within the scope of these guidelines. 29
Eggs have traditionally been used for the production of influenza vaccines from defined 30
influenza virus reassortants, but cell culture techniques and recombinant protein technologies 31
have also been established and licensed for seasonal influenza vaccine production with 32
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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international expectations for production and quality control specifications defined (17). The 1
principles outlined in this guidance should be applicable to these and other technology 2
platforms for pandemic vaccine development. 3
Most effort to date with candidate pandemic vaccine development has been targeted towards 4
inactivated vaccines; however, in both North America and Europe live attenuated pandemic 5
virus vaccines have been produced and a ‘mock dossier’ approach has been accepted by the 6
European Medicines Agency (EMA). This raises important issues beyond the risks to humans, 7
namely the potential for excreted viruses or their derivatives to infect and replicate in non-8
human species particularly in those raised for commercial purposes, which could have a 9
significant economic impact as well as ramifications for international trade. Developers and 10
regulators will need to assess both the human and the agricultural risk of live pandemic virus 11
vaccines for their ability to shed and to replicate in different hosts. Both inactivated and live 12
vaccines are therefore covered in the scope of these guidelines. 13
Technologies not covered by this guideline, although the general principles could be applied, 14
include new generation technology platforms that do not use live influenza vaccine viruses for 15
production such as expressed recombinant proteins, virus-like particles, DNA- and RNA-based 16
vaccines and vectored vaccines. 17
Furthermore, it is intended that the guidelines on containment measures should apply to all 18
facilities and laboratories that handle live vaccine virus. This includes not only the vaccine 19
manufacturing facility but also applies to the quality control laboratories of the manufacturer as 20
well as National Control Laboratories and specialist laboratories as appropriate. The transport 21
of live virus materials within and between sites should comply with international and national 22
specifications (18). 23
Finally, it should be noted that the risk assessments for vaccine manufacture will vary 24
according by whether production is occurring in an interpandemic period, in a pandemic alert 25
period (as for example early in 2004 when H5N1 was threatening to circulate extensively in 26
South East Asia) or in a pandemic period. These guidelines are intended to describe steps to 27
minimize risks involving vaccine manufacture with emphasis on the interpandemic period, 28
while indicating modifications that may be found appropriate during other periods. 29
3. Terminology 30
The definitions given below apply to the terms used in these guidelines. They may have 31
different meanings in other contexts. 32
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Aerosol: A dispersion of solid or liquid particles of microscopic size in a gaseous medium. 1
Airlock: Areas found at entrances or exits of rooms that prevent air in one space from entering 2
another space. These generally have two doors and a separate exhaust ventilation system. In 3
some cases a multiple-chamber airlock consisting of two or more airlocks joined together is 4
used for additional control. 5
Biosafety Committee: An institutional committee of individuals versed in the subject of 6
containment and handling of infectious materials. 7
Biosafety manual: A comprehensive document describing the physical and operational 8
practices of the laboratory facility with particular reference to infectious materials. 9
Biosafety officer: A staff member of an institution who has expertise in microbiology and 10
infectious materials, and has the responsibility for ensuring the physical and operational 11
practices of various biosafety levels are carried out in accordance with the standard procedures 12
of the institution. 13
Biosafety Level 2, enhanced or BSL3: A specification for the containment of pandemic 14
influenza during vaccine manufacture and quality control testing with specialized air handling 15
systems, waste effluent treatment, immunization of staff, specialized training, and validation 16
and documentation of physical and operational requirements. 17
Biosecurity: Laboratory biosecurity describes the protection, control and accountability for 18
valuable biological materials within laboratories, in order to prevent their unauthorized access, 19
loss, theft, misuse, diversion or intentional release. 20
Decontamination: A process by which an object or material is freed of contaminating agents. 21
EID 50: Egg Infetious Dose. A potency unit for measuring infectious activity of a biologic 22
product or infectious agent equal to a base-10 logarithm of amount of product or agent 23
preparation that causes infection in the 50% of embryos. 24
FFP3: A Mask that protect health professional against airborne particles and microorganisms 25
in pharmaceutical work. 26
Fumigation: The process whereby gaseous chemical is applied to an enclosed space for the 27
purpose of sterilizing the area. 28
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Good manufacturing practice (GMP): That part of quality assurance which ensures that 1
products are consistently produced as controlled to the quality standards appropriate to their 2
intended use and as required by the marketing authorization. 3
HEPA filter: A filter capable of removing at least 99.97% of all airborne particles with a mean 4
aerodynamic diameter of 0.3 micrometres. 5
Highly pathogenic avian influenza (HPAI) virus: Avian influenza viruses of subtypes H5 or 6
H7 containing a cleavage site in HA with multiple inserted amino acids (also referred to in the 7
literature as ‘multibasic’ cleavage site) and causing a systemic infection in poultry associated 8
with mortality of up to 100% (fowl plague). The designation “highly pathogenic” does not refer 9
to the virulence of these viruses in mammalian and human hosts. 10
Inactivation: To render an organism inert by application of heat, chemicals (e.g. formalin, 11
beta-propiolactone), UV irradiation or other means. 12
Low-pathogenic avian influenza (LPAI) virus: Avian influenza viruses containing an HA 13
with a single basic amino acid preceding the site of proteolytic cleavage (also referred to as 14
‘monobasic’ cleavage site) causing localized infections in poultry leading to mild or moderate 15
disease. The designation “low-pathogenic” does not refer to the virulence of these viruses in 16
mammalian and human hosts. 17
N95: A surgical mask, also known as a procedure mask, designed to catch the microorganisms 18
shed in droplets and aerosols from the wearer's mouth and nose with 95% efficiency at 19
removing particles 0.3 micron and larger when correctly fitted to the user. 20
Positive pressure laminar flow hood: An enclosure with unidirectional outflowing air, 21
generally used for product protection. 22
Primary containment: A system of containment, usually a biological safety cabinet or closed 23
container, which prevents the escape of a biological agent into the immediate working 24
environment. 25
Respirator hood: A respiratory protective device with an integral perimeter seal, valves and 26
specialized filtration, used to protect the wearer from toxic fumes or particulates. 27
Risk assessment: A formalized documented process for analysing risks involving a systematic 28
process of evaluating the potential risks that may be involved in a projected activity or 29
undertaking. 30
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TCID 50: median tissue culture infective dose 50%. The quantity of a virus suspension that 1
will infect 50% of tissue culture inoculated with the suspension, expressed as TCID50/ml. 2
Validation: The documented act of proving that any procedure, process, equipment, material, 3
activity, or system actually leads to the expected results. 4
4. Hazard identification 5
Hazards associated with pandemic vaccine manufacturing and laboratory testing are dependent 6
on the type of pandemic vaccine virus (reassortant or wild-type), method of production (egg-7
based or cell-based or other), whether it is an inactivated, live (attenuated) virus or a 8
recombinant virus-vectored vaccine, and whether or not there are any deliberate modifications 9
of the virus for attenuation or for enhanced immunogenicity and/or increased yield. For 10
recombinant virus-vectored vaccines, (e.g. MVA, adenovirus, or VSV) which use replicating 11
recombinant constructs based on viruses other than influenza, virus homogeneity, the nature of 12
the transgene, shedding and the potential for recombination are important factors to evaluate 13
(19, 20). 14
4.1 Candidate vaccine viruses (CVV) 15
CVVs for live and inactivated influenza vaccines are generally produced via reassortment with 16
well-defined backbones from established parent viruses, such as human virus A/Puerto 17
Rico/8/34 (PR8), A/Ann Arbor/6/60, or A/Leningrad/134/17/57 (A/Len/17); wt viruses may 18
also be used for vaccine production. Further, new donor strains for reassortment are being 19
developed and evaluated to enhance vaccine yields. It is likely that a high growth reassortant 20
will provide the basis for pandemic vaccine development, although it is conceivable that a wt 21
virus could be used. 22
4.1.1. Reassortants 23
The genome of influenza A viruses is composed of eight individual single-stranded RNA 24
segments of negative polarity. Segments 4 and 6 encode the two surface glycoproteins, HA and 25
neuraminidase (NA), respectively. The HA is the major surface antigen of the virus and 26
antibodies directed against HA can protect from infection. Antibodies to NA are not 27
neutralizing but can inhibit virus release from infected cells thereby reducing severity and 28
duration of disease and limiting viral shedding. The remaining six RNA segments ("internal 29
genes") encode internal and non-structural viral proteins. The segmented structure of the 30
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genome allows for the exchange of the individual RNA segments between influenza viruses, a 1
process defined as reassortment, upon coinfection of a single cell with two or more viruses. 2
The classical or conventional method for reassortment involves preparing CVVs by co-3
inoculation of a WHO recommended wt virus and a backbone donor virus with a high growth 4
phenotype in embryonated chicken eggs. Concomitant infection allows for reassortment of 5
genetic segments between the two viruses. Antiserum is used for negative selection against the 6
donor virus surface glycoproteins; amplification in eggs results in positive selection for growth. 7
The resulting high growth reassortant CVV must contain the HA and NA genes of the wt target 8
virus. This system takes time and is likely to take several weeks for the production of a high 9
growth reassortant. 10
An alternative approach is through the use of RG methodology to produce a reassortant vaccine 11
virus. (21). This process is based on incorporating the six RNA segments encoding the internal 12
genes of the (high growth) donor virus and the two segments encoding the HA and NA from 13
the circulating wt virus into plasmids. The plasmids are subsequently transfected into cells to 14
rescue the reassortant to be used for vaccine manufacturing. This system allows for the direct 15
genetic manipulation of the influenza gene segments and is generally faster and more accurate 16
than the use of classical reassortment. Moreover, if an HPAI virus is used in the RG process, 17
the HA gene can be easily modified to remove a specific motif of amino acids at the HA 18
cleavage site that is known to convey high pathogenicity in poultry (22). The reassortant can 19
thus be specifically designed to serve as a low pathogenic CVV. The RG system has been 20
reported to be able to produce a CVV within 9 to 12 days (23). 21
The distribution and receipt of the circulating wt virus as a source of RNA for construction of 22
RG HA and NA plasmids adds extra time to the reassortant process and can be bypassed if the 23
reassorting laboratories can produce plasmids by site‐directed mutagenesis of the template 24
DNA of an exisiting related virus, or by the use of synthetic DNA (24, 25). 25
4.1.1.1 Backbone donor viruses 26
PR8 is a common donor virus for generating reassortant vaccine viruses as it reaches a high 27
titre in embryonated chicken eggs. It was originally used in the late 1960s to produce “high 28
growth reassortants” and the use of such reassortants as vaccine viruses increases vaccine yield 29
many-fold. Moreover, PR8 has had over 100 passages in each of mice, ferrets and embryonated 30
chicken eggs. The result of such a passage history is complete attenuation of the virus rendering 31
it incapable of replication in humans (26). 32
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Improved donor viruses are under development in an effort to enhance vaccine yields from 1
CVVs used to manufacture inactivated influenza virus vaccines. Such donor viruses may be 2
new derivations of PR8 but also may have genes from viruses other than PR8, be synthetically 3
generated, and be optimized for specific HA and NA subtypes (23). Demonstration of adequate 4
attenuation for new/improved donor viruses will be needed. 5
Live attenuated seasonal influenza vaccines are licensed in some countries using reassortants 6
with a 6:2 gene constellation based on live attenuated donor viruses such as the A/Ann 7
Arbor/6/60 and A/Len/17 backbones. The attenuated A/Ann Arbor/6/60 virus has been used as 8
a backbone in 6:2 reassortant live attenuated vaccines in clinical studies for over 40 years using 9
approximately 30 different vaccine viruses, and the data demonstrate that the Ann Arbor/6/60 10
based reassortant vaccine viruses are attenuated for humans (27). To date, live attenuated 11
seasonal influenza vaccines derived from the A/Ann Arbor virus have been licensed in North 12
America and Europe while the A/Len/17 backbone has been licenced in Russia, China, 13
Thailand and India (28). These donors may also be used for the development of pandemic 14
influenza vaccine and an adequate level of attenuation has been shown for modified reassortant 15
viruses of various subtypes (29). For each candidate pandemic vaccine virus, this should be 16
verified by testing as described in section 5.1. H5N1-specific LAIV versions (LPAI) made 17
from A/Ann Arbor/6/60 reassortants have been licensed as pandemic preparedness vaccines in 18
several countries. 19
4.1.1.2 Gene segments from wild type viruses 20
Reassortants with a 6:2 gene constellation are considered to be ideal but are not always possible 21
to isolate by traditional co-cultivation methods and for seasonal vaccines some CVVs with 22
different gene constellations, such as 5:3, have been used. As the resultant gene constellation is 23
less predictable using classical methods there is a theoretical possibility of developing 24
reassortants with more than two wt parental genes or even of selection of a mutant (non-25
reassortant) wt virus with improved growth characteristics. The gene constellation of 26
reassortants derived by traditional co-cultivation methods should be determined. 27
4.1.1.3 Virulence factors associated with HA of wild type viruses 28
The CVV gene products derived from the wt virus will be, at a minimum, the HA and NA. For 29
reassortants derived from highly pathogenic H5 and H7 avian viruses by RG, the HA should be 30
modified so that the inserted amino acids at the HA cleavage site are reduced to a single basic 31
amino acid; for some H5 viruses additional nucleotide substitutions can be introduced in the 32
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Page 14
vicinity of the cleavage site in order to increase the genetic stability of the created monobasic 1
motif during virus amplification for large-scale manufacture. Also, the modification of the 2
cleavage site alone is not a guarantee of low pathogenicity as there are LPAI that are virulent in 3
humans due to the presence of other virulence factors (30). It should also be noted that this 4
procedure will increase the safety of the reassortants for avian species as cleavage site 5
modifications have consistently resulted in a reduction of their pathogenicity in avian embryos 6
and in poultry (31). Reassortants for vaccine production are expected to be of low 7
pathogenicity in poultry compared to the highly pathogenic wt parental viruses (32). 8
9
The hazards associated with reassortants depend in part on HA receptor specificity. If a 10
reassortant has a preference for avian cell receptors (i.e. α2,3-linked terminal sialic acid) the 11
hazard is considered to be minimal to humans; however, if a reassortant has a preference for 12
mammalian cell receptors (α2,6-linkages, e.g. human H2N2 pandemic virus from 1957), or 13
possesses both avian and mammalian receptor specificities (e.g. H9N2), there is a greater risk 14
of human infection. For H5 reassortants, the HA retains a preference for α2,3-linked terminal 15
sialic acid residues, so the ability of the H5N1 reassortants to bind to and replicate in human 16
cells should be reduced. It is envisaged that an H5N1 reassortant derived by RG according to 17
WHO guidance (33) would be attenuated for humans compared to the wt H5 virus. 18
Nevertheless, it should also be noted that the human lower respiratory tract contains α2,3-19
linked sialic acid receptors and exposure to high doses of H5N1 viruses represents a risk of 20
infection. Moreover, humans are immunologically naive to H5 and many other avian subtypes, 21
which is also an important risk factor. 22
4.1.1.4 Other factors associated with influenza virus virulence 23
Although it is clear from experience in south-east Asia from 1997 to the present that H5N1 24
influenza viruses that display preference for α2,3-linked sialic acids can still replicate in 25
humans, it must be noted that influenza virus pathogenicity does not depend solely on HA, but 26
is a polygenic trait. The 1997 H5N1 virus had unusual PB2 and NS1 genes that influenced 27
pathogenicity whereas the 2004 H5N1 viruses possessed complex combinations of changes in 28
different gene segments that affected pathogenicity in ferrets (34, 35). In these cases even a 29
virus with a poor affinity for the mammalian receptors was able to replicate in humans 30
(although it was not transmissible). Further, prior to the 2003 outbreak in The Netherlands, 31
only two cases of transmission of H7 viruses from birds to humans were documented (36). Also 32
during the many years of laboratory handling of high-titre avian viruses (of which 33
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Page 15
A/FPV/Dobson/27 is known to contain a gene which adapts it for replication in mammalian 1
cells (37, 38)), there has only been one report in the literature of a laboratory worker being 2
affected by these viruses. This was a laboratory worker in Australia who developed 3
conjunctivitis after accidentally being exposed to an H7N7 virus directly in the eye (39). The 4
PR8/H5N1 6:2 reassortants and the A/Ann Arbor/6/60 live attenuated 6:2 reassortants created 5
by RG for the production of H5N1 vaccine do not contain the gene constellation considered 6
necessary for pathogenicity in chickens, mice and ferrets and in contrast have internal genes 7
that are likely to confer sensitivity to the innate immune response. 8
Compared to HA, the NA protein of influenza viruses has a less prominent record as a 9
virulence factor. It is known that a balance of the counteracting activities of HA (receptor 10
binding) and NA (receptor destruction and virus release) is required for efficient viral 11
replication (40, 41). Further, specific adaptations in NA have been identified upon transmission 12
from wild aquatic birds to poultry. However, specific determinants for the adaptation to and 13
virulence in humans have so far not been detected in the NA protein, although there is some 14
evidence that the NA can mediate HA cleavage in rare H1N1 viruses (42, 43). Of note, 15
resistance to the viral inhibitors oseltamivir and zanamivir is caused by specific mutations in 16
either NA or HA, the latter reducing the affinity of HA to the receptor determinant. 17
4.2. Manufacture 18
Vaccine manufacture requires the establishment of both WHO Good manufacturing practices 19
(GMP) and appropriate biosafety requirements for biological products, as well as related 20
national regulations, technical standards, and guidelines (44, 45). GMP requires the protection 21
of the product from the operator and protection of the operator and the environment from the 22
infectious agent, thus minimizing the risk of any hazards associated with production. It should 23
be noted that reassortants derived from PR8 have been used routinely for the production of 24
inactivated influenza vaccines for the past 40 years. This work involves the production of many 25
thousands of litres of infectious egg allantoic fluids, which creates substantial aerosols of 26
reassortant virus within manufacturing plants. Most of the reassortants were made from wt 27
human influenza viruses. Although staff in the manufacturing facility may have some 28
susceptibility to infection with the wt virus, there have been no anecdotal or documented cases 29
of work-related human illness resulting from occupational exposure to the reassortants. 30
Similarly, reassortants derived from the A/Ann Arbor/6/60 virus have been used for the 31
production of LAIV for many years and no anecdotal or documented cases of work-related 32
human illness have been reported. While no conclusive study yet has been conducted to detect 33
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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silent infections for either the PR8 or live-attenuated viruses, the attenuation status of these 1
CVVs continues to be supported by their excellent safety record to date. 2
Furthermore, for pandemic human CVVs that express avian influenza genes there may be 3
potential consequences for agricultural systems. If influenza A viruses of H5 or H7 subtype or 4
any influenza A virus with an IVPI greater than 1.2 are introduced into poultry (46), the 5
presence of infection would be notifiable to OIE and should lead to implementation of 6
biosecurity measures that aim at preventing the spread of disease (46, 47). Infection with 7
influenza A viruses of high pathogenicity in birds other than poultry, including wild birds, 8
should be reported to OIE; however, a Member Country should not impose bans on the trade in 9
poultry and poultry commodities in response to such a notification, or other information on the 10
presence of any influenza A virus in birds other than poultry, including wild birds (46, 47). 11
4.2.1 Production in eggs 12
Influenza vaccine has been produced in embryonated hens’ eggs since the early 1940s. Much 13
experience has been gained and some facilities are capable of handling large numbers of eggs 14
on a daily basis with the aid of mechanized egg handling, inoculation and harvesting machines. 15
Hazards may occur during the production stages and quality control laboratory activities prior 16
to virus inactivation. During egg inoculation the virus used is dilute and of a relatively small 17
volume. The most hazardous production stage is egg harvesting when the eggs have to be 18
opened to harvest the allantoic fluid, as the open nature of the operations may lead to a greater 19
exposure to aerosols and spills. The allantoic fluid that is harvested from the eggs is invariably 20
manipulated thereafter in closed vessels and hazards arising from live virus during downstream 21
processing and during the virus inactivation process, if used, are therefore less than during virus 22
harvest. Collection and disposal of egg waste is potentially a major environmental hazard. Safe 23
disposal of the waste from egg-grown vaccines, both within the plant and outside, is therefore 24
critical. 25
4.2.2 Production in cell cultures 26
For pandemic influenza vaccines produced on cell cultures, the biosafety risks associated with 27
manufacturing will depend primarily on the nature of the cell culture system employed. Closed 28
systems, such as bioreactors, normally present little to no opportunity for exposure to live virus 29
during normal operation, but additional safety measures must be taken during procedures where 30
samples are introduced into or removed from the bioreactor. Roller bottles and cell culture 31
flasks used for virus production may allow exposure to live virus through aerosols, spills, and 32
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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other operations during virus production and, thereafter, additional risks are associated with the 1
inactivation and disposal of the large quantities of contaminated liquid and solid waste, 2
including cellular debris, generated by this method. 3
The possibility exists that genetic mutations may be selected in pandemic vaccine viruses 4
during passage in mammalian cells that render them more adapted to humans. Sequence 5
analysis of HA may be useful to detect such changes most likely occuring within or close to the 6
receptor-binding domain of the protein; however, it should be noted that little is known about 7
the relation between cell substrate and virus reversion or adaption. Beare et al. (48) tried to de-8
attenuate PR8 by multiple passage in organ cultures of human tissue, but failed, whereas 9
studies with MDCK cells demonstrated that human viruses that retained their α2,6 receptor 10
specificity (human-like) were likely to mutate to an α2,3 specificity (avian-like) as this 11
provided a replicative advantage on MDCK cells, rather than the reverse (49). Overall, hazards 12
arising from the inherent properties of a reassortant or wild type (wt) virus are likely to be far 13
greater than the probability of adaptation of the virus to a more human-like phenotype. 14
4.3 Hazards from the vaccine 15
Inactivated pandemic influenza vaccines present no biosafety risks provided that the results of 16
the inactivation steps show complete virus inactivation, as the viral vaccine is rendered 17
incapable of replication. 18
In an interpandemic or pandemic alert period, pilot-scale live attenuated pandemic influenza 19
vaccines may be developed for clinical evaluation. As there is some uncertainty concerning the 20
biosafety risks associated with shedding or other unintentional release into the environment 21
following vaccination, subjects participating in clinical trials in the interpandemic or pandemic 22
alert phase should be kept under appropriate clinical isolation conditions. If this is not done, 23
indirect hazards for humans could arise. 24
While it is unlikely that a reassortant will be harmful to humans, an indirect hazard may exist 25
through secondary reassortment with a human or animal influenza virus (50); however, recent 26
studies have shown that there is a clear time dependence of coinfections and the generation of 27
reassortants limiting this possibility (51). For secondary reassortants to be generated, several 28
events need to occur: 29
• Infection of production staff (or recipients of live attenuated vaccine in clinical trials) 30
with the reassortant virus 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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• A worker to simultaneously contract infection with a wt influenza virus and the 1
reassortant virus 2
• A reassortment event would have to take place. 3
Evidence to date indicates that the probability of generating secondary reassortants is low. For 4
example, manufacturers have more than 30 years of experience with large-scale production of 5
vaccines based on PR8 reassortants and there have been no reported cases of human illness. 6
Moreover, containment procedures have significantly improved over the last 30 years and 7
production staff can be vaccinated to reduce the chances of an infection with a circulating wt 8
virus thus minimising the risk for secondary reassortment and appropriate personal protective 9
equipment (PPE) can also be provided. 10
5. Safety testing of candidate vaccine viruses (CVVs) 11
CVVs can be developed by either a WHO laboratory, or by a laboratory approved by a national 12
regulatory authority. The following tests and specifications have been developed based on 13
experience gained with the evaluation of CVVs derived from viruses of various subtypes. The 14
required safety testing of different CVVs and proposed containment levels are summarised in 15
Table 1. The information summarised in this table should be considered as guidance and 16
changes to these requirements may be determined on a case-by-case basis by WHO and/or 17
national authorities. It is recommended that for CVVs developed from newly emerging viruses 18
of pandemic potential, an appropriate WHO expert group review the associated data from 19
safety testing and advise WHO; WHO will then provide further guidance as to the appropriate 20
biocontainment requirements through its expert networks such as GISRS. 21
The requirement to conduct or complete some or all of these tests prior to the distribution of a 22
CVV may be relaxed based on additional risk assessments; these assessments should take into 23
account WHO pandemic phase, evolving virological, epidemiological and clinical data as well 24
as national regulatory requirements applying to shipment and receipt of infectious substances. 25
26
5.1 Tests to evaluate pathogenicity of CVVs 27
The recommended tests for CVVs are dependent on the parental viruses from which they are 28
derived (Table 1). The parental viruses determine the appropriate biocontaiment level for 29
conducting tests. The tests are described in the following sections. 30
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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CVVs that are genetically similar (i.e. have HA and NA derived from the same or a genetically 1
very closely related wt virus and on the nominally same backbone) to a CVV that has already 2
undergone complete safety testing may not require full testing; it may be sufficient to confirm 3
sequence and genetic stability in this case. 4
5.1.1 Attenuation in ferrets 5
Ferrets were chosen because they have been used extensively as a good indicator of influenza 6
virus virulence for humans (52). Typically, seasonal influenza viruses cause mild to no clinical 7
signs in ferrets and virus replication is usually limited to the respiratory tract. PR8 virus has 8
been assessed in ferrets and found to cause few or no clinical signs, and virus replication is 9
limited to the upper respiratory tract. However, some wt HPAI viruses can cause severe and 10
sometimes lethal infections (53, 54). Thus, in the absence of human data, the ferret is the best 11
model to predict whether a virus will be pathogenic or attenuated in humans. 12
CVVs should be shown to be attenuated in ferrets in accordance with Table 1, except when 13
virus-specific risk assessments suggest a different approach (ie, performance of ferret test 14
where Table 1 does not require it or no ferret test where Table 1 requires it). These tests should 15
be conducted in well-characterized ferret models standardised by the use of common reference 16
viruses available from WHO CCs/ERLs for influenza. Detailed test procedures are described in 17
Appendix 1. Attenuated viruses would be those that meet the attenuation criteria as defined in 18
Appendix 1, with reference to the reference/standard viruses1. One or more laboratories may 19
have ferret pathogenicity data on parental wt viruses that could be used by all testing 20
laboratories as a further benchmark for comparison. Assessing the transmissibility of CVVs 21
between ferrets is not required. 22
5.1.2 Pathogenicity in chickens 23
For CVVs derived from HPAI H5 or H7 parental viruses determining the chicken intravenous 24
pathogenicity index (IVPI) is recommended and may be required by national authorities. The 25
procedure should follow that described in the OIE guidance. Any virus with an index greater 26
than 1.2 is considered an HPAI (55). 27
5.1.3 The ability to plaque in the presence or absence of added trypsin 28
1 Until the establishment of reference viruses for pathogenicity testing, CVVs are expected to be
compared to their respective wt parent virus; attenuation is then defined as relative to the pathogenicity of the wt virus.
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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HPAI viruses can replicate in mammalian cell culture in the absence of added trypsin, whereas 1
LPAI viruses generally do not. This test is recommended for all CVVs derived from HPAI H5 2
or H7 parental viruses. It is recommended that these tests be established and characterized by 3
use of known positive and negative control viruses. 4
5.1.4 The ability to cause chicken embryo death 5
HPAI viruses cause rapid chicken embryo death upon inoculation into eggs and this test has 6
been used to complement in vivo IVPI assays (55). This test is recommended for all CVVs 7
derived from H5 or H7 parental viruses. 8
5.1.5 Genetic sequence analysis and stability 9
Genetic sequencing is important to confirm identity and/or to verify the presence of attenuating 10
and other phenotypic markers, (e.g., cold adaptation and temperature sensitivity in the case of 11
LAIV CVVs). Genetic sequencing is recommended to verify retention (stability) of the markers 12
of relevant phenotypic traits, where such markers are known, after 10 passages beyond 13
production level in relevant substrates i.e., embryonated chickens’ eggs or cultured cells. These 14
tests should be conducted on all CVVs, including wt CVVs. 15
16
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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Table 1 1
Required safety testing of different CVVs and proposed containment levels for vaccine 2
production 3
4
aTest performed by WHO reference laboratory. 5
bWith deletion of additional residues at HA connecting peptide 6
c Highly pathogenic avian influenza virus 7
Candidate vaccine
virus
Tests needed on CVVsa Proposed containment for
vaccine production
Reassortantsb and
modified viruses derived
from H5 and H7 HPAIc
virusesd
Ferret, chickene, sequence,
plaquing, egg embryo,
genetic stability
BSL2 Enhanced (pandemic
influenza vaccine)
Reassortantsb and
modified viruses derived
from synthetic DNA
representing H5 and H7
HPAIc virusesf
Ferret, sequence, plaquing,
egg embryo, genetic
stability
BSL2 Enhanced (pandemic
influenza vaccine)
Reassortants and
modified viruses derived
from H5 and H7 LPAIg
viruses
Ferret, sequence, egg
embryo, genetic stability
BSL2 Enhanced (pandemic
influenza vaccine)
Reassortants and
modified viruses derived
from non-H5, H7 viruses
Ferret, sequence, genetic
stability
BSL2 Enhanced (pandemic
influenza vaccine)
Wild type H5, H7 HPAI
viruses
Sequence, genetic stabilityh BSL3 Enhanced (pandemic
influenza vaccine)
Wild type H5, H7 LPAI
viruses
Ferret, sequence, genetic
stabilitye, i
BSL2 Enhanced i (pandemic
influenza vaccine)
Wild type non-H5, H7
viruses
Ferret. sequence, genetic
stabilitye
BSL2 Enhanced (pandemic
influenza vaccine)
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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dThis category refers to viruses derived by reverse genetics technology using as starting material wt 1
HPAI virus (or RNA extracted from wt HPAI virus) 2
ethe requirement for performance of the chicken pathogenicity test (IVPI) is dependent on national 3
regulatory requirements which are currently under review in some countries and may change 4
fthis category refers to viruses derived by reverse genetics technology using as starting material 5
synthetic DNA; the nucleotide sequence of the synthetic DNA is equivalent or similar to wt HA and NA 6
genes from HPAI viruses, with the exception that additional residues at the at HA connecting peptide 7
have been removed 8
gLow pathogenic avian influenza viruses. 9
hif changes are identified pathogenicity tests may be required 10
iif a virus specific hazard assessment identifies that additional control measures are appropriate, 11
containment level may be increased, for example Asian-lineage H7N9, and additional pathogenicity 12
testing required 13
14
6. Risk assessment and management 15
6.1 Nature of the work 16
Production of influenza vaccines in embryonated chicken eggs or cell culture require 17
propagation of live virus. Modifications of some CVVs will result in viruses that are expected 18
to be attenuated in humans (12, 21). Several production steps have the potential to generate 19
aerosols containing live virus. The virus concentration in aerosols will depend on the 20
production step and is highest during the harvest of infected allantoic fluid. Small amounts of 21
virus containing liquid or very dilute virus suspensions are used during seed virus preparation 22
and egg inoculation. Appropriate biosecurity and biosafety measures, such as use of laminar air 23
flows, positive pressure laminar flow hoods, cleaning and decontamination of equipment, waste 24
management and spill kits, will be in place to prevent accidental exposure in the work 25
environment and the release of virus into the environment. 26
6.2 Health protection 27
6.2.1 Likelihood of harm to human health 28
Wild-type influenza viruses are able to infect humans and cause serious illness. Certain 29
phenotypic features that are associated with virulence can be modified and the resulting CVV 30
will have lower probability of causing harm to human health. 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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6.2.1.1 Reassortant CVVs 1
Reassortant CVVs containing a backbone of genes except segments 4 and 6 (HA and NA) from 2
PR8, A/Ann Arbor/6/60 or А/Len/17 have been widely used for production of seasonal 3
influenza vaccines and vaccines against the pandemic H1N1pdm09 influenza viruses. A 4
significant body of data is available, suggesting that a reassortant virus composed of RNA 5
segments coding for HA and NA derived from a pandemic virus and the genes coding for the 6
internal and non-structural proteins ("internal genes") from PR8, A/Ann Arbor/6/60 or 7
A/Len/17 will have only a low probability of causing harm to human health (12, 21). 8
Viruses that were rescued from plasmids will have the desired 6 "internal" genes of the helper 9
virus, however this is not always the case for reassortants prepared by mixed infection and 10
antibody selection ("classical reassortment"). Risks associated with RNA segments, other than 11
segments 4 and 6, that were derived from the pandemic virus must be carefully evaluated. 12
Efforts have been made to modify backbones that were derived from PR8 or other viruses to 13
achieve attenuation or to increase the yield during vaccine manufacturing. Until more field 14
experience has become available, the potential of vaccine viruses that contain modified 15
backbones to cause harm to human health needs careful assessment. 16
HA cleavage site: Most HPAI viruses contain a sequence of basic amino acids at the cleavage 17
site separating the HA1 and HA2 subunit. HPAI viruses in which the basic amino acids have 18
been removed from the HA by genetic engineering are likely to be attenuated. Modification of 19
the HA cleavage site is associated with reduced virulence in animal models and is expected to 20
result in attenuation in humans. Modification of the HA cleavage site only applies to HPAI H7 21
and H5 viruses. It should be noted that LPAI, e.g. H7N9 wt viruses also have caused serious 22
human illness (56). 23
Receptor specificity: Preferential binding of the HA to α2,6 receptors is associated with 24
transmissibility of pandemic influenza and circulating human viruses in humans (57,58). 25
However, it must be noted that viruses with a preference to bind to α2,3 receptors, such as 26
H7N9 viruses, have also been causing serious human illness (59). While receptor specificity 27
should be considered during risk assessment as a factor in reducing the risk for a virus to cause 28
harm to human health, it is not in itself sufficient for virus attenuation. 29
Secondary reassortment: It is conceivable that reassortment between a CVV, containing HA 30
and NA from a pre-/pandemic virus, and a seasonal human wt influenza virus could occur 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 24
during simultaneous infection of humans with both viruses. Such a reassortant may be 1
replication-competent in humans, while having pre-/pandemic surface proteins. The likelihood 2
that these events occur and lead to secondary reassortment is considered to be low. Laboratory 3
and production facilities have biosafety control measures in place to prevent exposure of staff 4
to live virus. In case of accidental exposure, it is is unlikely that a CVV would replicate 5
efficiently or transmit to human contacts. Virus shedding would be expected to be well below 6
the titres considered to be needed for human infection; however, although there is no known 7
precedent of secondary reassortment of using PR8 reassortants for vaccine manufacturing in 8
more than 40 years, the public health consequences of such an event could be serious. 9
6.2.1.2 Wild-type CVVs of low pathogenicity (LP) 10
Pathogenicity of wt viruses with low in vivo pathogenicity, without multiple basic amino acids 11
at the HA cleavage site is likely to be low in humans (60). However, it should be noted that 12
some H7N9 viruses that are LP in poultry have caused severe illness in humans. 13
Transmissibility of wt viruses with avian receptor specifity is likely to be low in humans, 14
whereas transmissibility of wt viruses with mammalian receptor specificity (e.g. human H2N2 15
and H9N2) is unknown. 16
In case of accidental exposure there is a risk of secondary reassortment with human viruses and 17
the resulting reassortant viruses may be able to replicate in humans. Appropriate biosafety 18
control measures will be in place during manipulation of these viruses to prevent exposure of 19
staff. 20
6.2.1.3 Wild-type CVVs of high pathogenicity (HP) 21
The use of highly pathogenic wt CVVs is restricted to cell-culture-based production, which 22
allows the use of closed systems. For diagnostic tests and vaccines for terrestrial animals, HPAI 23
viruses should not be used. Instead, CVVs produced by reverse genetics and containing the 24
haemagglutinin gene of an HPAI virus that had the cleavage site sequence altered to that of a 25
LPAI H5/H7 virus are preferred (47). Appropriate biosecurity and biosafety measures during 26
production, analytical testing and waste disposal are required to protect staff and prevent 27
release of infectious virus into the environment. 28
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6.2.1.4 Susceptibility of CVVs to neuraminidase inhibitors 1
Influenza viruses/CVVs that are sensitive to NA inhibitors (or other licensed drugs, once 2
available) should be used for production if at all possible. Sequence verification may be enough 3
to confirm drug susceptibility. 4
6.3 Environmental protection 5
6.3.1 Environmental considerations 6
Influenza A viruses are endemic throughout the world in some farm animals (pigs and horses) 7
and some populations of wild birds, specifically birds of the families Anseriformes (ducks, 8
geese and swans) and Charadriiformes (shorebirds) (61). 9
A number of influenza A viruses, such as H5, H7 and H9 can cause disease in domestic 10
poultry. H5 and H7 can be highly pathogenic in poultry, whereas H9 is less so. In addition, 11
sporadic infections by influenza A viruses have been reported in farmed mink, wild whales and 12
seals, dogs and captive populations of big cats (tigers and leopards) (62). In big cats, the 13
infections followed consumption of dead chickens infected with H5N1 viruses. Influenza A 14
virus infection in dogs were caused by H3N8 or H3N2 viruses closely related to endemic 15
equine and avian viruses, respectively. The H3N8 and H3N2 viruses continue to circulate in 16
dogs in North America (63). Recently, LPAI avian A(H7N2) virus was identified as the cause 17
of an outbreak of respiratory disease in domestic shelter cats in northeastern United States, 18
indicating yet another mammalian host for avian influenza viruses (64) 19
It is expected that many prepandemic viruses will have avian receptor specificity and thus birds 20
would theoretically be the species most susceptible. Several studies indicate that viruses with 21
PR8 backbones are attenuated in chickens (65). A reassortant containing HA (with a single 22
basic amino acid at the cleavage site) and NA from the 1997 Hong Kong H5N1 virus and the 23
genes coding for the internal and non-structural proteins of PR8 was barely able to replicate in 24
chickens and was not lethal (65). Similar studies have been performed with the 2003 25
Hong Kong H5N1 virus at the WHO Collaborating Centre, Memphis, USA (66), where the 6:2 26
PR8 reassortant did not replicate or cause signs of disease in chickens. The removal of the 27
multiple basic amino acids from the H5 x PR8 reassortants in both studies probably played a 28
major role in reducing the risk for chickens. 29
Hatta et al. (34) have demonstrated that acquisition of only one PR8 gene by an avian influenza 30
virus can abolish virus replication in ducks. 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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It is likely that the temperature sensitive phenotype of the cold-adapted vaccine virus would 1
also be attenuated in avian species due to the elevated body temperature of birds. 2
Pigs have both α2,3 and α-2,6 –linked sialic acid receptors in abundance (67) and must be 3
considered susceptible to most influenza viruses, including LAIV and CVVs with a backbone 4
of PR8 genes. 5
6.4 Assignment of containment level 6
The production of influenza vaccine reassortant CVVs from highly pathogenic wt viruses 7
should take place in BSL3 enhanced or BSL4 facilities, as advised by WHO (68) or national 8
authorities. WHO Collaborating Centres, other specialized laboratories and some vaccine 9
manufacturers provide characterized reassortant CVVs to all interested vaccine manufacturers 10
who may develop vaccine seeds and vaccines from these materials. 11
In consideration of the hazards associated with egg and cell culture vaccine production and 12
quality control with "classical" or "RG" derived reassortant or wt viruses of pandemic potential 13
which have been demonstrated to be attenuated in ferrets and/or have demonstrated low 14
pathogenicity in chickens where applicable, as specified in section 5.1, the assigned 15
containment level is BSL2 enhanced (pandemic influenza vaccine), as defined below. This 16
applies to both pilot-scale and large-scale production during the interpandemic phase and 17
pandemic alert period (68). Any subsequent relaxation of the levels of containment to the 18
standard used for seasonal production during the developing pandemic, should be authorized on 19
a case-by-case basis by the national competent authorities after careful evaluation of the risks. 20
Special consideration should be given to hazards associated with cell culture vaccine 21
production and quality control of HPAI or "classical" or "RG" reassortant pandemic viruses of 22
unknown pathogenicity. The assigned containment level is BSL3 Large Scale manufacturing as 23
defined below. This applies to both pilot-scale and large-scale production during the 24
interpandemic phase and pandemic alert period (68). The parts of the facility where such work 25
is done (both production and quality control) should additionally meet the OIE requirements for 26
containment, which include not only biosafety and biosecurity but also requirements to limit 27
the introduction and spread within animal populations (55). Any subsequent relaxation of the 28
levels of containment during the developing pandemic, should be authorized on a case-by-case 29
basis by the national competent authorities after careful evaluation of the risks. 30
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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In view of the open nature of large-scale egg-based vaccine production, it is not feasible to 1
operate at BSL3 enhanced (pandemic influenza vaccine). Therefore egg-based vaccine 2
production from HPAI H5 or H7 wt viruses is not recommended. 3
Containment conditions must be defined, based on an activity based risk assessment, taking 4
into account the scale of manipulations, the titres of live virus, and whether an activity involves 5
virus amplification. Biosafety control measures must be reconciled with rules and regulations 6
governing the manufacture and testing of medicinal products known as good manufacturing 7
paractices (GMP) (44). It should be noted that biosafety control measures apply to 8
manipulations involving live virus; they no longer apply once virus has been inactivated by a 9
validated process. 10
6.5 Environmental control measures 11
Appropriate containment measures to prevent release of live virus into the environment must be 12
in place. 13
Local biosafety/biosecurity regulations provide guidance on the disposal of potentially 14
infectious waste. Notably, contaminated waste from current production facilities may reach 15
high virus titres. All decontamination methods should be validated. If possible, 16
decontamination of waste should take place on site. Where this is not possible, it is the 17
responsibility of each manufacturer to ensure that procedures are in place to safely contain 18
material during transport prior to decontamination off site. Guidance on regulations for the 19
transport of infectious substances is available from WHO (18) and national competent 20
authorities. In all cases the procedures must be validated to ensure that they function at the 21
scale of manufacturing. 22
Medical surveillance program of staff should be established prior to vaccination. 23
Procedures should be in place to provide antiviral medicines, in case the situation warrants it, 24
e.g. in case of accidental exposure. Where antiviral medicines are only available as prescription 25
medicines, care should be taken to have stocks available in pharmacies. 26
Suitable PPE must be available to prevent exposure of staff to live virus. In order to further 27
reduce the risk of secondary reassortment in case of accidental exposure, the use of seasonal 28
influenza vaccines should be recommended to staff. For the same reason, the use of a pre-29
/pandemic vaccine can be considered if available and if marketing authorization has been 30
received. 31
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Page 28
Each manufacturer should also assess the risk of contamination of birds, horses, pigs or other 1
susceptible animals based on the likelihood of their being in the vicinity of the manufacturing 2
plant. Following occupational exposure, staff or other personnel entering the area potentially 3
exposed to live virus should avoid visiting pig, horse or bird facilities (e.g. farms, equestrian 4
events, bird sanctuaries) for at least 14 days following occupational exposure. If conjunctivitis 5
or respiratory signs indicating the potential development of influenza infection or disease 6
develop during this 14 day period this period, quarantine time should be extended to 14 days 7
after the symptoms have resolved. 8
Stringent measures to control rodents, other mammals and birds should be in place. 9
6.5.1 Specifications for "BSL2 enhanced (pandemic influenza vaccine) facilities" 10
Specifications for BSL2 enhanced (pandemic influenza vaccine) facilities include the following 11
in addition to the principles for BLS2 facilities as specified in the WHO Laboratory biosafety 12
manual (1). 13
6.5.1.1 Facility 14
The facility should be designed and operated according to the stage of the manufacturing 15
process to meet the demands of protection of the recipient of the vaccine, the staff producing 16
and testing the vaccine, the environment and the population at large. It is noted that different 17
solutions may be needed depending on the risks inherent in the operation(s) conducted in an 18
area. Specialized engineering solutions will be required that may include: 19
— use of relative negative pressure biosafety cabinets 20
— use of high-efficiency particulate air (HEPA) filtration of air prior to exhaust into public 21
areas or the environment 22
— use of positive pressure barrier and/or negative pressure in-line sinks prior to exhausting 23
to areas where no live virus is handled 24
In addition, the following decontamination procedures should take place: 25
— decontamination of all waste from BSL2 enhanced (pandemic influenza vaccine) areas 26
— decontamination of all potentially contaminated areas at the end of a production 27
campaign through cleaning and validated decontamination, for example gaseous 28
fumigation. 29
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 29
6.5.1.2 Personal protection 1
• Full-body protective laboratory clothing (for example, Tyvek® disposable overalls) 2
• If activities cannot be contained by primary containment and open activities are being 3
conducted, the use of respiratory protective equipment, such as N95, FFP3 (69) or 4
equivalent respirators is strongly recommended. Minimal specifications for the 5
filtering/absorbing capacity of such equipment should be met, and masks, if used, must be 6
fitted properly and the correctness of fit tested. 7
• All personnel, including support staff and others who may enter the production or QC areas 8
where pandemic viruses may be handled, should be instructed, in a written document to 9
which they sign their agreement, not to have any contact with susceptible animals such as 10
ferrets or farm animals, in particular birds, horses or pigs, for 14 days after departure from 11
the facility where vaccine has been produced. This period should be extended to 14 days 12
after the symptoms resolve if conjunctivitis or respiratory signs indicating the potential 13
development of influenza infection or disease develop during this 14 day period. Currently 14
the risks involved in contact with household dogs and cats are not considered to be 15
significant, but the available scientific evidence is sparse. 16
• Wherever possible, it is strongly recommended that staff should be prophylactically 17
vaccinated with seasonal inactivated influenza vaccines. 18
• It is anticipated that pilot lots of pandemic virus vaccine will have already been produced 19
before large scale vaccine production is attempted. Experimental vaccines inducing 20
protective antibody levels are recommended for use by staff before large scale vaccine 21
production commences, provided they are available and marketing authorization has been 22
received. 23
• Procedures should be in place to provide antiviral treatment in case the situation warrants it 24
(e.g. accidental exposure). 25
6.5.1.3 Monitoring of decontamination 26
Cleaning and decontamination methods need to be validated and reviewed periodically as part 27
of a master validation plan to demonstrate that the protocols, reagents and equipment used are 28
effective in the inactivation of pandemic influenza virus on facility and equipment surfaces, 29
garments of personnel and waste materials, and within cell growth and storage containers. Once 30
decontamination procedures for influenza virus have been fully described and validated, there 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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is no need to repeat them for each new virus. Validation studies using influenza viruses may be 1
supplemented by studies with biological (for example bacterial) markers selected to be more 2
difficult to inactivate than influenza. 3
6.5.2 Specifications for "BSL3 Large Scale Manufacturing" 4
It is assumed that ferret pathogenicity testing will be conducted on all strains of unknown 5
pathogenicity even given the assumptions above (Section 5.1) related to the low probability of 6
a PR8 reassorted virus being pathogenic in humans. This assumption is based on the experience 7
currently held relating to reassortants of HA subtypes other than H1 & H3; i.e., H5, H7, and 8
H9. 9
Under these circumstances, a facility will need to meet the requirements for protection of 10
personnel handling potentially dangerous micro-organisms found in the WHO Laboratory 11
Biosafety Manual, Third edition (1), which lays out a Risk Survey to be undertaken prior to 12
rating a laboratory space as either BSL- 1, 2 or 3. Similar requirements are found in the 13
European Directive 2000/54/EC (2007) (70) on the protection of workers from risks related to 14
exposure to biological agents at work and the US CDC’s Biosafety in Microbiological and 15
Biomedical Laboratories Guide (Edition 5) (71). 16
Following review of the requirements for Biosafety at Level BSL3 from the sources noted 17
above, it is proposed that a facility meeting the criteria detailed below, and with the noted 18
operator protection in place, would be suitable to manufacture vaccine, at large scale using a 19
virus seed prepared by RG or classical reassortant methodology, whilst pathogenicity remains 20
unknown. 21
6.5.2.1 Facility 22
The facility should be designed and operated to meet the demands of protection of the recipient 23
of the vaccine, the staff producing and testing the vaccine and of the environment. This will 24
require specialized engineering solutions that may include: 25
• Appropriate signage and labeling related to the activities being undertaken when a virus of 26
unknown pathogenicity is in use. 27
• The facility must be designed and constructed as a contained GMP space. The surfaces and 28
finishes must comply with GMP requirements which will ensure they are appropriately 29
sealed and easily cleaned and decontaminated. 30
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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• The air cascades within the facility should be such that any live virus is contained within 1
the work zones in which it is used. All work with infectious virus must be conducted within 2
these contained zones. 3
• Access to the contained areas must be gained via double door entry airlocks. The airlocks 4
should operate at a pressure either lower or higher than that on either side. In this way the 5
airlocks become either a sink or a pressure barrier, maintaining an airflow containment of 6
the facility. Note that in the case where the airlocks provide a low pressure ‘sink’ it will be 7
required that the entry and exit doors be interlocked or alarmed with a suitable delay or 8
alarm system to prevent both being opened at the same time. The air pressure cascade 9
within the negatively pressure ‘contained’ zone should allow for compliance with GMP (i.e. 10
higher pressures in cleanest zones) requirements for clean rooms. 11
• All supply and exhaust air must be passed through HEPA filtration, again maintaining the 12
required containment and GMP conditions. The air conditioning systems within the facility 13
must be the subject of rigorous risk assessment related to potential failure modes and ‘fail 14
safe’ systems implemented where deemed necessary. The facility should be under constant 15
monitoring related to environmental factors including the maintenance of appropriate room 16
pressure differentials. Should there be any undesirable fluctuation the situation should be 17
alarmed and appropriate action taken. 18
• All equipment to be reused is either cleaned in place, decontaminated by means of 19
autoclaving or otherwise cleaned and decontaminated by validated, dedicated systems prior 20
to washing for reuse. 21
• Areas of potential liquid spill should be assessed and bunded (dammed) to ensure 22
containment of any spill. This should include waste treatment plants and processes. 23
Procedures must be in place such that spills are contained, cleaned and the contaminated 24
materials properly disposed so as not to compromise the integrity of the facility. 25
• Materials entry to the ‘contained’ zone should be via separately HEPA filtered, interlocked, 26
double ended Pass Through Cabinets (PTC) or double-ended autoclaves. 27
• All facility waste, including egg waste, should be discarded via validated on-site waste 28
effluent systems or by autoclaving. The validation of waste systems should specifically 29
include viral inactivation testing. Any items which follow the process from the external 30
environment, through the manufacturing process and are returned to the external 31
environment must be the subject of particular attention (e.g. the plastic egg maché). 32
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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Dedicated washing and decontamination of equipment and/or procedures must be provided 1
which, again, must be fully validated for viral inactivation. 2
6.5.2.2 Personal protection 3
• All clothing worn outside the facility should be replaced by manufacturing facility garments 4
upon entry into the facility. 5
• It should be noted that gowning requirements in viral zones should always include full suit, 6
overshoes, eye protection and double gloves. 7
• The provision of full hood powered air-purifying respirators (PAPR) for all personnel 8
working within the containment areas of the manufacturing facility. These powered hoods 9
are to be worn at all times when the facility is in operation under these enhanced biosafety 10
requirements. Figure 1 shows the type of operator protection to be provided. 11
12
Figure 1: Operator Protection 13
• All facility clothing is to be removed on exit, with soiled clothing transferred out of the 14
facility via a decontamination autoclave or similar method. The respirator hoods may be 15
decontaminated with 70% alcohol or similar and stored in the ‘grey’ zone of the entry/exit 16
airlock. 17
• Specific operational procedures should be developed and implemented for operation under 18
enhanced biosecurity conditions. 19
• Wherever possible it is strongly recommended that staff should be prophylactically 20
vaccinated with a seasonal influenza vaccine. In the case of pandemic strains, it is 21
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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anticipated that before large scale vaccine production is attempted, pilot lots of vaccine will 1
have already been produced. If available and if marketing authorization has been received, 2
experimental vaccines inducing protective antibody levels are recommended for use by staff 3
before large scale vaccine production commences 4
• Procedures should be in place to provide antiviral treatment in case the situation warrants it. 5
(e.g. accidental exposure). 6
• Daily temperature monitoring before commencing work (with an exclusion policy for ill 7
workers) where this is possible 8
• On-site Occupational Health and Safety and medical support should be maximized with 9
such additional measures as: 10
o Medical consultation / training in recognition of ‘flu-like’ symptoms 11
o Out of hours referral plan to medical facilities with quarantine facilities 12
• Taking a full body shower upon exit from the BSL3 Large Scale Manufacturing 13
containment facility is recommended. It is mandatory following situations when staff may 14
have been exposed to vaccine virus. 15
• All personnel, including support staff and others who may enter the production or QC areas 16
where pandemic viruses may be handled, should be instructed, in a written document to 17
which they sign their agreement, not to have contact with animals, in particular farm 18
animals 14 days following departure from the facility where vaccine has been produced. 19
This period should be extended to 14 days after the symptoms resolve if conjunctivitis or 20
respiratory signs indicating the potential development of influenza infection or disease 21
develop during this 14 day period. Currently the risks involved in contact with household 22
dogs and cats are not considered to be significant, but the available scientific evidence is 23
sparse. 24
6.6 Biosafety management and implementation within a vaccine production facility 25
6.6.1 Management structure 26
The implementation of the biosafety levels described in these guidelines requires that the 27
institution employ a biosafety officer who is knowledgeable in large-scale virus production and 28
containment, but is independent of production in his or her reporting structure. The biosafety 29
officer is responsible for the independent oversight of the implementation of the biosafety 30
practices, policies and emergency procedures in place within the company or organization and 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 34
should report directly to the highest management levels within the company. A biosafety 1
officer is needed in addition to a qualified person who, in some countries, has overall 2
responsibility for a medicinal product. 3
There should also be a Biosafety Committee comprising representatives of virus production and 4
quality control that is responsible for reviewing the biosafety status within the company and for 5
coordinating preventive and corrective measures. The institutional biosafety officer must be a 6
member of the Committee. The chairperson should be independent of both the production and 7
quality control functions. The Committee should report to the executive management of the 8
manufacturing company to ensure that adequate priority and resources are made available to 9
the Committee to implement the required measures. 10
6.6.2 Medical surveillance 11
Occupational health departments at vaccine manufacturers of pandemic virus influenza 12
vaccines should provide training in recognizing the clinical signs of influenza infection to 13
company physicians, nurses and vaccine manufacturing staff including supervisors, who must 14
make decisions on the health of personnel associated with the manufacture and testing of 15
pandemic virus influenza vaccine. Local medical practitioners caring for personnel from the 16
manufacturing site should receive special training in the diagnosis and management of 17
pandemic influenza infection. Any manufacturer embarking on large-scale production should 18
have documented procedures for dealing with influenza-like illness in the staff involved, or 19
their family members, including diagnostic procedures and prescribed treatment protocols. 20
Manufacturers should ensure staff understand that they have an obligation to seek medical 21
attention and to report any influenza-like illness to the occupational health department or 22
equivalent. Manufacturers should ensure that procedures are in place to provide antiviral 23
treatment if the situation warrants it (e.g. accidental exposure) and have defined means or 24
arrangements of advising staff with any influenza–like illness as necessary. 25
6.6.3 Implementation 26
A detailed and comprehensive risk analysis should be conducted to define possible sources of 27
contamination of personnel or the environment that may arise from the production or testing of 28
live influenza virus within the establishment. For each procedure or system, this analysis 29
should take into account the concentration, volume and stability of the virus at the site, the 30
potential for inhalation or injection that could result from accidents, and the potential 31
consequences of a major or minor system failure. The procedural and technical measures to be 32
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 35
taken to reduce the risk to workers and the environment should be considered as part of this 1
analysis. The results of this risk analysis should be documented. 2
A comprehensive Biosafety Manual, or equivalent must be created and implemented to fully 3
describe the biosafety aspects of the production process and of the quality control activities. It 4
should define such items as emergency procedures, waste disposal, and the requirements for 5
safety practices and procedures as identified in the risk analysis. The manual should be made 6
available to all staff of the production and quality control units, with at least one copy present 7
in the containment area(s). The manual should be reviewed and updated when changes occur 8
and at least biannually. 9
Comprehensive guidelines outlining the response to biosafety emergencies, spills and accidents 10
should be prepared and made available to key personnel for information and for coordination 11
with emergency response units. Rehearsals of emergency response procedures are helpful. 12
These guidelines should be reviewed and updated annually. 13
The implementation of the appropriate biosafety level status in the production and testing 14
facilities should be verified through an independent assessment. National requirements 15
concerning verification mechanisms should be in place and complied with. 16
17
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
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Authors and acknowledgements 1
The first draft was prepared by Gary Grohmann (consultant) and TieQun Zhou (WHO), based 2
on input from the participants of the Working Group Meeting on Revision of WHO TRS 941, 3
Annex 5: WHO Biosafety Risk Assessment and Guidelines for the Production and Quality 4
Control of Human Influenza Pandemic Vaccines, held on 9-10 May 2017: 5
Othmar G Engelhardt, National Institute for Biological Standards and Control, Medicines and 6
Healthcare products Regulatory Agency, Potters Bar, United Kingdom; Glen Gifford; World 7
Organisation for Animal Health, Paris, France; Shigeyuki Itamura, Centre for Influenza Virus 8
Research, National Institute of Infectious Diseases, Tokyo, Japan; Jacqueline M. Katz, WHO 9
Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza, Centers for 10
Disease Control and Prevention, Atlanta, United States of America; Changgui Li, National 11
Institutes for Food and Drug Control, Beijing, People's Republic of China; John McCauley, 12
WHO Collaborating Centre for Reference and Research on Influenza, Crick Worldwide 13
Influenza Centre, The Francis Crick Institute, London, United Kingdom; Jennifer Mihowich, 14
Centre for Biosecurity, Health Security Infrastructure Branch, Public Health Agency of 15
Canada, Ottawa, Canada; Ralf Wagner, Paul-Ehrlich-Institut, Langen, Germany; Richard 16
Webby, WHO Collaborating Centre for Studies on the Ecology of Influenza in Animals, St. 17
Jude Children's Research Hospital, University of Tennessee, Memphis, United States of 18
America; Jerry Weir, Centre for Biologics Evaluation and Research, Food and Drug 19
Administration, Maryland, United States of America; Gert Zimmer, Institute of Virology and 20
Immunology, Mittelhäusern, Switzerland. Representatives of international federation of 21
pharmaceutical manufacturers & associations (IFPMA): Matthew Downham, 22
AstraZeneca/Medimmune; Lionel Gerentes, Sanofi Pasteur, France; Elisabeth Neumeier, GSK 23
Vaccines, Germany; Beverly Taylor, Seqirus, UK. Representatives of developing countries 24
vaccine manufacturers network (DCVMN): Pradip Patel, Cadila Healthcare Limited, 25
Ahmedabad, India; Kittisak Poopipatpol and Ponthip Wirachwong, Government 26
Pharmaceutical Organization, Bangkok, Thailand. WHO headquarters: Mustapha Chafai, 27
Prequalification Team, Essential Medicines and Health Products (EMP) Department, Health 28
Systems and Innovation (HIS) Cluster, World Health Organization (WHO), Geneva, 29
Switzerland; Gary Grohmann, HIS/WHO, Switzerland; Ivana Knezevic, Technologies 30
Standards and Norms (TSN) Team, EMP/HIS/WHO, Geneva, Switzerland; Wenqing Zhang, 31
High Threat Pathogens (PAT), Infectious Hazard Management (IHM), Health Emergencies 32
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 37
Programme (WHE), WHO, Geneva, Switzerland; TieQun Zhou, TSN/EMP/HIS/WHO, 1
Geneva, Switzerland. 2
3
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61. Swayne DE, Halvorson, DA. Influenza. In: Saif YM et al. eds. Diseases of Poultry. 11th 35
ed. Ames, Iowa, Iowa State University Press, 2003:135–160. 36
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62. Keawcharoen J et al. Avian influenza H5N1 in tigers and leopards. Emerging Infectious 1
Diseases, 2004, 10:2189–2191. 2
63. Pecoraro HL et al., Epidemiology and eclogy of H3N8 canine influenza viurses in US 3
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to-human transmission-New York City, 2016. Clin Infect Dis 2017;65:1927-29. 7
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virus vaccine candidate generated by plasmid-based reverse genetics. Virology (2003) 305: 9
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66. Webby RJ, Perez DR, Coleman JS, Guan Y, Knight JH, Govorkova EA, McClain-Moss 11
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Paulson JC, Webster RG, et al. Molecular basis for the generation in pigs of influenza A 16
viruses with pandemic potential. J Virol. 1998 Sep;72(9):7367–7373. 17
68. WHO global influenza preparedness plan. The role of WHO and recommendations for 18
national measures before and during a pandemic. WHO/CDS/CSR/GIP/2005.5. World Health 19
Organization, 2005. 20
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accessed 22 Nov 2017. 22
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to protect against particles — Requirements, testing, marketing. 24
70. European Directive 2000/54/EC (2007). 25
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https://www.cdc.gov/biosafety/, accessed 22 Nov 2017. 29
30
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Appendix 1 1
Testing for attenuation of influenza vaccine viruses in ferrets 2
3
Laboratories conducting testing for attenuation of influenza viruses in ferrets should make use 4
of a panel of standard/or reference viruses (‘pathogenicity standards’ in the following section) 5
and defined experimental outcomes for pathogenicity testing. The pathogenicity standards (to 6
be established by WHO laboratories) serve as benchmarks for the pathogenicity test in ferrets 7
and delineate the expected outcomes. The use of these standards will ensure that attenuation of 8
CVVs is being measured against common parameters independently of subtype. The CVV to 9
be tested must show parameters of pathogenicity below the predefined values of a high 10
pathogenicity standard and in line with those of an attenuation standard in order to be 11
designated as attenuated. Comparative attenuation in comparison with the parental wild-type 12
virus is not necessary in this case. However, laboratories that have the capacity to evaluate 13
attenuation of a CVV compared with the parental wild-type virus can continue to do so. To 14
account for the expected experimental variability of results across different laboratories, the 15
pathogenicity standards should be tested in ferrets at each testing laboratory according to the 16
experimental protocol shown below when establishing the ferret model for pathogenicity 17
testing and at regular intervals thereafter. The outcomes of these tests should fall within the 18
limits described for the pathogenicity standards. In cases of discrepancy, a review of the ferret 19
model should be conducted and advice should be sought from experienced WHO laboratories. 20
Test virus 21
The 50% egg or tissue culture infectious dose (e.g. EID50, TCID50) or plaque-forming units 22
(PFU) of the reassortant CVV or pathogencitiy standard will be determined. The infectivity 23
titres of viruses should be sufficiently high to allow infection with 107 to 106 EID50, TCID50 or 24
PFU of virus and diluted not less than 1:10. Where possible, the pathogenic properties of the 25
donor PR8 should be characterized thoroughly in each laboratory. 26
Laboratory facility 27
Animal studies with the CVV and the pathogenicity standards should be conducted in animal 28
containment facilities in accordance with the proposed containment levels shown in Table 1. 29
For untested CVVs, the biocontainment level to be used for the ferret safety test is the one 30
shown for the respective wild-type virus, except for ‘Reassortants and modified viruses derived 31
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 44
from synthetic DNA representing H5 and H7 HPAI viruses’, for which the containment level 1
proposed in Table 1 (BSL2 enhanced) can be used (1). An appropriate occupational health 2
policy should be in place (2). 3
Experimental procedure 4
Outbred ferrets 4-12 months of age that are serologically negative for currently circulating 5
influenza A and B viruses and the test virus strain are anesthetized by either intramuscular 6
administration of a mixture of sedatives (e.g. ketamine (25 mg/kg) and xylazine (2 mg/kg) and 7
atropine (0.05 mg/kg)) or by suitable inhalant anesthetics. A standard virus dose of 107 to106 8
EID50 (or TCID50 or PFU) in 0.5 to 1 ml of phosphate-buffered saline is used to inoculate 9
animals. The dose should be the same as that used for pathogenesis studies with the wild-type 10
parental virus, if used, or the pathogenicity standards previously characterised and regularly 11
assessed in the laboratory. The virus is slowly administered into the nares of the sedated 12
animals, reducing the risk of virus being swallowed or expelled. A group of four to six ferrets 13
should be inoculated. One group of two to three animals should be euthanized on day 3 or 4 14
after inoculation and samples should be collected for estimation of virus replication from the 15
following organs: spleen, intestine, lungs (samples from each lobe and pooled), brain (anterior 16
and posterior sections sampled and pooled), olfactory bulb of the brain, and nasal turbinates. If 17
gross pathology demonstrates lung lesions similar to those observed in wild-type viruses, 18
additional lung samples may be collected and processed with haematoxylin and eosin (H&E) 19
staining for histopathological evaluation. The remaining animals are observed for clinical signs, 20
which may include weight loss, lethargy [based on a previously published index (3)], 21
respiratory and neurologic signs, and increased body temperature. Collection of nasal washes 22
from animals anesthetized as indicated above should be performed to determine the level of 23
virus replication in the upper airways on alternate days after inoculation for up to seven days. 24
At the termination of the experiment on day 14 after inoculation, a necropsy should be 25
performed on at least two animals and organs collected. If signs of substantial gross pathology 26
are observed (e.g. lung lesions), the organ samples should be processed as described above for 27
histopathology. 28
Expected outcome 29
Clinical signs of disease such as lethargy and/or weight loss should be within the predefined 30
ranges of acceptable pathogenicity defined by the pathogenicity standards. Viral titres of the 31
vaccine strain in respiratory samples should be within the ranges of acceptable virus replication 32
WHO/INFLUENZA/DRAFT/NOVEMBER 2017
Page 45
defined by the pathogenicity standards. Replication of the CVV should be restricted to the 1
respiratory tract. Virus isolation from the brainis not expected. However, detection of virus in 2
the brain has been reported for seasonal H3N2 viruses (4); this is due to detection of virus in 3
the olfactory bulb. Therefore, should virus be detected in the anterior or posterior regions of 4
the brain (excluding the olfactory bulb) the significance of such finding may be confirmed by 5
performing immunohistochemistry to detect viral antigen and/or histopathological analysis of 6
brain tissue collected on day 14 after inoculation. Detection of viral antigen and/or neurological 7
lesions in brain tissue would confirm virus replication in the brain. Presence of neurological 8
signs and confirmatory viral antigen and/or histopathology in brain tissue would indicate a lack 9
of suitable attenuation of the CVV. 10
11
References for Appendix 1 12
13
1. World Organization for Anima Health (OIE). CHAPTER 2.3.4. AVIAN INFLUENZA, 14
Appendix 2.3.4.1- “Biosafety guidelines for handling highly pathogenic avian influenza viruses 15
in veterinary diagnostic laboratories” (adopted in May 2015), in Manual of Diagnostic Tests 16
and Vaccines for Terrestrial Animals 2017. http://www.oie.int/en/international-standard-17
setting/terrestrial-manual/access-online/, accessed 22 November 2017. 18
2. WHO laboratory biosafety guidelines for handling specimens suspected of containing avian 19
influenza A virus. 12 January 2005. 20
http://www.who.int/influenza/resources/documents/guidelines_handling_specimens/en/, 21
accessed 22 November 2017. 22
3. Reuman PD, Keely S, Schiff GM. Assessment of signs of influenza illness in the ferret 23
model. Laboratory Animal Science, 1989, 42:222–232. 24
4. Zitzow LA et al. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. Journal of 25
Virology, 2002, 76:4420–4429. 26
27
28