who/bs/11.2159 english only expert committee on …€¦ · 9 candidate tetravalent dengue virus...

1 WHO/BS/11.2159 2 ENGLISH ONLY 3

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EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION 7 Geneva, 17 to 21 October 2011 8

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Guidelines on the Quality, Safety and Efficacy of Dengue Tetravalent 11 Vaccines (Live, Attenuated) 12

13 Proposed replacement of TRS No. 932, Annex 1 14

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This document has been prepared for the purpose of inviting comments and suggestions on 17 the proposals contained therein, which will then be considered by the Expert Committee 18 on Biological Standardization (ECBS). The text in its present form does not necessarily 19 represent an agreed formulation of the Expert Committee. Comments proposing 20 modifications to this text MUST be received by 23 September 2011 and should be 21 addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: 22 Quality Safety and Standards (QSS). Comments may also be submitted electronically to 23 the Responsible Officer: Dr Jinho Shin at email: [email protected]. 24 25 The outcome of the deliberations of the Expert Committee will be published in the WHO 26 Technical Report Series. The final agreed formulation of the document will be edited to 27 be in conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). 28

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© World Health Organization 2011 30 All rights reserved. Publications of the World Health Organization are available on the WHO web site 31

(www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 32 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). 33 Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial 34 distribution – should be addressed to WHO Press through the WHO web site: 35 (http://www.who.int/about/licensing/copyright_form/en/index.html). 36

The designations employed and the presentation of the material in this publication do not imply the 37 expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status 38 of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or 39 boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full 40 agreement. 41

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The mention of specific companies or of certain manufacturers’ products does not imply that they are 1 endorsed or recommended by the World Health Organization in preference to others of a similar nature that are 2 not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial 3 capital letters. 4

All reasonable precautions have been taken by the World Health Organization to verify the information 5 contained in this publication. However, the published material is being distributed without warranty of any 6 kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the 7 reader. In no event shall the World Health Organization be liable for damages arising from its use. The named 8 authors alone are responsible for the views expressed in this publication 9 10 11 12 13

Recommendations and guidelines published by WHO are intended to be scientific and advisory in nature. It is recommended that modifications be made only on condition that the modifications ensure that the vaccine is at least as safe and efficacious as that prepared in accordance with the recommendations set out below. To facilitate the international distribution of vaccine made in accordance with these Guidelines, a summary protocol for the recording of results of the tests is given in Appendix 1.

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Table of contents 1

Introduction ··························································································································································5 2

General considerations ········································································································································5 3

Part A. Guidelines on manufacturing and control of dengue tetravalent vaccines (live, 4 attenuated)···························································································································································11 5

A.1 Definitions ··································································································································11 6

A.2 General manufacturing requirements·························································································14 7

A.3 Control of source materials········································································································15 8

A.4 Control of vaccine production····································································································21 9

A.5 Filling and containers ················································································································26 10

A.6 Control tests on final lot ·············································································································26 11

A.7 Records·······································································································································28 12

A.8 Samples·······································································································································28 13

A.9 Labelling·····································································································································28 14

A.10 Distribution and shipping···········································································································29 15

A.11 Stability, storage and expiry date ·······························································································29 16

Part B. Nonclinical evaluation of dengue tetravalent vaccines (live, attenuated)····································31 17

B.1 General remarks·························································································································31 18

B.2 Product development and characterization················································································33 19

B.3 Nonclinical immunogenicity and protective activity ··································································33 20

B.4 Nonclinical toxicity and safety ···································································································34 21

B.5 Environmental risk ·····················································································································37 22

Part C. Clinical evaluation of dengue tetravalent vaccines (live, attenuated)··········································38 23

C.1 General considerations for clinical studies················································································38 24

C.2 Immunogenicity ··························································································································39 25

C.3 Clinical studies ···························································································································42 26

C.4 Post-licensure investigations······································································································52 27

Part D. Environmental risk assessment of dengue tetravalent vaccines (live, attenuated) derived 28 by recombinant DNA technology·····················································································································54 29

D.1 Introduction································································································································54 30

D.2 Procedure for environmental risk assessment ············································································55 31

D.3 Special considerations for live recombinant dengue vaccines ················································56 32

Part E. Guidelines for national regulatory authorities ················································································60 33

E.1 General·············································································································································60 34

E.2 Release and certification ············································································································60 35

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Authors ································································································································································61 1

Acknowledgements·············································································································································63 2

Appendix 1: Model summary protocol for manufacturing and control of dengue tetravalent 3 vaccines (live, attenuated) ·································································································································64 4

1. Summary information on finished product (final vaccine lot) ······························································64 5

2. Summary Information on manufacture ·································································································65 6

3. Control of source materials ··················································································································65 7

4. Control of vaccine production ·············································································································71 8

5. Filling and containers···························································································································76 9

6. Control tests on final vaccine lot ··········································································································77 10

Appendix 2: Model certificate for the release of dengue vaccine (live, tetravalent) by national 11 regulatory authorities ········································································································································80 12

Appendix 3: Procedure for environmental risk assessment········································································81 13

Abbreviations······················································································································································84 14

References····························································································································································86 15

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Introduction 1

2 These Guidelines are intended to provide national regulatory authorities and vaccine 3 manufacturers with guidance on the quality, safety and efficacy of live-tetravalent dengue 4 vaccines currently under clinical development to facilitate their international licensure 5 and use. 6

7 These Guidelines update the Guidelines for the production and quality control of 8 candidate tetravalent dengue virus vaccines (live) [66]. They should be read in 9 conjunction with other WHO guidelines that are referred in each part. 10 11 These Guidelines cover dengue tetravalent vaccines (live, attenuated). Other types of 12 dengue virus vaccines under development, e.g. subunit or inactivated vaccines, are 13 outside the scope of these Guidelines. However, some guiding principles provided in 14 these Guidelines, e.g. Part C clinical evaluation, may be useful for the evaluation of other 15 types of dengue vaccines. For quality control aspects, guiding principles applicable to 16 other types of vaccines such as inactivated or subunit vaccines are available elsewhere if 17 the product in development shares similar manufacturing process. For example, 18 guidelines for human papillomavirus and hepatitis B vaccines may be useful for subunit 19 vaccines for dengue as well. 20 21 These Guidelines are based on experience gained from candidate live-attenuated dengue 22 tetravalent vaccines that have been developed, as described below, and will need to be 23 updated as new data becomes available from additional studies. 24

25 Part A sets out guidelines for manufacture and quality control. Guidelines specific to the 26 nonclinical, clinical evaluation and environmental risk assessment are provided in Parts B, 27 C, and D, respectively. Part E provides guidelines for national regulatory authorities. In 28 the following section, brief overviews of dengue disease and dengue vaccine 29 development at the time of preparing this document are provided as scientific basis for 30 each part. 31

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General considerations 33

Dengue viruses 34

Dengue is a mosquito-borne disease and represents a major public health problem 35 throughout the tropical world. The causative dengue viruses (DENVs) are members of 36 the genus Flavivirus, within the family Flaviviridae. There are four serotypes (termed 37 DENV-1 to -4) and at least 3 genetic groups (genotypes) within each serotype. 38 39 All flaviviruses are lipid-enveloped, positive-sense, single-stranded RNA viruses, 40 approximately 55 nm in diameter. The genome is capped at the 5’ terminus but does not 41 have a poly A tract at the 3’ terminus, and is approximately 11,000 nucleotides in length. 42 The virion RNA encodes a single open reading frame (ORF) that is flanked by a 5’ 43

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untranslated region (UTR) and 3’UTR. The ORF is translated into a polyprotein that is 1 co- and post-translationally cleaved to yield at least 10 proteins. Three structural proteins 2 are derived by cleavages of the amino-terminal one-third of the polyprotein: the capsid or 3 core protein forms a “nucleocapsid” complex with virion RNA that lies within the lipid 4 envelope. The premembrane (prM) and envelope (E) proteins are embedded in the lipid 5 envelope via carboxy-terminal transmembrane domains and are displayed on the surface 6 of virions. Cleavage of the carboxy-terminal two-thirds of the polyprotein yields seven 7 non-structural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS3 8 encodes a serine protease in the N-terminal 180 amino acids and helicase, nucleotide 9 triphosphatase, RNA 5'-triphosphatase activities in the C-terminal region while NS5 10 encodes two functions; the first one-third encodes a methyltransferase that sequentially 11 methylates the N7 and 2'-O positions of the viral RNA cap using S-adenosyl-l-methionine 12 as a methyl donor, and the remainder a RNA-dependent RNA polymerase. NS1 plays 13 various roles in the virus replication cycle while NS2A, NS2B, NS4A and NS4B are all 14 small hydrophobic proteins with the central region of NS2B required for the functioning 15 of the NS3 protease. 16

Host range and transmission 17

DENVs are most commonly transmitted to humans by the bite of infected Aedes (Ae.) 18 aegypti mosquitoes, which are highly domesticated and the primary mosquito vector; 19 however, Ae. albopictus can also sustain human-to-human transmission. The drastic 20 increase in the incidence of DENV infection in the Americas during the past 30 years is 21 primarily due to the geographical spread of Ae. aegypti following decline in vector-22 control efforts. The DENV that infects and causes disease in humans is maintained in a 23 human-to-mosquito-to-human cycle and does not require a sylvatic cycle in non-human 24 primates. Certain strains of DENV are known to be transmitted to non-human primates in 25 western Africa and Malaysia; however, transmission to humans via mosquitoes from non-26 human primates is believed to be very limited. 27

Clinical and pathologic manifestation in humans 28

Following infection by the bite of an infected mosquito, the virus is thought to replicate 29 in local dendritic cells. Subsequent infection of macrophages and lymphocytes is 30 followed by entry into the blood stream. Haematogenous spread is the likely mechanism 31 for seeding of peripheral organs and the occasional reports of central nervous system 32 (CNS) infections, which can lead to symptomatic illness. 33 34 Most DENV infections are either asymptomatic or only mildly symptomatic. The 35 incubation period of dengue can range from 3 to 14 days, but is generally 4 to 7 days. 36 Most symptomatic DENV infections present with a sudden onset of fever accompanied 37 by headache, pain behind the eyes, generalized myalgia and arthralgia, flushing of the 38 face, anorexia, abdominal pain and nausea. Rash is common in dengue and can be 39 macular, maculopapular, morbilliform, scarlatiniform or petechial in character. Rash is 40 most often seen on the trunk, insides of the arms and thighs, and plantar and palmar 41 surfaces. Laboratory abnormalities that can be observed in dengue infection include 42 leukopenia and thrombocytopenia. 43

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1 Dengue illness is classified as (i) dengue with or without warning signs and (ii) severe 2 dengue. A presumptive diagnosis of dengue can be made in a patient living in or traveling 3 from a dengue-endemic area who has fever and at least two of the following clinical signs 4 or symptoms: anorexia and nausea, rash, body aches and pains, warning signs, leukopenia, 5 and a positive tourniquet test. "Warning signs" include abdominal pain or tenderness, 6 persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy or 7 restlessness, liver enlargement of > 2 cm, or an increase in haematocrit concurrent with a 8 rapid decrease in platelet count. Dengue illness should be classified as severe in a patient 9 with presumptive dengue and any of the following: severe plasma leakage leading to 10 shock or respiratory compromise, clinically significant bleeding, or evidence of severe 11 organ involvement. Detailed case classification of dengue is provided in a separate 12 document (WHO/HTM/NTD/DEN/2009.1 or its update) [70]. 13

Non-human primate dengue virus infection 14

Natural hosts for DENV infection are humans and mosquitoes. Serological evidence 15 from nonhuman primate studies indicates a sylvatic DENV cycle involving several 16 species of mosquitoes and several monkey species exists. Although monkeys develop a 17 viremia and a neutralizing antibody response to DENV infection, they do not develop the 18 hematologic abnormalities seen in humans. However, in non-human primate (NHP) (and 19 AG129 mouse, see paragraph below) primary DENV infections cause a leukopenia. 20 21 Thrombocytopenia has been observed after a secondary infection [5, 8, 16, 19]. In the 22 rhesus macaque, viremia typically begins 2-6 days after infection and lasts for 3-6 days [5, 23 7, 16]. Virus spreads to regional lymph nodes and can be isolated from the skin, distant 24 lymph nodes, and rarely from spleen, thymus and other body organs. The NHP model for 25 DENV is useful to measure protection from viremia conferred by vaccination or 26 passively acquired antibody. Disadvantages of the NHP model include the lack of overt 27 clinical signs of disease [8, 16, 19, 51]. 28

Mouse dengue virus infection 29

Clinical isolates of DENV do not replicate well in genetically normal mice. However, 30 mouse-brain-adapted DENVs can induce fatal encephalitis after intracranial inoculation 31 of suckling mice. It has been demonstrated that adaptation of a DENV2 isolate to 32 neurovirulence in suckling mice correlated positively with attenuation of virulence in 33 humans [43]. Because of this ambiguity, the suckling mouse/encephalitis model is 34 probably not useful for studying candidate DEN vaccine safety or efficacy. Nevertheless, 35 it could be used to assess lot consistency (see A.3.2.5.5.2 and A.4.2.4.7). In recent years, 36 both chimeric mice that are transplanted with human cells and severely 37 immunocompromised strains of mice have been used to elucidate the immune response to 38 DEN infection and to study pathogenesis [8, 55, 76]. Interferon receptor -deficient 39 AG129 mice support replication of selected DENV strains which infect relevant cell and 40 tissue types comparable to human infection [47, 55]. AG129 mice have been used to 41 investigate antibody-mediated protection. A strain of DENV2 that has been adapted to 42

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AG129 mice by serial passage between mice and mosquito cells has a viscerotropic 1 phenotype, causing thrombocytopenia and vascular leakage in the infected animals. The 2 phenomenon of antibody-dependent enhancement (ADE) of virus infection was observed 3 in AG 129 mice following passive transfer of anti-DENV1 antibodies and challenge with 4 the adapted strain of DENV2 [2, 47, 55]. The relevance of such immunocompromised 5 mouse model may be however limited with regard to vaccine evaluation (see B.4.3). 6

Mosquito dengue virus infection 7

Vector competence refers to the efficiency with which the vector transfers infection 8 between hosts. Typically, this is a product of vector susceptibility to infection, replication 9 efficiency of the pathogen in the vector, and the sensitivity of the host to infection 10 transmitted by vector contact. Aedes aegypti mosquitoes exhibit global variation in 11 vector competence for flaviviruses. For example, in sub-Saharan Africa, a black “sylvan” 12 subspecies, Ae. formosus predominates. This mosquito has a low vector competence for 13 flaviviruses due primarily to a midgut infection barrier [3]. Once ingested in an infectious 14 blood meal, DENVs should replicate in the midgut and disseminate to the salivary glands 15 to facilitate transmission to a new host during feeding. In this process, the virus should 16 overcome any midgut barrier that would limit replication and prevent spread of the 17 virus to other tissues in the mosquito [19, 44, 45]. 18 19 None of the live DEN vaccine preparations currently in the clinical trial phase of 20 development is effectively transmitted by mosquito vectors [29, 45],because vaccine 21 viruses replicate poorly in mosquito midgut epithelium, and/or do not disseminate 22 efficiently to the salivary glands, thereby interdicting transmission to humans [19, 22, 50]. 23 In addition, the low peak titre and very short duration of viremia induced by these 24 candidates in humans has been shown to render vaccinees relatively non-infectious for 25 feeding mosquitoes. The net effect of these two phenomena is a drastic reduction in 26 vector competence. 27

At-risk populations and global health importance 28

Dengue is the most rapidly spreading mosquito-borne viral disease in the world. Since 29 1955, the incidence of dengue and severe dengue reported to WHO has increased 30 approximately 30-fold with increasing geographic expansion to new countries and from 31 urban to rural settings. Approximately 3.5 billion people live in dengue endemic 32 countries which are located in the tropical and subtropical regions of the world. An 33 estimated 50 million dengue infections occur annually and the number of cases reported 34 annually to WHO ranged from 0.4 to 1.3 million in the decade 1996 - 2005 [70]. 35

Justification for vaccine development 36

Prevention of dengue by vector control has proven to be very difficult and costly. While 37 vector control efforts should be sustained, vaccination holds substantive potential in the 38 control of the disease. Hence, there is an urgent need to develop dengue vaccines, 39 especially to protect people from disease in endemic countries. 40

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Development of candidate dengue vaccines 1

Efforts to develop a vaccine against dengue have primarily focused on live attenuated 2 viruses, derived by serial passage of virulent viruses in tissue culture or via recombinant 3 DNA technology that permits site-directed mutagenesis of the genome of a virulent 4 parent strain. Success in the development, licensure, and clinical use of live attenuated 5 flavivirus vaccines, such as yellow fever 17D vaccines and Japanese encephalitis SA14-6 14-2, suggests that a suitably attenuated live dengue vaccine could be highly efficacious. 7 Other vaccine candidates based on inactivated whole virus, subunits that include E 8 protein, virus-like particles comprised of prM and E proteins, and DNA vaccines that 9 induce expression of DENV prM/E proteins and are in preclinical or early clinical stages 10 of development. 11

12 There are no animal models that completely mimic the protean manifestations of dengue. 13 The lack of a suitable animal model makes it difficult to assess efficacy of vaccine 14 candidates and to identify/establish possible correlates of protection in vivo. Therefore, 15 the protective capacity of any vaccine candidate will be finally defined by its ability to 16 protect humans from dengue febrile illness (DFI). Results of non-clinical studies using 17 monkeys and susceptible mouse strains suggest, however, that protection from dengue is 18 best correlated with the presence of virus-neutralizing antibodies [1, 5, 17, 26, 27, 30, 42, 19 54]. Studies in which vaccinated volunteers were challenged with dengue viruses have 20 been conducted in the past but are not a required part of currently recommended clinical 21 development programs. 22 23 There is general agreement that DENV vaccines should ideally induce protective 24 neutralizing antibodies to each of the four serotypes simultaneously. In theory, a 25 tetravalent immune response would protect against all DFI and would also reduce or 26 eliminate the risk of a phenomenon termed antibody-dependent enhancement of disease, 27 which is thought to be one of the mechanisms that predispose to severe forms of dengue. 28 29 Several strategies have been employed to derive candidate live attenuated vaccines. There 30 are four candidates that are in clinical development at the present time. 31 32 The Walter Reed Army Institute of Research (WRAIR) developed attenuated DENV 33 strains by empirical serial passage in primary dog kidney (PDK) cells and produced 34 vaccine candidates in fetal rhesus lung (FRhL) cells. Tetravalent formulations of these 35 attenuated vaccine candidates have been evaluated in Phase I and Phase II clinical trials 36 conducted by WRAIR and GlaxoSmithKline [21, 23, 51, 52]. 37 38 The other three candidates were developed using recombinant DNA technology which 39 involves first the generation of a full-length DNA copy of the DENV genome. Site-40 specific mutations expected to affect virulence are then introduced into the DNA, and 41 mutant full-length DNAs can then be copied in vitro to produce infectious RNA 42 transcripts that can be used to generate mutant DENVs in tissue culture. The Laboratory 43 of Infectious Diseases of the U.S. National Institute of Allergy and Infectious Diseases 44 (NIAID) has thus derived a total of five candidate DEN vaccine viruses that have been 45

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tested in clinical trials. Two were generated by introduction of a 30-nucleotide deletion 1 (termed ∆30) into the 3’- UTR of the DENV-4 and DENV-1 genomes [21, 23, 51, 52]. 2 These DENV-1 and DENV-4 vaccine candidates were shown to be attenuated and 3 immunogenic in nonhuman primates. A DENV-2 candidate vaccine was developed by 4 replacing the gene segments encoding the prM and E proteins of the DEN4∆30 candidate 5 vaccine with those of DENV-2. An additional DENV-3 candidate vaccine was developed 6 by replacing the 3´ UTR of a DENV-3 wild-type virus with that of the DEN4∆30 UTR. A 7 third DENV-3 candidate vaccine was developed by introducing a 30 nucleotide deletion 8 into the 3´ UTR homologous to that of the DEN4∆30 vaccine virus and a second non-9 contiguous 31 nucleotide deletion, also in the 3´ UTR [6]. Phase I trials have been 10 conducted with “∆30” monovalent vaccines [7, 21, 51], and Phase I trials with the 11 tetravalent formulation were initiated in 2010. 12 13 Thai scientists at Mahidol University developed a candidate DENV-2 vaccine empirically 14 by 53 serial passages of the virus in PDK cells, designated DENV-2 strain PDK53, which 15 was found to be highly attenuated and immunogenic in Phase I and Phase II clinical trials. 16 The U.S. Centers for Disease Control and Prevention (CDC) determined that the 17 attenuation mutations of DENV-2 PDK53 virus were not located in the prM or E proteins, 18 and in collaboration with InViragen, Inc. used this genetic background to derive chimeric 19 DENV-1, -3 and -4 vaccines expressing the respective prM/E genes in the context of the 20 DENV-2 PDK53 genome “backbone” [21, 51]. A tetravalent formulation is in Phase I 21 clinical trials. 22 23 Finally, a candidate live vaccine was developed by Acambis/Sanofi Pasteur using the live 24 attenuated yellow fever virus (YFV) vaccine, 17D, as the backbone for chimeric DENV 25 vaccine candidate. In these viral genomes, the prM and E genes from each of the four 26 DENV serotypes, respectively, are substituted for those of YFV in the context of the 27 genetic background of the 17D vaccine. A tetravalent chimeric YFV-DENV vaccine has 28 been evaluated in Phase I and II clinical trials for safety and immunogenicity. Phase IIb 29 trials to investigate protective efficacy in children began late in 2009 [21, 51] and Phase 30 III trials have been in progress since late 2010. 31

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Part A. Guidelines on manufacturing and control of dengue 1

tetravalent vaccines (live, attenuated) 2

A.1 Definitions 3

A.1.1 International name and proper name 4

Although there is no licensed dengue vaccine, the provision of a suggested international 5 name at this early stage of development will aid harmonization of nomenclature if 6 licensure is obtained. The international name should be “dengue tetravalent vaccine, live 7 attenuated”. The proper name should be the equivalent to the international name in the 8 language of the country of origin. The use of the international name should be limited to 9 vaccines that satisfy the specifications formulated below. 10

A.1.2 Descriptive definition 11

A live attenuated tetravalent dengue virus vaccine defined in <A.1.1> should contain live 12 attenuated dengue viruses representing each of the four serotypes, or replication-13 competent viral vectors that express the major structural antigen genes of each of the four 14 dengue serotypes, that have been separately prepared in cell culture. It may be presented 15 as a sterile, aqueous suspension or as freeze-dried material. The preparation should 16 satisfy all of the specifications given below. 17

A.1.3 International reference materials 18

As the prospective vaccines are very different in type, no international reference material 19 for a candidate live dengue vaccine is available. However, an international reference 20 panel of human antisera against all four dengue serotypes is available from the National 21 Institute of Biological Standards and Control (NIBSC). The panel is intended to help 22 calibrate the response to vaccines. 23

A.1.4 Expression of dose related to vaccine potency 24

Potency of a live vaccine most often is expressed in terms of the number of infectious units 25 of virus contained in a human dose, using a specified tissue culture substrate and based on 26 results of phase I and II clinical trials. In the case of a tetravalent dengue vaccine, potency 27 will have to be assessed in terms of the individual titers of each of the four serotypes of 28 vaccine virus contained in a human dose. When international reference standards for the 29 vaccine type under production become available, the dose related to vaccine potency 30 should be calculated against the international standard and expressed in international unit 31 (IU) to reduce variation between laboratories. Until then, the use of PFU, IFU or CCID50 32 to express the potency and doses of vaccine can be an alternative. The dose should also 33 serve as a basis for establishing parameters for stability and for the expiry date. 34

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A.1.5 Terminology 1

The definitions given below apply to the terms as used in these Guidelines. They may 2 have different meanings in other contexts. 3

Adventitious agents 4

Contaminating microorganisms of the cell culture or source materials including bacteria, 5 fungi, mycoplasmas/spiroplasmas, mycobacteria, rickettsia, protozoa, parasites, 6 transmissible spongiform encephalopathies (TSE) agents, and viruses that have been 7 unintentionally introduced into the manufacturing process of a biological product. 8

Cell bank 9

A cell bank is a collection of appropriate containers whose contents are of uniform 10 composition stored under defined conditions. Each container represents an aliquot of a 11 single pool of cells. 12

Cell culture infectious dose 50% (CCID50) 13

A measure of the number of infectious viruses per unit volume of virus suspension as 14 determined in an end-point dilution assay in monolayer cell cultures. One CCID50 / mL 15 corresponds to the quantity of viruses capable of producing a cytopathic effect in 50% of 16 replicate cell cultures per mililitre of virus suspension. 17

Cell seed 18

A quantity of vials containing well-characterized cells derived from a single tissue or cell 19 of human or animal origin stored frozen in liquid nitrogen in aliquots of uniform 20 composition, one or more of which would be used for the production of a master cell 21 bank. 22

Cell substrates 23

Cells used for the production of a vaccine. Cells are derived from a master cell bank or a 24 working cell bank. 25

Dengue febrile illness (DFI) 26

DFI is defined by the detection of dengue virus in patients with two days of fever 27 irrespective of severity of illness. 28

Final lot 29

A collection of sealed final containers of finished vaccine that is homogeneous with 30

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respect to the risk of contamination during filling and freeze-drying. All the final 1 containers should, therefore, have been filled from one vessel of final tetravalent bulk and 2 freeze-dried under standardized conditions in a common chamber in one working session. 3

Final tetravalent bulk 4

The homogeneous finished tetravalent vaccine prepared from one or more virus harvest 5 pools in the vessel from which the final containers are filled. 6

Genetically modified organism (GMO) 7

An organism in which the genetic material has been altered in a way that does not occur 8 naturally by mating and /or natural recombination. 9

Immunofocus-forming unit (IFU) 10

A measure of the number of infectious viruses capable of forming plaques that can be 11 detected using dengue-specific antisera and a counter-stain in monolayer cell cultures per 12 unit volume of virus suspension. 13

Master cell bank (MCB) 14

A quantity of well-characterized cells of animal or other origin, derived from a cell seed 15 at a specific population doubling level (PDL) or passage level, dispensed into multiple 16 containers, cryopreserved, and stored frozen under defined conditions, such as the vapour 17 or liquid phase of liquid nitrogen in aliquots of uniform composition. The master cell 18 bank is prepared from a single homogeneously mixed pool of cells. It is considered best 19 practice that the master cell bank is used to derive working cell banks. 20

Virus master seed (VMS) 21

A suspension of vaccine virus that has been aliquoted into identical vials and stored at a 22 temperature and under conditions deemed to stabilize the virus in each container. The 23 VMS is used as a source of infectious virus for generation of each virus working seed lot. 24

Monovalent virus pool 25

A monovalent virus pool is a suspension of single serotype of dengue virus and may be 26 the result of one or more single harvests or multiple parallel harvests of the same virus 27 serotype collected into a single vessel before clarification. 28

Multiple parallel harvest 29

A pool of harvests coming from multiple cultures that are initiated in parallel from the 30

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same ampoule of the same working cell bank infected together by the same virus 1 suspension of the same virus working seed lot 2

Plaque-forming unit (PFU) 3

A measure of the number of infectious viruses capable of forming plaques, as detected by 4 virtue of a cytopathic effect, in monolayer cell cultures per unit volume of virus 5 suspension. 6

Production cell culture (PCC) 7

A collection of cell cultures used for biological production that have been prepared 8 together from one or more containers from the working cell bank or, in case of primary 9 cell cultures, from the tissues of one or more animals. 10

Single harvest 11

A quantity of virus suspension harvested from production cell cultures inoculated with 12 the same working seed lot and processed together in a single production run. 13

Working cell bank (WCB) 14

A quantity of well-characterized cells of animal or other origin, derived from the master 15 cell bank at a specific PDL or passage level, dispensed into multiple containers, 16 cryopreserved, and stored frozen under defined conditions, such as in the vapour or liquid 17 phase of liquid nitrogen in aliquots of uniform composition. The working cell bank is 18 prepared from a single homogeneously mixed pool of cells. One or more of the WCB 19 containers is used for each production culture. 20

Virus working seed (VWS) 21

A quantity of virus of uniform composition, well characterized, derived from a virus 22 master seed lot (see above) by passage, in an approved production cell line. The working 23 seed lot is used for the production of single harvest. 24 25

A.2 General manufacturing requirements 26

The general requirements for manufacturing establishments contained in the WHO Good 27 manufacturing practices for biological products [58] should be applied by establishments 28 manufacturing dengue tetravalent vaccine. 29

Separate manufacturing areas for each of the four dengue serotypes as well as tetravalent 30 vaccine formulation may be used. Alternatively, manufacturing areas may be used on a 31 campaign basis with adequate cleaning between campaigns to ensure that cross-32 contamination does not occur. 33

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Production steps and quality-control operations involving manipulations of live virus 1 should be conducted under the appropriate biosafety level as agreed with the national 2 regulatory authority and country biosafety laws. 3

4

A.3 Control of source materials 5

A.3.1 Cell cultures for virus production 6

A.3.1.1 Conformity with WHO recommendations 7

Dengue viruses used in producing tetravalent dengue vaccine should be propagated in 8 cell substrates which meet the WHO Recommendations for the evaluation of animal cell 9 cultures as substrates for the manufacture of biological medicinal products and for the 10 characterization of cell banks [74] and approved by the national regulatory authority. All 11 information on the source and method of preparation of the cell culture system used 12 should be made available to the national regulatory authority. 13

A.3.1.2 Types of cell culture 14

Dengue vaccine candidates have been produced in fetal rhesus lung diploid cells and in 15 continuous cell lines. For fetal rhesus lung diploid cells and continuous cells, sections 16 A.3.1.3 and A.3.1.4 apply. 17

A.3.1.3 Cell banks 18

The use of a cell line such as fetal rhesus lung diploid cells or Vero cells for the 19 manufacture of dengue vaccines should be based on the cell bank system. The cell seed 20 should be approved by the national regulatory authority. The maximum number of 21 passages or population doubling (PDL) allowable between the cell seed, the WCB and 22 the production passage levels should be established by the manufacturer and approved by 23 the national regulatory authority. Additional tests may include: 24

— Propagation of the MCB or WCB cells to or beyond the maximum in vitro age 25 for production; and 26 — Examination for the presence of retroviruses and tumorigenicity in an animal 27 test system [74]. 28

WHO has established a bank of Vero cells, designated as WHO Vero reference cell bank 29 (RCB) 10-87 that has been characterized in accordance with the WHO Requirements for 30 continuous cell lines used for biologicals production, WHO Technical Report Series 745, 31 1987 [57] . The cell bank is available to manufacturers as a well-characterized starting 32 material for preparation of their own master and working cell banks on application to the 33 Coordinator, Quality, Safety and Standards, WHO, Geneva, Switzerland [74]. 34

35 In normal practice a master cell bank is expanded by serial subculture up to a passage 36 number (or population doubling, as appropriate) selected by the manufacturer and 37

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approved by the national regulatory authority, at which point the cells are combined to 1 give a single pool distributed into ampoules and preserved cryogenically to form the 2 WCB. 3 4 The manufacturer’s WCB is used for the preparation of production cell culture, and thus 5 for production of vaccine batches 6

A.3.1.4 Characterizations of cell banks 7

The cell seed (if applicable), master and working cell banks and end of production cells 8 (EOPC) or extended cell bank (ECB) should be characterized according to the WHO 9 Recommendations for the evaluation of animal cell cultures as substrates for the 10 manufacture of biological medicinal products and for the characterization of cell banks 11 [74]. 12

A.3.1.5 Cell culture medium 13

Serum used for propagating cells should be tested to demonstrate freedom from bacteria, 14 fungi and mycoplasmas, according to the requirements given in Part A, sections 5.2 and 15 5.3 of the General requirements for the sterility of biological substances (Requirements 16 for biological substances, no. 6) [56, 61], and from infectious viruses. 17

Detailed guidelines for detecting bovine viruses in serum for establishing MCB and WCB 18 are given in Appendix 1 of the WHO Recommendations for the evaluation of animal cell 19 cultures as substrates for the manufacture of biological medicinal products and for the 20 characterization of cell banks [74]. The principles outlined in the cell substrate 21 recommendations should be applied as appropriate, and the guidelines for detecting 22 bovine viruses in serum for establishing the cell banks may be applicable to production 23 cell cultures as well. In particular, validated molecular tests for bovine viruses might 24 replace the cell-culture tests of bovine sera if agreed by the national regulatory authority. 25 As an additional monitor of quality, sera may be examined for freedom from phage and 26 endotoxin. Gamma-irradiation may be used to inactivate potential contaminant viruses, 27 recognizing that some viruses are relatively resistant to gamma-irradiation. 28

The sources of animal components used in culture medium should be approved by the 29 national regulatory authority. These components should comply with current guidelines 30 in relation to animal transmissible spongiform encephalopathies [63, 67]. 31

Human serum should not be used. If human albumin is used, it should meet the revised 32 Requirements for the collection, processing and quality control of blood, blood 33 components and plasma derivatives (Requirements for biological substances no. 27) [60], 34 as well as current guidelines in relation to human transmissible encephalopathies [63, 67]. 35

The use of human albumin as a component of a cell culture medium requires careful 36 consideration due to potential difficulties with the validity period of albumin (which is 37 based on the length of time for which it is suitable for use in clinical practice) in relation 38 to the potential long-term storage of monovalent bulks of each dengue serotype. In 39 addition, if human albumin is used, it should be tested according to the WHO 40 Recommendations for the evaluation of animal cell cultures as substrates for the 41

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manufacture of biological medicinal products and for the characterization of cell banks 1 [74]. 2

Penicillin and other beta-lactams should not be used at any stage of the manufacture. 3 Other antibiotics may be used at any stage in the manufacture provided that the quantity 4 present in the final product is acceptable to the national regulatory authority. Nontoxic 5 pH indicators may be added, e.g. phenol red at a concentration of 0.002%. Only 6 substances that have been approved by the national regulatory authority may be added. 7

If porcine or bovine trypsin is used for preparing cell cultures, it should be tested and 8 found free of cultivable bacteria, fungi, mycoplasmas and infectious viruses as 9 appropriate [74]. The methods used to ensure this should be approved by the national 10 regulatory authority. 11

The source(s) of trypsin of bovine origin, if used, should be approved by the national 12 regulatory authority and should comply with current guidelines in relation to animal 13 transmissible spongiform encephalopathies [63, 67]. 14

A.3.2 Virus seeds 15

A.3.2.1 Vaccine virus strains 16

The strains of DENV 1-4 viruses attenuated either by serial passage in cell cultures or by 17 recombinant DNA technology used in the production of candidate tetravalent dengue 18 vaccine should be thoroughly characterized. This will include their historical records, 19 such as information on the origin of the strain, cell culture passage history, method of 20 attenuation, results of preclinical and clinical studies demonstrating attenuation, whether 21 the strains have been biologically or molecularly cloned prior to generation of the master 22 seed; their genome sequence; and the passage level at which clinical trials were 23 performed, as well as the results of the clinical studies. 24

Only strains approved by the national regulatory authority should be used. 25

Strains of dengue recombinant viruses used for master and working seeds to produce 26 vaccine candidates should comply with the additional specifications given below (section 27 A.3.2.2 Strains derived by molecular methods). 28

A.3.2.2 Strains derived by molecular methods 29

If vaccine seeds derived by recombinant DNA technology are used, and because this is a 30 live attenuated vaccine, the candidate vaccine is considered a GMO in several countries 31 and should comply with the regulations of the producing and recipient countries 32 regarding GMOs. An environmental risk assessment should be undertaken according to 33 Part D of these Guidelines. 34

The nucleotide sequence of any cDNA clone used to generate vaccine virus stocks should 35 be determined prior to transfection of DNA-derived RNA into the cell substrate. The cell 36 substrate used for transfection to generate the virus should be appropriate for human 37 vaccine production and approved by the national regulatory authority. 38

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Pre-seed lot virus stocks derived from passaging of the primary virus stock should also be 1 sequenced as part of preclinical evaluation. 2

Viral vaccine seeds that are directly re-derived from RNA extracted from virus, to reduce 3 the risk of previous contamination by TSE or other adventitious agents, are considered as 4 new vaccine seeds and should be appropriately characterized to demonstrate 5 comparability with the starting virus seed. 6

A.3.2.3 Virus seed lot system 7

The production of vaccine should be based on the master and working seed lot system to 8 minimize the number of tissue culture passages needed for vaccine production. Seed lots 9 should be prepared in the same type of cells using the same conditions for virus growth 10 (other than scale) as those used for production of final vaccine. 11

The virus working seed lot should be prepared by defined number of passages from the 12 virus master seed lot by a method and a passage level from the original virus seed that is 13 established through clinical and vaccine development studies. Once the passage level of 14 the working seed lot is established, it may not be changed without approval from the 15 national regulatory authority. 16 17 Virus seed lots should be stored in a dedicated temperature-monitored freezer at a 18 temperature that ensures stability upon storage. It is recommended that a large working 19 virus seed lot be set aside as the basic material for use by the manufacturer for the 20 preparation of each batch of vaccine. 21 22 Full in vitro and in vivo testing for detecting adventitious agents should be conducted on 23 either master or working seed lots. 24

A.3.2.4 Control cell cultures for virus seeds 25

In agreement with national regulatory authorities, tests on control cell cultures may be 26 required and performed as described in section A.4.1. 27

A.3.2.5 Tests on virus master and working seed lots 28

A.3.2.5.1 Identity 29

Each virus master and working seed lot should be identified as the appropriate dengue 30 virus serotype by immunological assay or by molecular methods. 31

A.3.2.5.2 Genetic/phenotypic characterization 32

Different live dengue vaccine viruses may have significantly different properties. Such 33 differences may influence the tests to be used to examine their genetic and phenotypic 34 stability relevant to consistency of production. The applicable tests will be identified in 35 the course of the nonclinical evaluation of the strains. Each seed should be characterized 36 by full-length nucleotide sequence determination and by other relevant laboratory and 37 animal tests, which will provide information on the consistency of each virus seed. 38

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Mutations introduced during derivation of each vaccine strain should be maintained in the 1 consensus sequence, unless spontaneous mutations induced during tissue culture passage 2 were shown to be innocuous in non-clinical and small-scale clinical trials. Some 3 variations in the nucleotide sequence of virus population on passage are to be expected, 4 but what is acceptable should be based on experience in production and clinical use. 5

For any new virus master and working seeds, it is recommended that the first three 6 consecutive consistency bulk vaccine lots be analysed for consensus sequence changes 7 from the virus master seed. The nucleotide sequence results should be used to 8 demonstrate the consistency of the production process. 9

Routine nucleotide sequence analysis of bulk vaccine is not recommended. 10

A.3.2.5.3 Tests for bacteria, fungi mycoplasmas and mycobacteria 11

Each virus master and working seed lot should be shown to be free from bacterial, fungal 12 and mycoplasmal contamination by appropriate tests as specified in Part A, sections 5.2 13 and 5.3, of the General requirements for the sterility of biological substances 14 (Requirements for biological substances, no. 6) [56, 61]. Nucleic acid amplification 15 techniques (NAAT) alone or in combination with cell culture, with an appropriate 16 detection method, might be used as an alternative to one or both of the compendial 17 mycoplasma detection methods after suitable validation and agreement from the national 18 regulatory authority [74]. 19

Seed lots should be shown to be free from mycobacteria by a method approved by the 20 national regulatory authority. NAAT might be used as an alternative to mycobacteria 21 microbiological culture method and/or to the in vivo guinea-pigs test for the detection of 22 mycobacteria after suitable validation and agreement from the national regulatory 23 authority [74]. 24

A.3.2.5.4 Tests for adventitious agents 25

Each virus working seed lot and/or master seed lot should be tested in cell cultures for 26 adventitious viruses relevant to the passage history of the seed virus. Where antisera are 27 used to neutralize dengue virus or the recombinant dengue virus, the antigen used to 28 generate the antisera should be produced in cell culture from a species different from that 29 used for the production of the vaccine and free from extraneous agents. Simian and 30 human cell cultures inoculated with the virus-antibody mixture should be observed 31 microscopically for cytopathic changes. For virus grown in simian or human cells, the 32 neutralized virus is tested on a separate culture of these cells. If other cell systems are 33 used, cells of that species, but from a separate batch, are also inoculated. At the end of the 34 observation period, the cells should be tested for haemadsorbing viruses. 35

Each virus master seed lot or working seed lot should also be tested in animals that 36 include guinea pigs, adult mice, and suckling mice. For test details, refer to the section 37 B.11 of the WHO Recommendations for the evaluation of animal cell cultures as 38 substrates for the manufacture of biological medicinal products and for the 39 characterization of cell banks [74]. Additional testing for adventitious viruses may be 40 performed using validated NAAT. 41

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New molecular methods with broad detection capabilities are being developed for 1 adventitious agent detection. These methods include degenerate NAAT for whole virus 2 families with analysis of the amplicons by hybridization, sequencing or mass 3 spectrometry; NAAT with random primers followed by analysis of the amplicons on 4 large oligonucleotide micro-arrays of conserved viral sequencing or digital subtraction of 5 expressed sequences; and high throughput sequencing. These methods might be used in 6 the future to supplement existing methods or as alternative methods to both in vivo and in 7 vitro test after appropriate validation and agreement from the national regulatory 8 authority. 9

A.3.2.5.5 Tests in experimental animals 10

A.3.2.5.5.1 Tests in non-human primates 11

All vaccine candidates should be evaluated at least once during pre-clinical development 12 for neurotropism in non-human primates, as detailed in Part B (nonclinical evaluation). 13 Candidate vaccines that are homogeneously “dengue” in terms of genetics are not 14 expected to be neurotropic in such a test, but where attenuation has been achieved by 15 recombination of dengue genes with those of a different virus species that itself displays 16 neurotropism (e.g. dengue/yellow fever recombinants); the master seed should be tested 17 in non-human primates. If these tests were not performed at the master seed level, they 18 should be performed at working seed level. 19

National regulatory authorities may decide that such testing does not need to be repeated 20 each time a novel Working seed lot is derived, if results of a well conducted monkey 21 neurovirulence assay on the Master seed lot are negative. Recent data suggest that certain 22 small animal models for neurovirulence may serve as a surrogate for non-human 23 primates, at least where viruses expressing YF strain 17D genes are concerned [38]. 24 National regulatory authorities may eventually wish to consider accepting results of such 25 studies as a surrogate for studies using non-human primates to evaluate neurovirulence of 26 novel dengue vaccines. 27

28 Test for neurotropism/neurovirulence 29

30

To provide assurance that a candidate vaccine virus is not unexpectedly neurovirulent, 31 each vaccine strain of each serotype should be tested for neurovirulence in monkeys by 32 inoculation of Macaca mulatta (rhesus), Macaca fascicularis (cynomolgus) or other 33 susceptible species of monkey in the course of preclinical evaluation. 34

Prior to testing for neurotropism, the neutralizing antibody test should be used to assess 35 the immune status of non-human primates to both dengue and yellow fever viruses. Tests 36 should follow the WHO Recommendations to assure the quality, safety and efficacy of 37 live attenuated yellow fever vaccines [75]. 38

A.3.2.5.5.2 Tests in suckling mice 39

The virulence of different vaccine candidates in mice will depend on the strains of virus 40

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and mouse. Novel vaccines that reach the clinical phase of development in many cases 1 were tested for neurovirulence in suckling and adult mice during the pre-clinical phase 2 of development. 3

While mice are not considered a good model for dengue, suckling and adult mice have 4 been used to assess the neurovirulence of dengue/yellow fever recombinant vaccines 5 [17, 18]. A mouse test might be considered in order to demonstrate consistency of 6 characteristics of dengue/yellow fever recombinant viruses during production (see 7 A.4.2.4.7). 8

A.3.2.5.6 Virus titration for infectivity 9

Each virus master and working seed lot should be assayed for infectivity in a sensitive 10 assay in cell culture. Depending on the results obtained in preclinical studies, plaque 11 assays, CCID50 assays, IFU assays or CCID50 with a molecular readout such as 12 quantitative polymerase chain reaction (QPCR) may be used. All assays should be 13 validated. 14

A.4 Control of vaccine production 15

A.4.1 Control of production cell cultures 16

Where the national regulatory authority requires the use of control cells, the following 17 procedures should be followed. From the cells used to prepare cultures for production of 18 vaccine, a fraction equivalent to at least 5 % of the total or 500 mL of cell suspension, or 19 100 million cells, should be used to prepare uninfected control cell cultures. These 20 control cultures should be observed microscopically for cytopathic and morphologic 21 changes attributable to the presence of adventitious agents for at least 14 days at a 22 temperature of 35-37 °C after the day of inoculation of the production cultures, or until 23 the time of final virus harvest, whichever comes last. At the end of the observation 24 period, supernatant fluids collected from the control culture should be tested for the 25 presence of adventitious agents as described below. Samples that are not tested 26 immediately should be stored at -60 °C or lower, until such tests can be conducted. 27

If adventitious agent testing of control cultures yields a positive result, the harvest of 28 virus from the parallel vaccine virus-infected cultures should not be used for production. 29

For the test to be valid, not more than 20 % of the control culture flasks should have been 30 discarded, for any reason, by the end of the test period. 31

A.4.1.1 Test for haemadsorbing viruses 32

At the end of the observation period, a fraction of control cells comprising not less than 33 25% of the total should be tested for the presence of haemadsorbing viruses, using 34 guinea-pig red blood cells. If the guinea pig red blood cells have been stored prior to use 35 in the haemadsorption assay, the duration of storage should not have exceeded 7 days, 36 and the storage temperature should have been in the range of 2–8 °C. 37

In some countries, the national regulatory authority requires that additional tests for 38

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haemadsorbing viruses be performed using red blood cells from other species including 1 those from humans (blood group O), monkeys, and chickens (or other avian species). For 2 all tests, readings should be taken after incubation for 30 minutes at 0–4 °C, and again 3 after a further incubation for 30 minutes at 20–25 °C. The test with monkey red cells 4 should be read once more after an additional incubation for 30 minutes at 34–37 °C. 5

For the tests to be valid, not more than 20 % of the culture vessels should have been 6 discarded for any reason by the end of the test period. 7

A.4.1.2 Tests for cytopathic, adventitious agents in control cell fluids 8

Supernatant culture fluids from each of control cell culture flasks or bottles collected at 9 the time of harvest should be tested for adventitious agents. A 10-mL sample of the pool 10 should be tested in the same cell substrate, but not the same cell batch as that used for 11 vaccine production, and an additional 10-mL sample of each pool should be tested in both 12 human and continuous simian cells. 13

Each sample should be inoculated into cell cultures in such a way that the dilution of the 14 pooled fluid in the nutrient medium does not exceed 1:4. The surface area of the flask 15 should be at least 3 cm2 per mL of pooled fluid. At least one flask of the cells should 16 remain uninoculated, as a control. 17

The inoculated cultures should be incubated at a temperature of 35–37 °C and should be 18 examined at intervals for cytopathic effects over a period of at least 14 days. 19

Some national regulatory authorities require that, at the end of this observation period, a 20 subculture is made in the same culture system and observed for at least an additional 7 21 days. Furthermore, some national regulatory authorities require that these cells should be 22 tested for the presence of haemadsorbing viruses. 23

For the tests to be valid, not more than 20 % of the culture vessels should have been 24 discarded for any reason by the end of the test period. 25

A.4.1.3 Identity test 26

At the production level, the cells should be identified by means of tests approved by the 27 national regulatory authority. Suitable methods are, but are not limited to, biochemical 28 tests (e.g. isoenzyme analyses), immunological tests (e.g. major histocompatibility 29 complex assays), cytogenetic tests (e.g. for chromosomal markers),and tests for genetic 30 markers (e.g. DNA fingerprinting). 31

A.4.2 Production and harvest of monovalent virus 32

A.4.2.1 Cells used for vaccine production 33

On the day of inoculation with the working seed virus, each production cell culture flask 34 (or bottle, etc) and/or cell culture control flask should be examined for cytopathic effect 35 potentially caused by infectious agents. If such examination shows evidence of the 36 presence in any flask of an adventitious agent, all cell cultures should be discarded. 37

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If animal serum is used in growth medium, the medium should be removed from the cell 1 culture either before or after inoculation of working virus seed. Prior to beginning virus 2 harvests, the cell cultures should be rinsed and the growth medium replaced with serum-3 free maintenance medium. 4

Penicillin and other beta-lactam antibiotics should not be used at any stage of 5 manufacture. 6 Minimal concentrations of other suitable antibiotics may be used if approved by the 7 national regulatory authority. 8

A.4.2.2 Virus inoculation 9

Cell cultures are inoculated with dengue working seed virus at an optimal and defined 10 multiplicity of infection. After viral adsorption, cell cultures are fed with maintenance 11 medium and incubated at a temperature within a defined range and for a defined period. 12

The multiplicity of infection, temperature range and duration of incubation will depend 13 on the vaccine strain and production method, and specifications should be defined by 14 each manufacturer. 15

A.4.2.3 Monovalent virus harvest pools 16

Vaccine virus is harvested within a defined period post-inoculation. A monovalent 17 harvest may be the result of one or more single harvests or multiple parallel harvests. 18 Samples of monovalent virus harvest pools should be taken for testing and stored at a 19 temperature of -60 °C or below. The sponsor should submit data to support the 20 conditions chosen for these procedures. 21

The monovalent virus harvest pool may be clarified or filtered to remove cell debris and 22 stored at a temperature that ensures stability before being used to prepare the tetravalent 23 final bulk for filling. The sponsor should provide data to support the stability of the bulk 24 over the duration of the chosen storage conditions as well as to support the choice of 25 storage temperature . 26

Harvests derived from continuous cell lines should be subjected either to further 27 purification to minimize the amount of cellular DNA and/or treatment with DNase to 28 reduce the size of the DNA. 29

A.4.2.4 Tests on monovalent virus harvest pools 30

A.4.2.4.1 Identity 31

Each monovalent virus harvest pool should be identified as the appropriate dengue virus 32 serotype by immunological assay on cell cultures using specific antibodies or by 33 molecular methods (see section A.6.1) approved by the national regulatory authority. 34

A.4.2.4.2 Tests for bacteria, fungi, mycoplasmas, and mycobacteria 35

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Each monovalent virus harvest pool should be shown to be free from bacterial, fungal, 1 mycoplasmal, and mycobacterial contamination by appropriate tests. Sterility tests are 2 specified in Part A, sections 5.2 (bacteria and fungi) and 5.3 (mycoplasmas), of the 3 General requirements for the sterility of biological substances (Requirements for 4 biological substances, no. 6) [56, 61]. 5 NAAT alone or in combination with cell culture, with an appropriate detection method, 6 might be used as an alternative to one or both of the pharmacopoeial mycoplasma 7 detection methods after suitable validation and the agreement of the national regulatory 8 authority [74]. 9 For testing mycobacteria, the test method should be approved by the national regulatory 10 authority. NAAT might be used as an alternative to mycobacteria microbiological culture 11 method after validation and agreement of the national regulatory authority [74]. 12

A.4.2.4.3 Tests for adventitious agents 13

Each monovalent virus harvest pool should be tested in cell culture for adventitious 14 viruses by inoculation into continuous simian kidney cells, cell lines of human origin, and 15 the cell line used for production, but from another batch. Where antisera are used to 16 neutralize dengue virus or the recombinant virus, the antigen used to generate the antisera 17 should be produced in cell culture from a species different from that used for the 18 production of the vaccine and free from extraneous agents. The cells inoculated should be 19 observed microscopically for cytopathic changes. At the end of the observation period, 20 the cells should be tested for haemadsorbing viruses. 21 Additional testing for adventitious viruses may be performed using validated NAAT. 22

23

New molecular methods with broad detection capabilities are being developed. These 24 methods include degenerate NAAT for whole virus families with analysis of the 25 amplicons by hybridization, sequencing or mass spectrometry; DNA amplification with 26 random primers followed by analysis of the amplicons on large oligonucleotide micro-27 arrays of conserved viral sequencing or digital subtraction of expressed sequences; and 28 high throughput sequencing. These methods might be used in the future to supplement 29 existing methods or as alternative methods to in vitro test after appropriate validation and 30 agreement from the national regulatory authority. 31

A.4.2.4.4 Virus titration for infectivity 32

The titre for each monovalent virus harvest should be determined in a sensitive assay in 33 cell culture. Depending on the results obtained in preclinical studies, plaque assays, 34 CCID50 assays, immunofocus formation assays or CCID50 with a molecular readout such 35 as QPCR may be used. 36

A.4.2.4.5 Tests for host cell proteins 37

The host cell protein profile should be examined as part of characterization studies [74]. 38

A.4.2.4.6 Tests for residual cellular DNA 39

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For viruses grown in continuous cell line cells, the monovalent harvest pool should be 1 tested for the amount of residual cellular DNA, and the total amount of cell DNA per 2 dose of vaccine should be not more than the upper limit agreed by the national regulatory 3 authority. If this is technically feasible, the size distribution of the DNA should be 4 examined as a characterization test, taking into account the amount of DNA detectable 5 using state of the art methods [74] as approved by the national regulatory authority. 6

A.4.2.4.7 Test for consistency of virus characteristics 7

The dengue virus in the monovalent harvest pool should be tested to compare it with the 8 working seed virus, or suitable comparator, to ensure that the vaccine virus has not 9 undergone critical changes during its multiplication in the production culture system. 10

11

Relevant assays should be identified in preclinical studies and may include virus yield in 12 tissue culture, plaque phenotype, or temperature sensitivity as examples. Other 13 identifying characteristics might also be applicable. 14

Assays for the attenuation of dengue/yellow fever recombinants and others as appropriate 15 include tests in suckling mice. Intracerebral inoculation of suckling mice with serial 16 dilutions of vaccine and yellow fever 17D is followed by the determination of the 17 mortality ratio and survival time. The results obtained with the vaccine are compared to 18 the yellow fever 17D control results. 19

The test may be omitted as a routine test once the consistency of the production process 20 has been demonstrated on a significant number of batches in agreement with the national 21 regulatory authority. Where there is a significant change in the manufacturing process, 22 the test should be reintroduced. 23

A.4.2.5 Storage 24

Monovalent virus harvest pools should be stored at a temperature that ensures stability 25 until tetravalent vaccine formulation. 26

A.4.3 Final tetravalent bulk lot 27

A.4.3.1 Preparation of final tetravalent bulk lot 28

The final tetravalent bulk lot should be prepared from monovalent virus pools of the four 29 dengue virus subtypes using a defined virus concentration of each component. 30 The operations necessary for preparing the final bulk lot should be conducted in such a 31 manner as to avoid contamination of the product. 32

33

In preparing the final bulk, any excipients (such as diluents or stabilizer) that is added to 34 the product should have been shown to the satisfaction of the national regulatory 35 authority not to impair the safety and efficacy of the vaccine in the concentration used. 36

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A.4.3.2 Tests on the final tetravalent bulk lot 1

A.4.3.2.1 Residual animal serum protein 2

If appropriate, a sample of the final bulk should be tested to verify that the level of serum 3 is less than 50 ng per human dose. 4

A.4.3.2.2 Sterility 5

Except where it is subject to in-line sterile filtration as part of the filling process, each 6 final bulk suspension should be tested for bacterial and fungal sterility according to Part 7 A, section 5.2 of the General requirements for the sterility of biological substances 8 (Requirements for biological substances, no. 6) [56], or by a method approved by the 9 national regulatory authority. 10

A.4.3.3 Storage 11

Prior to filling, the final bulk suspension should be stored under conditions shown by the 12 manufacturer to retain the desired viral potency. 13 14

A.5 Filling and containers 15

The requirements concerning good manufacturing practices for biological products [58] 16 appropriate to a vaccine should apply. 17

Care should be taken to ensure that the materials from which the container and, if 18 applicable, the closure are made do not adversely affect the infectivity (potency) of the 19 vaccine under the recommended conditions of storage. 20

A final filtration could be included during the filling operations. 21

The manufacturer should provide the national regulatory authority with adequate data to 22 prove the product is stable under appropriate conditions of storage and shipping. 23 24

A.6 Control tests on final lot 25

The following tests should be carried out on the final lot. 26

A.6.1 Vaccine 27

A.6.1.1 Inspection of final containers 28

Each container in each final lot should be inspected visually, and those showing 29 abnormalities should be discarded. 30

A.6.1.1.1 Appearance 31

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The appearance of the freeze-dried or liquid vaccine should be described with respect to 1 its form and colour. In the case of freeze-dried vaccines, a visual inspection should be 2 performed of the freeze-dried vaccine, the diluent, and the reconstituted vaccine. 3

A.6.1.2 pH 4

The pH of the final lot should be tested in a pool of final containers and an appropriate 5 limit set to guarantee virus stability. In the case of freeze-dried vaccines, pH should be 6 measured after reconstitution of the vaccine with the diluent. 7

A.6.1.3 Identity 8

Each monovalent component of a tetravalent dengue vaccine lot should be identified as 9 dengue or recombinant virus type-1, -2, -3 or -4 by immunological assay using specific 10 antibodies or by molecular methods. The methods used for the potency assay (A.6.1.5) 11 may serve as the identity test. 12

A.6.1.4 Sterility 13

Vaccine should be tested for bacterial and fungal sterility according to the requirements 14 in Part A, section 5.2 of the General requirements for the sterility of biological 15 substances (Requirements for biological substances, no. 6) [56] by acceptable methods 16 approved by the national regulatory authority. 17

A.6.1.5 Potency 18

At least three containers of each tetravalent vaccine lot should be assayed for infectivity 19 in a validated assay in appropriate cell culture. The assay should include a working 20 reference preparation to control the accuracy and reproducibility of the testing system. 21 The titre of each individual serotype should be determined. 22

A.6.1.6 Thermal stability 23

The thermal stability test is to demonstrate consistency of production. Additional 24 guidance on evaluation of vaccine stability is provided in the WHO Guideline on stability 25 evaluation of vaccines [71]. At least three containers of tetravalent vaccine should be 26 incubated at the appropriate elevated temperature for appropriate time (e.g. 37 °C for 7 27 days) depending on products. The geometric mean infectious virus titre (GMT) of the 28 containers for each individual virus components that have been exposed should not have 29 decreased during the period of exposure by more than an amount (e.g. 1 log) justified by 30 production data and approved by the national regulatory authority. Titration of non-31 exposed and exposed containers should be done in parallel. A validity control reagent of 32 each of the four virus components should be included in each assay to validate the assay. 33

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A.6.1.7 General safety 1

Each filling lot should be tested for unexpected toxicity (sometimes called abnormal 2 toxicity) using a general safety test approved by the national regulatory authority. 3 This test may be omitted for routine lot release once consistency of production has been 4 established to the satisfaction of the national regulatory authority and when good 5 manufacturing practices are in place. Each lot, if tested, should pass a general safety test. 6

A.6.1.8 Residual moisture (if appropriate) 7

The residual moisture in each freeze-dried lot should be conducive to the stability of the 8 product and the upper limit of the moisture content should be approved by the national 9 regulatory authority based on results of stability testing. 10

A.6.1.9 Residual antibiotics (if applicable) 11

If any antibiotics are added in the vaccine production, the content of the residual 12 antibiotics should be determined and be within limits approved by the national regulatory 13 authority. 14

A.6.2 Diluent 15

The recommendations given in the Good manufacturing practices for pharmaceutical 16 products [62] should apply for the manufacturing and control of diluents used to 17 reconstitute live-attenuated dengue vaccines. An expiry date should be established for the 18 diluent based upon stability data. For lot release of the diluent, tests for identity, 19 appearance, pH, volume, sterility, and the content of key components should be done. 20

A.7 Records 21

The recommendations of the Good manufacturing practices for biological products [58] 22 pp. 27–28, should apply, as appropriate for the level of development of the candidate 23 vaccine. 24

A.8 Samples 25

A sufficient number of samples should be retained for future studies and needs. Vaccine 26 lots that are to be used for clinical trials may serve as reference materials in the future, 27 and a sufficient number of vials should be reserved and appropriately stored for that 28 purpose. 29

A.9 Labelling 30

The recommendations of the Good manufacturing practices for biological products [58] 31 pp. 26–27, appropriate for a candidate vaccine should apply, with the addition of the 32 following: 33 34

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The label on the carton enclosing one or more final containers, or the leaflet 1 accompanying the container, should include: 2

— a statement that the candidate vaccine fulfils Part A of these Recommendations; 3

— a statement of the nature of the preparation, specifying the designation of the 4 strains of dengue or recombinant viruses contained in the live attenuated tetravalent 5 vaccine, the minimum number of infective units per human dose, the nature of any 6 cellular systems used for the production of the vaccine, and whether the vaccine 7 strains were derived by molecular methods; 8

— a statement of the nature and quantity, or upper limit, of any antibiotic present in 9 the vaccine; 10

— an indication that contact with disinfectants is to be avoided; 11

— a statement concerning the photosensitivity of the vaccine, cautioning that both 12 lyophilized and reconstituted vaccine should be protected from light; 13

— a statement indicating the volume and nature of diluent to be added to reconstitute 14 the vaccine, and specifying that the diluent to be used is that supplied by the 15 manufacturer; and 16

— a statement that after the vaccine has been reconstituted, it should be used without 17 delay, or if not used immediately, stored between 2 °C and 8 °C and protected from 18 light for a maximum period defined by stability studies. 19

A.10 Distribution and shipping 20

The recommendations given in the Good manufacturing practices for biological products 21 [58] appropriate for a candidate vaccine should apply. 22

Shipments should be maintained at temperatures of 8 °C or below, and packages should 23 contain cold-chain monitors [73]. 24

A.11 Stability, storage and expiry date 25

The recommendations given in the Good manufacturing practices for biological products 26 [58] and the Guidelines on stability evaluation of vaccines [71] appropriate for a candidate 27 vaccine should apply. The statements concerning storage temperature and expiry date that 28 appear on the primary or secondary packaging should be based on experimental evidence 29 and should be submitted for approval to the national regulatory authority. 30

A.11.1 Stability testing 31

Stability testing should be performed at different stages of production, namely on single 32 harvests, purified bulk, final bulk and final lot. Stability-indicating parameters should be 33 defined or selected appropriately according to the stage of production. It is advisable to 34 assign a shelf-life to all in-process materials during vaccine production, in particular 35 stored intermediates such as single harvests, purified bulk and final bulk. 36

The stability of the vaccine in its final container and at the recommended storage 37

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temperatures should be demonstrated to the satisfaction of the national regulatory 1 authorities on at least three lots of final product. Accelerated thermal stability tests may 2 be undertaken on each final lot to give additional information on the overall stability of a 3 vaccine (see section A.6.1.6). 4

The formulation of vaccine and adjuvant (if used) should be stable throughout its shelf-5 life. Acceptable limits for stability should be agreed with national authorities 6

A.11.2 Storage conditions 7

Before being distributed by the manufacturing establishment or before being issued from 8 a storage site, the vaccine should be stored at a temperature shown by the manufacturer to 9 be compatible with a minimal loss of titre. The maximum duration of storage should be 10 fixed with the approval of the national regulatory authority and should be such as to 11 ensure that all quality specifications for final product including the minimum titre 12 specified on the label of the container (or package) will still be maintained until the end 13 of the shelf-life. 14

A.11.3 Expiry date 15

The expiry date should be defined on the basis of shelf-life and supported by the stability 16 studies with the approval of the national regulatory authority and should relate to the date 17 of the last satisfactory determination, of potency (infectious titer), performed in 18 accordance with section A.6.1.5, i.e. the date on which the cell cultures were inoculated. 19 If the vaccine is stored at a temperature lower than that used for stability studies and 20 intended for release without re-assay, the expiry date is calculated from the date of 21 removal from cold storage. The expiry dates for the vaccine and the diluent may be 22 different. 23 24

Part B. Nonclinical evaluation of dengue tetravalent vaccines 1

(live, attenuated) 2

B.1 General remarks 3

Nonclinical evaluation of a live dengue vaccine includes in vitro and in vivo testing 4 required prior to initiation of the clinical phase of the vaccine development program. 5 This testing should yield information suggesting the safety and potential for efficacy of a 6 dengue vaccine candidate. Testing may continue in parallel with the clinical phase of 7 product development. Tests should include product characterization at each stage of 8 manufacture (including quantification of contaminants such as cellular proteins and 9 DNA), proof of concept/immunogenicity studies (including dose ranging in animals, etc.), 10 toxicology if required by the national regulatory authority, establishment of a test for 11 potency to be used throughout, and safety testing in animals (see Table B1). These 12 Guidelines should be read in conjunction with the WHO Guidelines on nonclinical 13 evaluation of vaccines [65] and are specifically aimed at nonclinical evaluation of a live, 14 attenuated dengue vaccine. 15 16 Although there is no animal model that precisely mimics dengue disease in humans, 17 animal models have been and are being used in studies on immunogenicity, protective 18 activity, toxicology, and safety. Animal models are briefly reviewed at the time of 19 preparing these Guidelines to highlight the latest developments and to provide better 20 understanding of their use in vaccine development. 21 22

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Table B1. Nonclinical evaluation of dengue vaccines 1 Area of nonclinical evaluation

Primary concern Scope of nonclinical evaluation

In vitro nonclinical evaluation Product characterization

Product risks are appropriate for the anticipated use

Mutations in the genome may impact infection efficiency and growth capacity in different cell types including cells of a monocyte lineage. Virus structural protein profiles, serotype identity. Consistency of manufacturing process; genetic stability of vaccine candidates.

Process development, quality control and quality assurance

Process meets all good manufacturing practices standards

Sources of all media, cells, and seed viruses; purification and virus concentration procedures; sources of all animal sera used to cultivate viruses and cells; demonstrate efficiency of purification processes; titration of virus dose; safety of excipients; standardize lab assays to measure immunogenicity, etc.

In vivo nonclinical evaluation

Immunogenicity and protective activity in an animal model.

Demonstrate that the vaccine can protect from some aspect of DEN infection; estimate dose range for humans

DENV attenuated vaccines are immunogenic in nonhuman primates; candidate should induce limited viremia and should protect against viremia following wt DENV challenge in NHP. Interference between DENV serotypes may be evaluated in mice or NHP, but data may not always correlate with human.

Toxicity and Safety

Product risks are appropriate for the anticipated use

Focus on unexpected consequences of the effect of the vaccine dose and direct effects due to vaccine virus replication and tissue tropisms. The evaluation includes scoring and statistical analysis for histopathogical legions and clinical signs between treatment and control groups.

2 3

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B.2 Product development and characterization 1

It is critical that vaccine production processes are standardized and controlled to ensure 2 consistency of manufacture in support of nonclinical data suggesting potential safety and 3 efficacy in humans. This is a pre-requisite for entering the clinical trial phase. 4 5 Each of the attenuated virus candidates in the tetravalent dengue vaccine formulation 6 should be characterized to define as far as is practical the critical genetic and phenotypic 7 markers associated with attenuation. Each vaccine virus should also be evaluated to 8 determine whether the genetic basis of attenuation is stable enough to reduce the risk of 9 reversion to wild-type virus (and consequently virulence), either during manufacture or 10 during replication in a vaccinee following inoculation, using available in vivo and in vitro 11 approaches. To this end, laboratory and animal studies should define genetic changes in 12 the virus genome that are associated with phenotypic attenuation markers. 13 14 It is helpful when possible also to identify in vitro phenotypic markers that correlate with 15 attenuation. Both types of markers are useful to detect reversion events and to 16 differentiate vaccine strains from wild-type virus strains in epidemiological surveillance 17 following human immunization. 18 19 Qualification of each attenuated vaccine strain should include obtaining the consensus 20 nucleotide sequence of the entire genome of the vaccine candidate, using the consensus 21 nucleotide sequence of the genome of the parent virus as a comparator. This is essential 22 to map the sites of attenuating mutations. It may also be possible thereby to determine 23 the genetic basis of any in vitro phenotypic markers that correlate with attenuation. Such 24 markers include but are not limited to plaque size, replication efficiency in mosquito 25 vectors, induction of viremia in non-human primates, suckling mouse neurovirulence, 26 virulence in any other animal model, and temperature sensitivity [8, 11, 12, 25, 26]. 27 Developers should bear in mind that consensus genome sequencing is unsuitable for 28 identifying minor or quasi-species genomes in a vaccine seed or batch [19]. 29

30

B.3 Nonclinical immunogenicity and protective activity 31

Assessment of innate and adaptive immune responses in animals can provide evidence 32 that the DEN vaccine has replicated in the host. Animals, particularly mice, have also 33 been valuable in assessing the various elements of the immune response to DENV. 34 Although there is no specific immune correlate of protection, antibodies directed against 35 the virus E protein neutralize the virus and have been shown to protect animals when 36 actively induced by experimental vaccines or when passively administered, prior to 37 challenge. Based on the accumulated data, it is generally accepted that protection in 38 humans should require a DENV-specific neutralizing antibody response. However, a 39 correlation between the titre of neutralizing antibodies in serum, as determined in an in 40 vitro neutralizing antibody assay (e.g. PRNT50), and protection has not been established 41 for any of the four serotypes of virus. 42 43

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While protective activity in an animal model does not necessarily predict the protective 1 effect in humans, it provides useful information regarding the immunological potency of 2 the vaccine. 3 4 The immune response or protective activity to each of the four serotypes in a tetravalent 5 DENV vaccine should be assessed, including the quality of response and any potential 6 virological/immunological interference between types. 7

8

B.4 Nonclinical toxicity and safety 9

B.4.1 Considerations 10

General guidance on the nonclinical safety assessment and design of preclinical studies 11 that apply to dengue vaccines is provided in the WHO Guidelines on nonclinical 12 evaluation of vaccines [65]. The term “toxicity” is generally associated with the untoward 13 consequences of the administration of a nonreplicating drug or biological that relate to its 14 direct dose-dependent effect in the test animal. Thus toxicity studies entail the careful 15 analysis of all major organs, as well as tissues near to and distal from the site of 16 administration, to detect unanticipated direct toxic effects typically of a drug or 17 nonreplicating biological agent, over a wide range of doses, including doses sufficiently 18 exceeding the intended clinically relevant dose amount. It is generally expected that if a 19 live attenuated vaccine does not replicate in the test animal, direct toxic effects are very 20 unlikely to be detected. However, single dose and repeated dose toxicity studies, which 21 are recommended for inactivated vaccines (sometimes in combination with adjuvants) 22 should be conducted to confirm the absence of direct toxicity and could be combined 23 with nonclinical safety evaluation (e.g. distribution, tropism, shedding). For live vaccines 24 the emphasis is on the demonstration of nonclinical safety as a consequence of vaccine 25 virus replication. 26

27 Nonclinical safety studies of live vaccines should be required for live-attenuated vaccines 28 in certain stages of development. Such studies are designed with the primary purpose to 29 demonstrate the vaccine(s) are less “virulent’ in the animal host than comparable wild-30 type viruses and that the vaccine does not exhibit any unexpected harmful tissue tropism 31 and damage or the capacity to elicit a harmful immune response. There is no animal 32 model that replicates human dengue disease adequately (see B.2.1 and B.2.2). However 33 non human primates and mice may provide useful information to characterize the viruses 34 (see B.5.2 and B.5.3). The design of preclinical safety studies should reflect route and 35 frequency of administration as proposed in the protocol to support clinical trials [65]. 36 37 If the live attenuated DENV vaccine is intended to be used to immunize women of 38 childbearing age, developmental/reproductive toxicity studies should be performed 39 according to WHO guidelines [65]. 40

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B.4.2 Assessment in the non-human primate 1

B.4.2.1 Neurotropism/neurovirulence in non-human primates 2

The consensus of current opinion is that all live dengue vaccines should be tested once 3 for neurovirulence and/or neurotropism (whether vaccine viruses cause diseases in the 4 CNS or they have affinity to nerve tissues). At this time, the most well-established model 5 for vaccine neurotropism is the NHP, which has been historically used to evaluate new 6 seeds of YF vaccines (17D substrains 17D204- or 17DD-derived) and live polio vaccines. 7 Novel rodent (hamster and mouse) models for YF vaccine virulence are currently under 8 development. A rodent model could eventually be considered in lieu of NHP testing [38] 9 (see section A.3.2.5.5.1). 10 11 Involvement of the CNS in cases of dengue fever and dengue hemorrhagic fever has 12 usually been diagnosed as secondary to vasculitis with resultant fluid extravasation . The 13 rarity of reports of patients with dengue encephalitis suggest that the virus does not 14 typically cross the blood-brain barrier and infect neuronal cells [32]. However, since 15 dengue vaccine viruses are genetically altered compared to their wildtype parent viruses, 16 it is advisable to ensure that candidate vaccines have not acquired a neurotropic 17 phenotype as an unintended consequence of the attenuation process. This is a particular 18 issue as regards dengue vaccine viruses that contain YF 17D chimeric genomes, and it 19 would be of similar special importance in the future, if novel dengue vaccines are derived 20 from the genomes of any other known neuropathic viruses. This evaluation could be 21 done once at an early stage of development, using a master seed or working seed lot of 22 the vaccine. National regulatory authorities would need to decide whether each 23 component of the tetravalent formulation needs to be tested separately for the property of 24 neurovirulence or whether the tetravalent formulation could be tested initially, in which 25 case no further testing of the individual vaccines would need to be done if results of the 26 initial tests were within predefined specifications. 27 28 Testing for neurotropism/neurovirulence in the NHP model via the intracerebral route 29 should follow the WHO recommendations for neurotropism testing of YF vaccines [31, 30 75]. A brief testing and evaluation procedure is provided below. In addition, neurotropism 31 should be also evaluated as part of nonclinical safety study for distribution and tropism. 32 33 Groups of at least 10 monkeys, determined to be non-immune to DENV and YFV prior to 34 inoculation with the DENV master seed, should be inoculated intracerebrally in the 35 frontal lobe. A control group of 10 monkeys, also demonstrated to be non-immune to 36 DENV and YFV, should receiveYF-17D as the control group. All monkeys should be 37 observed for 30 days for signs of encephalitis, prior to necropsy. If the number of 38 monkeys, the observation period and/or time point(s) for necropsy for histological 39 examination are different from these recommendations, they should be justified and in 40 agreement with the national regulatory authority. Clinical scores, and the scores of 41 histological lesions in the central nervous system should be recorded. Advanced 42 histological scoring method such as automated image analysis [33] may be implemented 43 to provide quantitative assessment of virus-induced histopathology in brain tissues if the 44

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method has been properly validated and is acceptable by the national regulatory authority. 1 The overall mean clinical and histological scores of test group should not exceed the 2 scores of the control YF vaccine group. The significance level in statistical difference 3 between test and control groups should be in agreement with the national regulatory 4 authority. 5 6

B.4.2.2 Viremia in non-human primates 7

Non-human primates, humans and mosquitoes are the only natural hosts of DENV [8, 19, 8 51]. NHPs have been widely used to evaluate replication and immunogenicity of 9 candidate dengue vaccines [5, 16, 55]. Primary infection of macaques with wildtype 10 DENV results in moderate lymphadenopathy and a robust immune response [8, 19]. The 11 NHP model has traditionally been used as an important guide for selecting vaccine strains 12 for further development. In such studies, reduced peak titres and duration of viremia 13 induced by a candidate vaccine, compared to those induced by the non-attenuated parent 14 virus, is often, but not always, a correlate of attenuation. Consequently, if a dengue 15 vaccine candidate causes viremia in NHPs comparable to that caused by its wild-type 16 parent virus the vaccine, developers may wish to consider discontinuing further 17 development. 18

B.4.3 Assessment in mouse models 19

DENV infection has been studied in many different mouse models [2, 8, 47, 55, 76]. 20 When appropriate, a mouse model may be selected to evaluate the potential of a 21 candidate vaccine to cause disease in comparison to its wild-type parent virus. In such an 22 experiment, the titres of virus in blood, spleen, liver, lymph nodes, lungs, brain, and other 23 tissues at various time points post-infection [8]. The AG129 interferon receptor-deficient 24 mouse will support replication of DENVs of all serotypes [25, 26]. A DENV2 strain 25 adapted to replicate in the AG129 induces a physiologically relevant disease in that strain 26 [55]. At present, the AG129 mouse seems most suitable for safety studies, but National 27 regulatory authorities should be aware of the pitfalls of interpreting results, since these 28 animals do not possess an intact innate immune response. For this same reason, as 29 mentioned earlier, it would not be advisable to use AG129 mice for classic toxicology 30 studies. Other inbred mouse strains with genes knocked-out are under investigation as 31 models of DENV infection and disease. One or more of these may have applicability to 32 vaccine development in the future. 33

B.4.4 DENV replication in vector mosquitoes 34

Transmission of DENV to arthropod vectors from humans is essential to maintain the 35 virus in nature. As noted previously, none of the DENV attenuated candidate vaccines 36 studied to date induce a viremia in vaccinees that is sufficient in magnitude to infect 37 feeding mosquitoes [19, 44, 50]. Further, if mosquitoes are infected with dengue vaccines, 38 the viruses do not replicate sufficiently to permit transmission of the virus. For these two 39 reasons, Ae. Aegypti mosquitoes are not expected to transmit dengue vaccine viruses [19, 40 22, 50]. As a measure of attenuation and safety, future novel candidate vaccines should 41

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be shown to have reduced ability to replicate and disseminate in Ae aegypti mosquitoes 1 that have been infected in a controlled laboratory setting, using parent strains as controls 2 [19, 22, 36, 45]. 3 4

B.5 Environmental risk 5

For live dengue vaccines, the primary environmental risks relate to their capacity to be 6 spread from human to human by vector mosquitoes and the risk that prolonged or 7 repeated cycles of replication in mosquitoes could permit reversion to virulence. As 8 previously noted, live vaccines currently under development have been shown to 9 replicate poorly both in vaccinees and in mosquitoes, such that the risk for transmission 10 by the mosquito vector is very low, if any risk exists at all [3, 22, 28, 44, 45, 50]. These 11 factors should markedly reduce the chance that any of these vaccines could revert in 12 mosquitoes to a virulent phenotype when used in a mass vaccination campaign in an 13 endemic area. In addition, genetic stability during multiple sequential passages in 14 mosquitoes has also been demonstrated for most existing live dengue vaccine candidates. 15 For future candidate novel live vaccines, similar studies would need to be done. 16 17 Recently, some investigators have raised the issue, as regards live dengue vaccines, that 18 vaccine viruses could revert to virulence in mosquitoes via intragenic recombination with 19 endogenous wild-type flaviviruses. Such a phenomenon would seem to be highly 20 unlikely due to factors noted above plus the controversial question of whether 21 flaviviruses are able to undergo recombination even under ideal conditions in vitro. For 22 further details see Part D guidelines for environmental risk assessment.23

Part C. Clinical evaluation of dengue tetravalent vaccines (live, 1

attenuated) 2

C.1 General considerations for clinical studies 3

The following should be read in conjunction with the: 4

— WHO Guidelines on clinical evaluation of vaccines: regulatory 5 expectations (TRS 924, Annex 1) [64] and 6

— Guidelines for the clinical evaluation of dengue vaccines in 7 endemic areas (WHO/IVB/08.12) [69]. 8

9

C.1.1 Objectives of the clinical development programme 10

The clinical evaluation of a candidate live dengue tetravalent vaccine should aim to 11 demonstrate that it: 12

— Elicits immune responses against all four dengue serotypes 13

— Prevents DFI of any severity caused by any of the serotype 1, 2, 3 14 and 4 viruses over an appropriate minimum period of observation 15

— Has an acceptable safety profile 16 17

In addition, the program should: 18

— Gather preliminary evidence that the immediate and longer-term 19 immune response to a candidate dengue vaccine does not predispose 20 vaccinated individuals to develop severe DFI (e.g. including 21 haemorrhagic manifestations and systemic shock) during natural 22 infections 23

— Attempt to examine the association between neutralizing antibody and 24 protection against clinical disease (referred to as a surrogate marker 25 for efficacy in this document) 26

— Attempt to identify a neutralizing antibody titre that predicts (in the 27 short or longer-term) protection against clinical disease (referred to 28 as an immunological correlate of protection in this document). 29

30

C.1.2 Outline of the clinical development program 31

In the initial clinical studies (i.e. Phase 1 studies) it is expected that relatively small 32 numbers of healthy adults are vaccinated with investigational vaccine formulations and 33 that the primary focus is on assessing safety. These studies may include exploration of 34 immune responses to ascending doses of the four DENV serotypes when administered 35 alone and in combination. 36

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The subsequent clinical studies (i.e. Phase 2 studies) should be designed to select a dose 1 of each DENV serotype for use in the tetravalent candidate vaccine formulation and to 2 identify an appropriate primary immunization schedule for further study. 3 4 It is not currently possible to license candidate dengue vaccines based only on safety and 5 immunogenicity data. because: 6

— No dengue vaccine has yet been licensed 7

— There is no established surrogate marker for protection and no 8 immunological correlate of protection. 9

10 Therefore, candidate tetravalent dengue vaccines should be evaluated for protective 11 efficacy against DFI. 12 13 Sponsors may decide to conduct at least one preliminary study of protective efficacy (i.e. 14 sometimes referred to as a Phase 2b study) in order to identify a final candidate vaccine 15 and immunization schedule for further study. Alternatively, depending on the data 16 already accumulated (e.g. based on demonstration of a robust neutralizing antibody 17 response), sponsors may consider it appropriate to omit such a study. 18

19 The selected candidate vaccine should be evaluated in at least one adequately sized study 20 of protective efficacy (i.e. Phase 3 study) that compares numbers of cases of laboratory-21 confirmed DFI between groups of vaccinated and unvaccinated subjects. The total DFIs 22 counted should include those due to any of the four DENV serotypes and of any degree 23 of clinical severity that occur within a defined observation period. 24

25 Section C.3 gives more details of study designs and populations to be enrolled into studies 26 conducted at each phase of development. 27

28

C.2 Immunogenicity 29

C.2.1 Measurement of immune responses to vaccination 30

Current evidence suggests that neutralizing antibody against each DENV serotype is 31 likely to be the best surrogate marker for efficacy. 32

33 It is recommended that the methodology for determination of DENV serotype-specific 34 neutralizing antibody titres should follow the WHO Guidelines for the plaque reduction 35 neutralization test (PRNT) (WHO/IVB/07.07) [68]. If alternative methods for 36 determining neutralizing antibody (e.g. high throughput microneutralization assays) are 37 developed these should be validated against the PRNT. 38

39 Reference virus strains and cell substrates are available from the WHO and titres should 40 be expressed in IU calibrated against the WHO reference sera. In-house strains may be 41 used provided that the reference serum is employed and results expressed in IU. In 42 addition, a comparison with results obtained with the WHO reference strains is advised. 43

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1 An assessment of neutralizing antibody titres against a range of DENV serotypes and 2 strains of those serotypes, including recent wild-type isolates, is encouraged. This would 3 be valuable information to obtain due to worldwide strain diversity and because 4 neutralizing antibody titres against specific isolates will be variable. Such additional 5 assays could be applied to subsets of sera collected from vaccinees that have been 6 selected randomly or based on a scientific justification (e.g. to select sera known to cover 7 a range of neutralizing antibody titres against the reference or in-house strains). 8 9 The assay of DENV-specific antibody other than neutralizing antibody (e.g. IgM and IgG 10 ELISA) may be of interest but is not considered to be essential for the assessment of 11 potential vaccine efficacy. 12

13 It is considered unlikely that data on cell-mediated immunity (CMI) will provide an 14 immunological correlate of protection. However, the exploration of CMI is encouraged 15 since specific CMI assays may be useful for the assessment of immunological memory 16 and durability of protection. Assessments of cytokine responses may assist in the 17 evaluation of vaccine safety and provide some indication of the potential risk that 18 vaccination could predispose subjects to develop severe DFI during subsequent natural 19 infection [49]. 20 21

C.2.2 Investigation and interpretation of immune responses to 22 vaccination 23

There is no established immunological correlate of protection against any DENV 24 serotype. In the initial clinical studies of safety and immunogenicity, including the dose- 25 and regimen-finding studies, it is essential to fully describe the pre- and post-vaccination 26 neutralizing antibody titres that are observed against each of the four DENV serotypes 27 (see also section C.3.1). Adequate data should be generated to describe the kinetics of the 28 neutralizing antibody response in the short-term. Longer-term antibody persistence data 29 may be collected in these and/or in later studies, as described below. 30 31 In a non-endemic population with no detectable pre-vaccination antibody in the majority 32 of subjects a comparison of percentages with a detectable titre post-vaccination (which 33 may be defined as seroconversion in such a population) against each DENV serotype 34 should be made. The analyses should also look at proportions that seroconvert (in 35 accordance with an appropriate definition of seroconversion stated in the protocol) to 36 multiple serotypes (i.e. two, three or all four serotypes). 37 38 In an endemic population in which very high proportions of subjects are already 39 seropositive with respect to at least one dengue type a comparison of pre- and post-40 vaccination geometric mean titres (GMTs) with respect to those types will be informative 41 in addition to seroconversion rates. 42 43

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In endemic and non-endemic populations detailed consideration of reverse cumulative 1 distribution curves is important. For example it may be informative to compare 2 percentages achieving a pre-defined high titre of neutralizing antibody. 3

4 In protective efficacy studies neutralizing antibody against DENV serotypes should be 5 determined and followed over time in pre-defined subsets of the study population, 6 including an assessment of antibody persistence after the protocol-defined period for the 7 primary evaluation of protective efficacy. It is preferable that subsets of subjects to be 8 included in these detailed immunogenicity evaluations should be identified at the time of 9 randomization with stratification for age and any other factors that might have an 10 important impact on immune responses to vaccination. In any case the data should be 11 analysed according to pre-defined subsets. Immune responses should be determined for 12 vaccinated and unvaccinated subjects so that the effects of background exposure to 13 DENVs during the study period can be assessed. 14 15 Depending on the specific vaccine construct and taking into account any pertinent results 16 of non-clinical studies, sponsors may wish to undertake some exploratory investigations 17 of antibody against other antigens (e.g. those associated with the attenuated yellow fever 18 virus backbone in a chimeric vaccine). 19 20 Long-term storage of sera is encouraged since future developments in the field and/or 21 emerging data on longer-term safety or efficacy might point to a need for additional 22 investigations that cannot be predicted at the time of conducting the study. 23 24 Subsets of subjects should also be identified for collection of peripheral blood 25 mononuclear cells (PBMCs), taking into account feasibility issues, such as the blood 26 volumes required from different age groups to produce adequate cell numbers for study 27 and accessibility to adequate sample processing and storage facilities. 28 29 For the analysis of the relationship between neutralizing antibody titres and protection 30 against laboratory-confirmed DFI, sera should be collected at timed intervals from a 31 substantial cohort of subjects (and preferably the entire study population if feasible). 32 Once the protocol-defined double-blind observation period has been completed, the 33 analysis of the relationship between immune response and protection against DFI should 34 follow according to: 35

36 a) A cohort study in which one or more measures of immune response are related to 37 disease in all or a large subset of immunized subjects. This design is potentially the more 38 informative if there are enough cases among immunized subjects to allow for a 39 quantitative estimation of disease risk as a function of immune response(s); 40 OR 41 b) A case-control study in which immune responses are compared between immunized 42 subjects who developed disease and a subset of immunized subjects who did not develop 43 disease. This design enables establishment of an association between disease and 44 measure(s) of immune response but it does not allow for a quantitative estimation of 45 disease risk as a function of immune response. 46

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1 The use of serology to help identify infections with dengue viruses (whether or not 2 clinically apparent) is a quite separate issue that is discussed in section C.3.3. 3

4

C.3 Clinical studies 5

C.3.1 Phase 1 studies 6

The Phase 1 studies should be designed to provide an early indication of whether severe 7 local and/or systemic adverse events (AEs) may occur commonly after vaccination. 8 These studies may also provide preliminary data on immune responses to assist in the 9 selection of DENVs (or constructs) and doses to be included in candidate tetravalent 10 vaccine formulations for further study. 11

12 Subjects enrolled into these initial studies should be healthy adults who are naïve to 13 flaviviruses (based on medical and vaccine history and serological studies). It is preferred 14 that subjects are resident in non-endemic areas so that they are not at risk of natural 15 infection with dengue or other flaviviruses. Eligible subjects should not be in need of 16 vaccination against other flaviviruses at least throughout the duration of the study. 17 18 Sponsors may choose to commence studies with monovalent (i.e. containing a single live 19 attenuated DENV serotype) before progressing to evaluate multivalent versions (which 20 may include bivalent, trivalent and then tetravalent formulations) of a candidate dengue 21 vaccine. 22

23 If a candidate tetravalent vaccine formulation elicits a much lower antibody titer to one 24 (or more than one) DENV serotype than to others it is important that consideration is 25 given to modification of the vaccine (e.g. by modifying the infectious titers of serotypes) 26 and/or the immunization schedule due to the potential implications for safety and efficacy. 27 28 If a likely candidate tetravalent vaccine is identified it may be appropriate that a 29 preliminary exploration of safety and immunogenicity should be conducted in healthy 30 adult residents of an endemic area (i.e. including subjects with evidence of some pre-31 existing immunity to dengue or other flaviviruses). Such a study could provide further 32 reassurance regarding the ability of the candidate vaccine to elicit immune responses to 33 all four DENV serotypes before progressing to studies in larger numbers of subjects. 34

35


The Phase 2 studies should extend the information on safety and immunogenicity of 37 candidate tetravalent vaccine formulations. They should include studies in residents of 38 endemic areas who are therefore at risk of natural infection with dengue and may have 39 some degree of pre-existing immunity to one or more DENV serotypes and to other 40 flaviviruses. 41 42

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While the first data may be obtained in adults there should be a plan to move down to 1 younger age groups in a stepwise fashion. The age range should reflect that proposed for 2 the evaluation of protective efficacy of the tetravalent vaccine candidate. 3 4 Depending on the findings of the Phase 1 studies, the first Phase 2 studies may further 5 explore dose-response relationships. The data generated on safety and immunogenicity 6 should be sufficient to support the selection of one or more candidate tetravalent vaccines 7 and immunization schedules (i.e. number of doses and dose intervals) for further 8 evaluation. 9 10 If the sponsor chooses to undertake a preliminary (i.e. Phase 2b) study of safety and 11 efficacy this should be of an appropriate design and of adequate size to support a robust 12 decision regarding the vaccine formulation and schedule to be further evaluated (see 13 C.3.3). Even in a Phase 2b study it is recommended that subjects should be followed up 14 for approximately 3-5 years from the time of completion of vaccination to collect data on 15 safety and to document antibody to DENV serotypes in subsets of each treatment group. 16 17 The total number of subjects enrolled into Phase 2 studies should be sufficient to describe 18 at least common adverse reactions to vaccination with some degree of confidence. 19 Therefore it is expected that several hundred subjects should have been exposed to 20 candidate tetravalent vaccines containing the final or near final doses of DENVs of each 21 serotype. If any unusual, severe or serious adverse reactions are documented it may be 22 appropriate that further studies include the assessment of safety as one of the primary 23 objectives provided that these reactions would not preclude further vaccine development. 24

25


It is not currently possible to license dengue vaccines based only on safety and 27 immunogenicity data because there is no licensed dengue vaccine and no immunological 28 correlate of protection has been established for any DENV serotype. Each tetravalent 29 candidate vaccine should be evaluated in at least one study that is of an appropriate 30 design and adequate size to estimate vaccine efficacy (VE). This situation may change in 31 the future (see section C.3.3.7). 32 33

C.3.3.1 General issues for study design 34

VE is estimated by comparing the total numbers of laboratory–confirmed cases of DFI of 35 any degree of severity and due to any of the four DENV serotypes between the 36 vaccinated and unvaccinated (control) groups. The primary analysis of VE should be 37 conducted at the conclusion of a protocol-defined double-blind observation period. Each 38 study should be of sufficient size and duration to provide a robust estimation of VE and 39 to provide preliminary evidence that the vaccine does not predispose recipients to 40 develop one of the severe forms of DFI following natural infection. 41 42

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Studies of protective efficacy should be performed in endemic areas where a proportion 1 of the population is likely to have some naturally-acquired immunity to one or more of 2 the four DENV serotypes and/or other flaviviruses. It is assumed that in most, if not all, 3 cases each study will evaluate a single tetravalent candidate dengue vaccine and 4 immunization schedule. However, the study design may be adapted as necessary if more 5 than one possible active vaccination group is to be included. 6

7 Studies that involve vaccination of a large proportion of subjects at any one study locality 8 carry the potential to significantly interrupt DENV transmission during the observation 9 period. The result could be a reduced likelihood of demonstrating a difference in the 10 numbers of laboratory-confirmed cases of DFI between the vaccine and control groups. 11 Consideration should be given to this possibility when designing the study. 12

13 Randomization should be performed using a centralized system. When using a 1:1 14 randomization ratio the block size should be selected with the aim of enrolling 15 approximately equal numbers in test and control groups at each of the study sites so that 16 subjects in each group are at the same risk of developing mild and severe DFI throughout 17 the observation period. It is also possible to consider the use of unbalanced randomization 18 (e.g. 3 : 2 or 2 : 1 vaccine : control) provided that care is taken to ensure the desired ratio 19 is applied at each study site (or geographically localized sites) and the sample size is 20 calculated to provide adequate power. 21

22 The decision to use unbalanced randomization should take into consideration the possible 23 advantages and disadvantages. Advantages include a larger safety database and possibly 24 easier enrolment due to the greater chance that any one subject would receive the 25 candidate dengue vaccine. Disadvantages include the possibility that a larger proportion 26 vaccinated against dengue could increase the risk of achieving a reduction in DENV 27 transmission sufficient to impact on the chance of obtaining a conclusive study result. 28 29 Whenever possible, subjects randomized to the control group should receive an 30 alternative active vaccine (selected to provide an anticipated benefit to study participants) 31 rather than injections of placebo. If the active control vaccine cannot be given at the same 32 schedule as the candidate dengue vaccine, then placebo injections can be used within the 33 schedule as necessary to maintain a double-blind design. If the active control vaccine has 34 a different presentation or appearance to the candidate dengue vaccine then study 35 personnel who administer the vaccinations should not have any other involvement in the 36 conduct of the study. Vaccine recipients should not be allowed to observe preparation of 37 the vaccines for injection (e.g. any reconstitution steps that may or may not be necessary) 38 to avoid the risk of them sharing this information and so identifying themselves with one 39 of the study groups. 40 41 If there is no suitable active control vaccine that can be given by the same route of 42 administration as the candidate dengue vaccine then the use of a placebo control is 43 necessary to achieve a double blind design. In such cases, the protocol could plan to 44 administer a suitable licensed vaccine to all subjects in the study (i.e. those who do and 45 do not receive the candidate dengue vaccine) at some time after completion of the 46

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assigned study treatments and during the double-blind follow-up period. In this way all 1 study subjects can derive some potential benefit from participation in the study without 2 compromising the study integrity. 3 4 It is expected that several different production lots of vaccine will be used during 5 protective efficacy studies. Whether or not a formal lot-to-lot consistency study should be 6 built into the protocol, with the specific aim of comparing safety and immunogenicity 7 between subjects who receive different lots (usually 3 of the total used) according to pre-8 defined criteria, must be decided on a case by case basis. If such a formal comparison is 9 to be made then additional measures will be needed to ensure that adequately sized 10 subsets of subjects are randomized to receive each of the vaccine lots identified for this 11 comparison. 12

C.3.3.2 Study location and duration 13

The geographic areas selected for study should have background rates of DFI that are 14 sufficient to provide enough cases in the control group during the observation period to 15 facilitate the estimation of VE. To assess background rates efficacy studies should be 16 preceded by the collection of epidemiological information to document the expected 17 incidences of DENV serotype-specific and all DFI preferably over several years. The data 18 should include information on seasonality of disease to identify periods of transmission 19 and case demographics (e.g. age and gender) so that populations at highest risk of DFI 20 can be targeted for enrolment. 21 22 There should also be an assessment of the likely extent of exposure of the population to 23 other species of flaviviruses at potential study sites because such exposure may confound 24 the interpretation of dengue-specific serological data and possibly affect the clinical 25 course of DFI. This assessment should take into account any available epidemiological 26 data, serological studies and information on rates of vaccination against other flaviviruses. 27

28 Study sites should be endemic for one or more DENV serotypes with transmission 29 expected each season during the observation period, which would likely range from 1-3 30 years from the time of the first vaccination. Nevertheless, even if the study is conducted 31 over several seasons and at geographically dispersed study sites there may not be 32 sufficient numbers of cases of DFI to support an estimation of serotype-specific VE for 33 some or all of the four serotypes. Additional evidence for protective efficacy against 34 individual DENV serotypes should be sought from post-licensure (i.e. effectiveness) 35 studies as discussed in sections C.3.3.7 and C.4. 36

37 There should be a plan to follow-up subjects for safety and efficacy for at least 3-5 years 38 from the time of completion of primary vaccination. During this period it is possible that 39 an efficacious dengue vaccine may be offered to subjects originally assigned to the 40 control group with potential implications for interpretation of the data that can be 41 collected (see C.3.3.7). 42

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C.3.3.3 Study population 1

Since protective efficacy studies should be performed in endemic areas there is a need to 2 consider that the ultimate target group for vaccination may range from a subgroup (e.g. 3 infants) to the entire population. However, even in areas where a substantial proportion of 4 hospitalized cases of severe DFI are aged less than one year there are likely to be 5 concerns regarding inclusion of infants in protective efficacy studies because of the 6 possible risk of DFI that has been reported in association with waning maternal antibody 7 against one or more DENV serotypes and the unknown effects of vaccination in the 8 presence of maternal antibody. 9 10 Therefore it is expected that protective efficacy studies would likely exclude subjects 11 aged less than one year but should enrol children across a wide age range subject to 12 satisfactory results from the safety and immunogenicity studies. Section C.3.3.7 considers 13 bridging the observed VE to populations that were not included in efficacy studies. 14

15

C.3.3.4 Objectives, endpoints and analyses 16

The primary objective of a protective efficacy is to estimate VE against DFI. The primary 17 analysis should seek to demonstrate superiority for the vaccinated group versus the 18 control group in terms of the total numbers of cases of laboratory-confirmed DFI in 19 subjects who have been fully vaccinated in accordance with the protocol and have been 20 followed up for the required time with no major protocol deviations. In this analysis 21 counting of cases should commence from a designated time point after the last dose of 22 protocol-assigned doses has been administered. 23 24 VE is estimated by comparing the total numbers of laboratory–confirmed cases of DFI 25 (i.e. summation of cases due to any DENV serotype and of any degree of severity) that 26 occur in vaccinated and unvaccinated (control) groups during a protocol-defined double-27 blind observation period. VE should be calculated using the standard formula VE (%) = 28 100 x (1- r1/ro) [where r1 = incidence rate in the vaccine group and r0 = incidence rate in 29 the control group]. 30 31 The assessment of DENV serotype-specific VE should be a major secondary objective 32 and the subject of a planned secondary analysis. It is not expected that the study would be 33 powered to support a formal statistical analysis of DENV serotype-specific efficacy. 34 35 The statistical analysis plan should explain how multiple episodes of DFI in any one 36 study participant will be handled in the analyses. 37

38 Other important secondary analyses should include the estimation of: 39

— Efficacy based on counting all DFI that occur after administration of the 40 first dose of protocol-assigned treatment; 41

— Efficacy in all vaccinated subjects regardless of protocol deviations 42 (including those with incomplete vaccination courses and missing data); 43

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— Efficacy according to pre-vaccination flavivirus serological status, which 1 might be determined in a randomized subset of enrolled subjects who are 2 followed serologically; 3

— Efficacy according to severity of laboratory-confirmed DFI (with 4 adequate protocol definitions); 5

— Efficacy that includes prevention of “possible” or “probable”dengue 6 infection (e.g. applied to patients in whom serology is used as the basis for 7 dengue diagnosis without a virologically-confirmed diagnosis). The 8 justification for this secondary analysis is based on expectation that a 9 dengue vaccine may reduce the viremia so making it more difficult to 10 detect in patients who may also have abbreviated clinical signs and 11 symptoms. Thus, serological secondary end-points may help assess 12 overall efficacy assuming that serological assays are equally sensitive and 13 specific to DENV (but not to individual serotypes) in detecting dengue 14 infection in vaccine and control groups; or 15

— The effect of vaccination on the duration of hospitalization and/or need 16 for specific interventions to manage the clinical illness. 17

18 If more than one study of protective efficacy is performed with a single candidate vaccine 19 (e.g. perhaps covering different geographical regions) using the same or a very similar 20 study protocol, it may be appropriate to pre-define a pooled analysis of the data. This 21 pooled analysis could provide additional insight into serotype-specific VE and the risk of 22 severe DFI in vaccine and control groups. 23

24 Each study should have in place a data and safety monitoring board (DSMB) consisting 25 of persons with no involvement in study conduct and analysis and including a statistician. 26 The DSMB charter should enable it to unblind treatment assignments as necessary and to 27 recommend that enrolment is halted or the study is terminated based on pre-defined 28 criteria designed to protect subjects from harm. In addition, studies may include one or 29 more planned interim analyses with pre-defined stopping rules. 30

31

C.3.3.5 Case definitions 32

The case definitions for the primary and for the various secondary analyses, with details 33 of the criteria to be met, should be defined in the protocol and should be in accordance 34 with the latest WHO recommendations (WHO/HTM/NTD/DEN/2009.1) [70]. 35 36

Clinical diagnosis 37

The most commonly diagnosed form of clinically apparent dengue virus infection is 38 characterized by the sudden onset of fever lasting at least two and up to seven days. Fever 39 is commonly accompanied by severe headache, pain behind the eyes, gastrointestinal 40

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symptoms, muscle, joint and bone pain and a rash. These cases are usually self-limiting 1 and result in complete recovery. 2 3 For the purposes of classification of cases it is important to characterize the severity of 4 each DFI. The criteria used to assess severity should be those described by WHO that are 5 current when the protocol is finalized (WHO/HTM/NTD/DEN/2009.1 and updates of this 6 guidance) [70]. These criteria should be used to determine the features of DFI that are 7 captured in the case report form. 8 9

Virological diagnosis 10

All methods used for the virological component of the case definition should be fully 11 validated. Virological confirmation of the clinical diagnosis can be based on direct 12 detection of dengue viremia by isolation. However, the use of alternative virological 13 methods to confirm the diagnosis (e.g. detection of NS1 to demonstrate the presence of 14 DENV and the use of reverse transcription-polymerase chain reaction (RT-PCR) to 15 determine the serotype) is acceptable. The standardization of viral diagnostic methods is 16 encouraged. Every effort should be made to conduct testing in one or a small number of 17 designated central laboratories with appropriate expertise. 18

19 Obtaining specimens to attempt virological confirmation of the diagnosis should be 20 triggered by a set of clinical features laid down in the study protocol that aim to identify 21 all potential cases of DFI of any severity and as early as possible, taking into account the 22 observation that virological diagnostic methods (including virus isolation and PCR-based 23 assays) are more sensitive during the first five days of infection. 24

Serological diagnosis 25

There is at least a theoretical possibility of a reduced likelihood of obtaining virological 26 confirmation of the clinical diagnosis in vaccinated subjects due to levels of viremia that 27 are below detection limits. If this were to occur in a significant proportion of clinical 28 cases there could be a bias in numbers of virologically-confirmed DFI in favour of the 29 vaccinated group. 30 31 Commercial and/or in-house serological assays (e.g. enzyme immunoassay [EIA], 32 immunofluorescence and virus neutralization tests) may be performed on paired acute 33 and convalescent sera. The results may be used to identify possible cases of DFI in which 34 a virological diagnosis was not confirmed and the numbers may be compared between 35 vaccinated and control groups in an additional secondary analysis of vaccine efficacy. 36 37 For example, using the IgM capture EIA an acute primary infection may be implied from 38 a rise in IgM levels during the first two weeks post-infection. The results need to be 39 interpreted with some caution because the IgM response to acute infection may be 40 blunted in vaccinated subjects and in those infected previously by wildtype DENV or by 41 another flavivirus. Depending on the timing of the illness, the results may also be 42 confounded by the fact that IgM and IgG responses may reflect recent dengue vaccination 43

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rather than acute infection with wild type dengue. In this regard, the ratio of IgM and IgG 1 may assist in the differentiation of primary and secondary infections. 2 3 The interpretation of serological data is also complicated by cross-reacting antibody 4 among flaviviruses. In those instances where cross-reaction with other flaviviruses does 5 not occur, a four-fold or greater rise in dengue neutralizing antibodies makes it possible 6 to attribute recent infection to a dengue virus presumptively, but not definitively. 7

8

C.3.3.6 Case detection and description 9

It is essential that there is adequate surveillance to detect any possible case of DFI as 10 early as possible to optimize the chances of virological confirmation of the diagnosis. The 11 surveillance mechanisms (e.g. including arrangements for periodic home visits or 12 telephone calls and involvement of hospitals serving the study catchment areas) should be 13 tested before the study is initiated at each study site. It is essential that study subjects are 14 educated regarding the need to contact or directly present to the designated study 15 healthcare facilities whenever they develop signs or symptoms that may be indicative of 16 DFI. A check list of these signs and symptoms should be provided to all study 17 participants at the time of enrolment. 18 19 In addition, measures should be in place to follow each possible case of DFI for any 20 change in disease course (e.g. progression from mild to severe DFI, onset of 21 complications) and to document the outcome, including the time to recovery or death. In 22 case of death before collection of specimens or in the absence of virological confirmation 23 of the diagnosis permission should be sought to perform needle puncture of the liver for 24 virological study and a post-mortem examination. 25

26 In some study sites that are otherwise considered suitable it may not be possible to 27 identify a local healthcare facility willing to participate in the study. These sites should 28 not be initiated unless there is at least agreement from local healthcare providers to notify 29 study staff of possible cases of DFI within a sufficient timeframe to allow for specimens 30 to be collected and transported for virological diagnosis. Subjects should carry a study 31 participant card with contact names and numbers to ensure that study personnel are 32 alerted and can arrange for the collection of all the necessary clinical data and the 33 transport of specimens for virological diagnosis. 34 35 Despite taking the steps described, there will still be some cases of possible DFI that are 36 not confirmed virologically and for which serological testing is inconclusive. In addition, 37 some subjects may not comply with the study requirement to present to a designated 38 healthcare facility when they have signs and symptoms indicative of a possible DFI or 39 they may be so ill that they are immediately admitted to a hospital not directly 40 participating in the study and/or may die without notification of study personnel in time 41 to collect data and specimens. It is important that as much information as possible is 42 collected on these cases whenever and however they come to light and that they are taken 43

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into account in a “worse-case scenario” analysis of vaccine efficacy that counts all cases 1 (proven and unproven and regardless of protocol deviations). 2

3

C.3.3.7 Need for additional studies of efficacy 4

Once the efficacy of at least one candidate dengue vaccine has been satisfactorily 5 demonstrated (and perhaps already licensed and introduced into the routine vaccination 6 program in at least one country) there will be a need to reassess the content of clinical 7 development programs for other candidate dengue vaccines. In particular, there will be a 8 need to consider the appropriateness and/or feasibility of conducting studies in which 9 subjects are randomized to a control (i.e. unvaccinated) group. 10

11 It is not currently possible to make a definitive recommendation regarding what could or 12 should be required in this scenario. Much will depend on the findings reported from the 13 first completed efficacy study of a candidate vaccine or from ongoing studies with other 14 candidate vaccines. 15 16 Some issues to be taken into account include the following: 17

18 i) Once a vaccine has been licensed and introduced into the routine 19 vaccination program in one or more countries the inclusion of an 20 unvaccinated group in subsequent studies in these (and possibly many 21 other) countries is likely to be considered unethical. In addition, studies 22 that include an unvaccinated group are not likely to be feasible in some 23 regions after approval of the first vaccine (with or without introduction 24 into the routine vaccination program) because subjects are unlikely to risk 25 assignment to an unvaccinated group. However, if there remains 26 considerable uncertainty about vaccine efficacy against specific DENV 27 serotype(s) then another study with an unvaccinated control group might 28 be possible in a region where such serotype(s) are predominant; 29 30 ii) The use of licensed dengue vaccine(s) in the routine vaccination program 31

in a country or region may reduce the incidence of DFI down to levels that 32 are too low to permit the estimation of VE from further studies of 33 reasonable and manageable size and duration; 34

35 iii) There should be a careful scientific evaluation of the arguments for and 36

against extrapolation of the efficacy observed for a particular vaccine to 37 other populations. For example, to other age groups, to subjects at highest 38 risk of dengue and severe dengue, to geographical areas with predominant 39 circulating DENV serotypes for which serotype-specific efficacy may not 40 have been established and to populations with high rates of immunity to 41 other flaviviruses; 42

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iv) It is very possible that a definitive immunological correlate of protection 1

cannot be established from pre-licensure protective efficacy studies. 2 Moreover, accumulating evidence suggests that a neutralizing antibody 3 titer that seems to correlate with protection against DFI due to one DENV 4 serotype may not predict protection against DFI caused by other dengue 5 serotypes. Therefore, there needs to be a discussion of the validity of using 6 immunogenicity data to bridge the efficacy of a dengue vaccine that was 7 demonstrated in one test population to other populations, with or without 8 identification of correlates of protection; 9

10 v) There also needs to be a discussion regarding the use of bridging studies to 11

support the extrapolation of efficacy observed with one vaccine to 12 subsequent candidate vaccines that may or may not be live dengue 13 vaccines. For reasons already discussed above, there may be no alternative 14 to the use of bridging studies because the following approaches are 15 unlikely to be feasible; 16

17 vi) To continue to request evaluation of subsequent candidate dengue 18

vaccines in pre-licensure protective efficacy studies that include an 19 unvaccinated group; 20

21 vii) To request a relative efficacy study i.e. that compares rates of febrile 22

dengue illness in groups assigned to either a licensed or candidate vaccine; 23 or 24

25 viii) After consideration of all these issues it is expected that at some future 26

time point, which will depend on the safety and efficacy that is observed 27 with the first or first few vaccines to be licensed, the inclusion of 28 unvaccinated control groups in efficacy studies will no longer be possible. 29 Once this time point is reached there will have to be recommendations 30 made regarding: 31

— Additional data to be obtained after initial approval of a vaccine for 32 which VE has been estimated based on numbers of laboratory-33 confirmed DFI in vaccinated versus unvaccinated groups. 34

— Data that should be generated before and after licensure of subsequent 35 candidate vaccines. 36

37 In both instances it is important that vaccines should be evaluated in post-38 approval studies of safety and effectiveness as discussed in section C.4. 39

40

C.3.3.8 Documentation of safety during pre-licensure studies 41

The routine monitoring of safety during all pre-licensure clinical studies should follow 42 the usual principles taking into account issues relevant to live attenuated vaccines. In 43

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addition to providing study-specific safety data there should be an analysis of safety data 1 pooled across all study groups that received the final selected vaccine formulation. 2 3 In particular, there is a need to assess whether DFI (which could be of any degree of 4 severity, including a very mild illness) may be caused by vaccine strains. In studies 5 conducted in non-endemic areas there is a risk that vaccine-associated DFI, which 6 would be expected to occur only within about 30 days post-dose, could be confused with 7 an intercurrent infection while in studies in endemic areas there is a need to distinguish 8 vaccine-associated from naturally-occurring DFI. Therefore in all cases it is essential 9 that laboratory studies should be undertaken to determine whether or not the clinical 10 picture is associated with vaccine virus viremia. 11 12 The safety considerations also include the possible risk that vaccination could 13 predispose recipients to develop a severe form of DFI. The monitoring and investigation 14 of all subjects who develop signs or symptoms potentially indicative of DFI during pre-15 licensure studies in endemic regions should provide a preliminary assessment of this 16 risk. If no undue risk is identified and the vaccine is licensed it is essential that there is 17 adequate follow-up of study subjects together with further assessment of the risk in the 18 post-licensure period (see section C.4). 19 20

The total safety database derived from all pre-licensure studies should be sufficient to 21 describe uncommon adverse reactions. Based on considerations outlined in section 22 C.3.3.7, it may be that vaccines that are developed subsequent to the approval of the first 23 vaccine(s) will not be evaluated in pre-licensure studies of protective efficacy, in which 24 case the pre-licensure safety database would usually be much smaller. In these instances 25 the minimum acceptable safety database should be determined according to the nature of 26 the vaccine and in the light of experience amassed with other similar vaccines. 27

28

C.4 Post-licensure investigations 29

As with all medicinal products, including all vaccines, there is a need to ensure that 30 adequate surveillance is in place and is maintained to detect adverse reactions during the 31 post-licensure period in accordance with requirements of the countries in which approval 32 has been obtained. 33 34 The need for, design and extent of specific studies of safety and/or effectiveness 35 following approval of a dengue vaccine should be given careful consideration by 36 sponsors and national regulatory authorities. However, at least in the case of the first 37 dengue vaccines that reach the market there will be a clear need for well-designed studies, 38 which should include long-term extension periods of pre-licensure efficacy studies as 39 well as newly-initiated studies, to collect information at least on the following: 40

41

— Long-term evaluation of breakthrough cases of DFI to detect any waning 42 of protective immunity against one or more DENV serotypes and the 43 possible need for booster doses; 44

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1

— Antibody persistence; 2 3

— Responses to booster doses, which may be planned for pre-defined subsets 4 enrolled into studies or may be instituted as/when disease surveillance 5 indicates a possible need; 6

7

— The possible increased risk of severe DFI in vaccine recipients; and 8 9

— Vaccine effectiveness 10 11 Depending on the countries/regions in which any one licensed vaccine is introduced into 12 routine vaccination programs it is desirable that attempts should be made to estimate 13 DENV serotype-specific effectiveness and effectiveness in populations that were not 14 included in pre-licensure studies. There are several possible study designs and methods 15 for estimating vaccine effectiveness and it is essential that expert advice is sought. In 16 addition, it is likely that such studies would need to be performed in close liaison with 17 Public Health Authorities. 18 19 Sponsors and national regulatory authorities should consider the need to assess immune 20 interference and safety on co-administration of a dengue vaccine with other vaccines that 21 may be given at the same health care visits for convenience. In addition, if licensure is 22 sought in non-endemic areas with the intent to protect travellers to endemic areas, 23 sponsors and national regulatory authorities may consider it necessary to obtain 24 additional safety and immunogenicity data with the final vaccine and schedule in 25 flavivirus-naïve subjects. 26 27 Some or all post-licensure studies may be conducted as post-approval commitments made 28 to individual national regulatory authorities. In this regard, both sponsors and national 29 regulatory authorities that have approved a vaccine should communicate and co-operate 30 to ensure that studies are well-designed to answer the questions posed and to avoid 31 demands for numerous studies in individual countries that are likely to be too small to 32 provide reliable results. Provisional plans for appropriate post-licensure studies should be 33 submitted with the application dossier and these should be refined during the assessment 34 of the national regulatory authority and as necessary after initial approval. 35 36

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Part D. Environmental risk assessment of dengue tetravalent 1

vaccines (live, attenuated) derived by recombinant DNA 2

technology 3

D.1 Introduction 4

D.1.1 Scope 5

Some countries have legislation covering the environmental and other issues related to 6 the use of live vaccines derived by recombinant DNA technology since they may be 7 considered genetically modified organisms (GMO) including vaccines. Similar issues 8 may be raised by live vaccines derived by other methods. 9 10 This section of the guideline considers the environmental risk assessment (ERA) that may 11 be performed during DENV vaccine development. The ERA assesses the risk to public 12 health and the global environment. It does not assess the risk to the intended recipient of 13 the vaccine; this is assessed through clinical studies of the vaccine. Nor does it assess the 14 risk to laboratory workers. 15

16 The environmental impact is not usually the responsibility of the national regulatory 17 authority but of other agencies. Nonetheless the national regulatory authority should 18 receive a copy of the ERA and any associated decisions taken for information and to 19 ensure that the appropriate procedures have been followed. 20

21

D.1.2 Principles and objectives 22

Live DENV vaccines in which the genetic material has been genetically modified by 23 recombinant DNA technology, should be considered to be GMO. The manufacture, use 24 and transboundary shipping of such live recombinant vaccines, for research or 25 commercial use, should comply, when applicable, with any relevant legislation or 26 regulations of the producing and recipient countries regarding GMOs. In some regulatory 27 regimes, in order to comply with environmental regulations, an ERA should be 28 undertaken if the live vaccine is being tested in a clinical trial or if it is placed on the 29 market. It should be noted that this guidance on the ERA of live recombinant DENV 30 vaccines does not intend to replace existing GMO legislation when already in place in 31 certain countries. 32 33 Generally, the objective of an ERA is to identify and evaluate, on a case-by-case basis, 34 potential adverse effects of a GMO on public health and the environment, direct or 35 indirect, immediate or delayed. This means that for each different live recombinant 36 dengue vaccine a case-by-case ERA should be performed. Direct effects refer to primary 37 effects on human health or on the environment which is a result of the GMO itself and 38 which occur through a short causal chain of events. Indirect effects refers to effects 39 occurring through a more extended causal chain of events, through mechanisms such as 40

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interactions with other organisms, transfer of genetic material, or changes in use or 1 management. Immediate effects are observed during the period of the release of the GMO, 2 whereas delayed effects refer to effects which may not be observed during the period of 3 release of the GMO, but become apparent as a direct or indirect effect either at a later 4 stage or after termination of the release. 5 6 The ERA should be performed in a scientifically sound and transparent manner based on 7 available scientific and technical data. Important aspects to be addressed in an ERA 8 include the characteristics of: (a) the parental organism, (b) the recipient organism, (c) 9 viral vector characteristics, (d) the donor sequence, (e) genetic modification, (f) the 10 intended use and (g) the receiving environment. The data needed to evaluate the ERA do 11 not have to derive solely from experiments performed by the applicant; data available in 12 the scientific literature can also be used in the assessment. Regardless of the source of the 13 data, it should be both relevant and of an acceptable scientific quality. The ERA could be 14 based on data of experiments previously performed for other purposes, such as product 15 characterization tests, toxicity and nonclinical safety studies. 16 17 Ideally, the ERA is based on quantitative data and expressed in quantitative terms. 18 However, much of the information that is available for an ERA may be qualitative for the 19 reason that quantification is often hard to accomplish, and may not be necessary to make 20 a decision. The level of detail and information required in the ERA is also likely to vary 21 according to the nature and the scale of the proposed release. Information requirements 22 may differ between licensure and clinical development and whether studies will be 23 carried out in a single country or multiple countries. 24

25 Uncertainty is inherent in the concept of risk. Therefore, it is important to identify and 26 analyse areas of uncertainty in the risk assessment. Since there is no universally accepted 27 approach for addressing uncertainty, risk management strategies may be considered. 28 Precise data on the environmental fate of the live vaccine in early clinical trials will in 29 most cases be insufficient or lacking. However, at the market registration stage, the level 30 of uncertainty is expected to be lower as identified gaps in available data should already 31 have been addressed. 32 33 The need for risk management measures should be based upon the estimated level of risk. 34 If new information on the GMO becomes available the ERA may need to be re-35 performed to determine whether the estimated level of risk has changed. This also holds 36 true if the risks for the participating subjects have changed as these aspects can be 37 translated to other individuals. It should be noted that the ERA will not deal with medical 38 benefit for the subject or scientific issues such as proof-of-principle. 39

40

D.2 Procedure for environmental risk assessment 41

Risk assessment involves identification of novel characteristics of the GMO that may 42 have adverse effects (hazard), evaluating the consequences of each potential adverse 43 effect, estimation of the likelihood of adverse effects occurring, risk estimation, risk 44

WHO/BS/11.2159

management and, in some methodologies, estimation of the overall risk for the 1 environment. These processes should identify the potential adverse effects by comparing 2 the properties of the GMO with non-modified organisms under the same conditions, in 3 the same receiving environment. The principles and methodology of an ERA should be 4 applicable irrespective of the geographic location of the intended environmental release 5 of the GMO. Although the ERA should take into account the specificities associated with 6 the mosquito vector being endemic or non-endemic in the region in which vaccine trials 7 will be carried out, and/or where licensure is being requested. Depending upon local 8 regulatory requirements, the ERA may be undertaken by the applicant or by the local 9 competent authority on the basis of data supplied. In all cases, the local competent 10 authority should use the ERA as a basis for deciding whether any identified 11 environmental risks are acceptable. However, the decision on whether any identified 12 risks are acceptable may vary from country to country. 13 14 There are several national and multinational documents addressing ERA issues, such as: 15

— EU Directive 2001/18/EC [14] (Annex II) and Commission Decision 16 2002/623/EC [15]; 17

— Risk Analysis Framework of the Australian Office of the Gene 18 Technology Regulator [40]; 19

— New Substances Notification Regulations of Canadian Environmental 20 Protection Act, 1999 [13]; 21

— Recombinant DNA Safety Guidelines of Genetic Engineering Approval 22 Committee in India; and 23

— Normative Resolution No.5 (12 March 2008) comprising provisions on 24 rules for commercial release of GMOs and their derivatives in Brazil. 25

26 As an example, a detailed description of a six-step ERA module of EU Directive 27 2001/18/EC is described in the Appendix 3 of these Guidelines. 28

29

D.3 Special considerations for live recombinant dengue 30 vaccines 31

32 The ERA of live recombinant DENV vaccines should be conducted according to the 33 general principles described above taking into consideration in particular the vector 34 responsible for disease transmission. The following aspects which could be developed 35 include: the genetic stability of the live recombinant virus including reversion and 36 recombination, potential transmission of vaccine virus among hosts by the vector and the 37 immune status of the population, and these are further outlined below. 38 39

WHO/BS/11.2159

D.3.1 Genetic stability 1

DENV vaccines currently under clinical evaluation are attenuated DENV strains, 2 intertypic chimeric vaccines or DENV/YF-17D vaccine chimeras. In the intertypic 3 approach, the structural genes of an attenuated strain of DENV of a given serotype are 4 replaced by the corresponding genes of a different DENV serotype. In the dengue/yellow 5 fever chimeras, the prM/E structural genes of the dengue virus are cloned into the 6 backbone of the YF-17D vaccine, in replacement of the corresponding structural YF-17D 7 genes. 8

9

D.3.1.1 Reversion 10

After vaccination, there is the potential of reversion of attenuated live dengue virus 11 vaccines to a virulent form of the dengue virus, although this has not been seen in clinical 12 trials so far. The potential reversion is based on the stability of the attenuating mutation(s), 13 the number of attenuating mutations, and the nature of attenuating mutation. Attenuating 14 mutations that are dependent upon a single base change may be more susceptible to 15 reversion than a mutation that is stabilized by multiple base substitutions. In addition, 16 attenuating mutations that are derived by deletions of segments of RNA are generally 17 more stable against reversion. Changes in virus genotype have the potential to influence 18 disease transmission, tropism of vector vaccine, virulence, and/or patterns of disease, 19 resulting in a virus with a previously unknown combination of properties. However, the 20 likelihood of such a reversion depends on the number of attenuation mutations present 21 and the viral genes involved in the vaccine virus [9] 22

23

D.3.1.2 Recombination 24

Whether or not recombination takes place among flaviviruses is controversial. 25 Theoretically, recombination between live DENV vaccines and wild type flaviviruses 26 could produce a virus with an altered phenotype, but currently there is no evidence to 27 support this [24, 34, 35, 37, 41, 46, 48] . 28 29 The potential for recombination issues within and between flaviviruses has been widely 30 discussed and challenged in the past years, based on existing literature [24, 34, 35, 37, 41, 31 48], and also from data obtained in specific experiments. In particular, a "recombination 32 trap", has been recently designed to allow the products of rare recombination events to be 33 selected and amplified, in the case of West Nile (WN), tick-borne encephalitis (TBE) and 34 Japanese encephalitis (JE) viruses [48]. Intergenomic but aberrant recombination was 35 observed only in the case of JE virus, and not for WN or TBE viruses. Moreover, its 36 frequency appeared to be very low and generated viruses with impaired growth properties. 37 38 While their likelihood of appearance is very low, as stated above, the potential adverse 39 effects of recombined DENVs should be evaluated in the ERA. In this respect, "worst-40 case" scenario chimeras have been constructed to address that risk [34, 35, 41]. 41

WHO/BS/11.2159

1 These different studies showed that such recombinants constructed artificially from a 2 wild type flavivirus and a chimeric vaccine [41], or from two wild type viruses, such as 3 highly virulent YF Asibi virus and wild type DEN4 virus [35], were highly attenuated 4 compared to their parental viruses. Attenuation was shown in culture in vitro, in mosquito 5 vectors and in susceptible animal models, including monkeys. These data provide 6 experimental evidence that the potential of recombinants, should they ever emerge, to 7 cause disease or spread would probably be very low. Dual infection laboratory studies 8 between vaccine and wildtype strains are not recommended because the predictive 9 clinical value of such studies would be low. 10

11

D.3.2 Vector transmission 12

The presence of the DENV vectors such as Ae. aegypti and Ae. albopictus play a key role 13 in the transmission of flaviviruses and potentially live DENV vaccines from the 14 vaccinated subject to other individuals. Dengue does not usually spread directly from 15 person to person, except via blood transfusion. Transmission of the dengue vaccine in 16 regions in which the vector is absent is therefore highly unlikely. Dengue is currently 17 restricted to the tropical regions. Due to climate change there is an increasing incidence 18 of the detection of mosquito species not normally found in regions with traditionally 19 milder climates. This could lead to the introduction of dengue in these areas and 20 potentially change disease transmission and population immune status. 21 22 Recombination between live DENV vaccines and wild type flaviviruses could 23 theoretically occur in the vaccinee (see above), but possibly also within an infected 24 mosquito although neither has been reported. A recombined DENV could potentially, for 25 instance in combination with climate change, use new vectors for transmission leading to 26 previously unknown transmission characteristics. Therefore, the presence of a relevant 27 mosquito vector and a ‘dengue favorable climate’ in the vaccination region should be 28 taken into account in the ERA of live DENV vaccines. 29 30 To assess the likelihood of effective transmission of the vaccine from a vaccinated 31 individual, one has to take into consideration two parameters: the level of viremia in the 32 vaccinated hosts, and the ability of the mosquito vectors to transmit the live DENV 33 vaccine to new hosts. The blood titer required for effective transmission is difficult to 34 define. If the candidate vaccine strains replicate at low levels (1-2 logs) then possible 35 initiation of the transmission cycle (mosquito infection) is reduced. For instance, in the 36 case of the YFV17D chimeric vaccine, viremia in the hosts after tetravalent vaccination is 37 very low [39]. In case the viremia titer is high enough and virus is intrinsically incubated 38 in the mosquito, then the ability to transmit the virus to other hosts will be critical. For the 39 recombinant live attenuated DENV vaccines currently in clinical trial, the likelihood of 40 occurrence appears to be low to negligible. These viruses replicate to titers thousands-41 fold lower than that which results in transmission to mosquitoes. In addition, the ability 42 of these viruses to infect, replicate, and escape the midgut is impaired [4, 6, 22, 28, 50, 53, 43 54]. 44 45

WHO/BS/11.2159

The outcome of the ERA for clinical trials in regions in which the vector is absent, is that 1 the environmental risk is negligibly small. The mosquito vector is not present and 2 therefore the vaccine or possible created recombinants cannot be transmitted to other 3 people. However, in endemic areas, it is important to perform an ERA as the vector is 4 present, thus there is a possibility of transmission. 5

D.3.3 Immune status 6

Live DENV vaccines are able to replicate and form new viral particles in vaccinated 7 persons. The immune status of the vaccinee and the rest of the population could influence 8 the environmental risk of a live DENV vaccine. In general, the presence of pre-existing 9 immunity due to earlier exposure to DENVs will reduce the extent and duration of 10 vaccine virus replication and biodistribution. The potential for transmission of the 11 vaccine is therefore considered to be greater in naïve or immunocompromised individuals. 12

13 An unvaccinated population with no pre-existing immunity will respond differently upon 14 exposure to the vaccine compared to a population in which dengue is endemic. The 15 immune status should therefore be taken into account in the ERA as it can influence the 16 environmental impact of the vaccines, and the potential occurrence of adverse effects in 17 contacts of the vaccinees. There is a potential for pre-existing heterotypic antibody to 18 cause higher levels of vaccine virus. Enhanced illness in vaccine recipients who have 19 pre-existing DENV antibody (antibody-dependent enhancement) has not been observed 20 in clinical trials of live attenuated dengue vaccines to date and was not observed in a 21 clinical trial of live attenuated dengue vaccines designed to address this possibility [10, 22 20]. 23

Part E. Guidelines for national regulatory authorities 1

E.1 General 2

The general recommendations for national regulatory authorities and national control 3 laboratories given in the Guidelines for national authorities on quality assurance for 4 biological products [59] should apply. In addition, the general recommendations for 5 national regulatory authorities and national control laboratories provided in the 6 Guidelines for independent lot release of vaccines by regulatory authorities [72] should 7 be followed These Guidelines specify that no new biological substance should be 8 released until consistency of manufacturing and quality as demonstrated by a consistent 9 release of batches has been established. The detailed production and control procedures 10 and any significant changes in them should be discussed with and approved by the 11 national regulatory authority. The national regulatory authority should obtain the working 12 reference from manufacturers to establish a national working reference preparation until 13 an international reference reagent is available. 14 15

E.2 Release and certification 16

A vaccine lot should be released only if it fulfils the national requirements and/or Part A 17 of the present Guidelines. A protocol based on the model given in Appendix 1, signed by 18 the responsible official of the manufacturing establishment, should be prepared and 19 submitted to the national regulatory authority in support of a request for release of 20 vaccine for use. 21

22 A statement signed by the appropriate official of the national regulatory authority should 23 be provided if requested by a manufacturing establishment and should certify whether or 24 not the lot of vaccine in question meets all national requirements, as well as Part A of 25 these Guidelines. The certificate should also state the lot number, the number under 26 which the lot was released, and the number appearing on the labels of the containers. In 27 addition, the date of the last satisfactory determination of antigen concentration as well as 28 assigned expiry date on the basis of shelf life should be stated. A copy of the official 29 national release document should be attached. The certificate should be based on the 30 model given in Appendix 2. The purpose of the certificate is to facilitate the exchange of 31 dengue virus vaccines between countries. 32

33

Authors 1

2 The scientific basis for the revision of the Guidelines for the production and quality 3 control of candidate tetravalent dengue virus vaccines (live) published in WHO TRS no. 4 932 was discussed at the meeting of the WHO working group on technical specifications 5 for manufacture and evaluation of dengue vaccines, Geneva, Switzerland, 11–12 May 6 2009 attended by the following people: Professor Alan Barrett, University of Texas 7 Medical Branch (UTMB), Galveston, Texas, USA; Dr Diederik Bleijs, National Institute 8 for Public Health and the Environment (RIVM), Bilthoven, Netherlands; Dr Françoise 9 Denamur, GlaxoSmithKline Biologicals, Rixensart, Belgium; Dr Anna Durbin, Johns 10 Hopkins Bloomberg School of Public Health (JHSPH), Baltimore, Maryland, USA; Dr 11 Kenneth Eckels, Walter Reed Army Institute of Research (WRAIR), Silver Spring, 12 Maryland, USA; Professor Robert Edelman, University of Maryland School of Medicine 13 (UMSM), Baltimore, Maryland, USA; Dr Donald Francis, Global Solutions for 14 Infectious Diseases, South San Francisco, California, USA; Dr Marcos Freire, Instituto 15 Oswaldo Cruz, Manguinhos, Rio de Janeiro, Brazil; Dr Joachim Hombach, WHO, 16 Geneva, Switzerland; Dr Houda Langar, WHO Office for the Eastern Mediterranean, 17 Cairo, Egypt; Dr Ivana Knezevic, WHO, Geneva, Switzerland; Dr Corinne Lecomte, 18 GlaxoSmithKline Biologicals, Wavre, Belgium; Dr Laurent Mallet, Sanofi Pasteur, 19 Marcy l'Étoile, France; Dr Philip Minor, National Institute of Biological Standards and 20 Control (NIBSC), Potters Bar, Hertfordshire, UK (Chair); Dr Harold Margolis, 21 International Vaccine Institute, SNU Research Park, Seoul, Republic of Korea; Dr 22 Lewis Markoff, Center for Biologics Evaluation and Research (CBER), Food and Drug 23 Administration (FDA), Bethesda, Maryland, USA; Dr Sérgio Nishioka, WHO, Geneva, 24 Switzerland; Dr Keith Peden, CBER, FDA, Bethesda, Maryland, USA; Dr Mair Powell, 25 Medicines and Healthcare Products Regulatory Agency (MHRA), London, UK; Dr 26 James Robertson, NIBSC, Potters Bar, Hertfordshire, UK; Dr John Roehrig, Centers for 27 Disease Control and Prevention (CDC), Fort Collins, Collorado, USA; Dr Alain 28 Sabouraud, Sanofi Pasteur, Marcy l'Étoile, France; Dr Jinho Shin, WHO, Geneva, 29 Switzerland; Mrs Prapassorn Thanaphollert, Food and Drug Administration, Ministry of 30 Public Health, Nonthaburi, Thailand; Dr Dennis Trent, UTMB, Texas, USA 31 (Rapporteur); Dr David Wood, WHO, Geneva, Switzerland. 32 33 The first draft of these Guidelines was developed by the following lead authors for the 34 part indicated: (1) Part A - Dr Laurent Mallet, Sanofi Pasteur, Canada and Dr Philip 35 Minor, NIBSC, Potters Bar, Hertfordshire, UK; (2) Part B - Dr Dennis Trent, UTMB, 36 Galveston, Texas, USA; (3) Part C - Dr Mair Powell, MHRA, London, UK; (4) Part D - 37 Dr Diederik Bleijs, RIVM, Bilthoven, The Netherlands and Dr James Robertson, NIBSC, 38 Potters Bar, UK. 39 40 The first draft was discussed in the meeting of the working group held on 29-30 April 41 2010, Geneva, Switzerland attended by: Professor Alan Barrett, UTMB, Galveston, 42 Texas, USA; Dr Diederik Bleijs, RIVM, Bilthoven, Netherlands; Dr Françoise 43 Denamur, GSK Biologicals, Rixensart, Belgium; Dr Anna Durbin, JHSPH, Baltimore, 44 Maryland, USA; Dr Kenneth Eckels, WRAIR, Silver Spring, Maryland, USA; 45

WHO/BS/11.2159

Professor Robert Edelman, UMSM, Baltimore, Maryland, USA; Dr Donald Francis, 1 Global Solutions for Infectious Diseases, South San Francisco, California, USA; Dr 2 Marcos Freire, Instituto Oswaldo Cruz, Manguinhos, Rio de Janeiro, Brazil; Dr Neuza 3 Gallina, Buntan, Sao Paulo, Brasil; Mr Marcos Galves, National Agency of Health 4 Surveillance (ANVISA), Brasilia-DF, Brazil; Mrs Fabienne Garnier, French Agency for 5 Safety of Health Products (AFSSAPS), Lyon, France; Dr Joachim Hombach, WHO, 6 Geneva, Switzerland; Dr Ivana Knezevic, WHO, Geneva, Switzerland; Dr Laurent Mallet, 7 Sanofi Pasteur, Canada; Dr Philip Minor, NIBSC, Potters Bar, Hertfordshire, UK (Chair); 8 Dr Lynn Morgan, Sanofi Pasteur, Marcy l'Étoile, France; Van Phung Le, National 9 Institute for Control of Vaccine and Biologicals, Hanoi, Viet Nam; Dr Lewis Markoff, 10 CBER, FDA, Bethesda, Maryland, USA (Rapporteur for clinical breakgroup); Dr 11 Jehanara Korimbocus, AFSSAPS, Lyon, France; Dr Sérgio Nishioka, WHO, Geneva, 12 Switzerland; Dr Keith Peden, CBER, FDA, Bethesda, Maryland, USA (Rapporteur for 13 manufacture, nonclinical and environmental risk assessment breakgroup); Dr Mair 14 Powell, MHRA, London, UK; Dr Vincent Quivy, GSK Biologicals, Wavre, Belgium; 15 Dr John Roehrig, CDC, Fort Collins, Collorado, USA; Ms Melanie Saville, Sanofi 16 Pasteur, Marcy l'Étoile, France; Mrs Prapassorn Thanaphollert, Thai FDA, Ministry of 17 Public Health, Nonthaburi, Thailand; Dr Cynthia Thomson, Inviragen, Capricorn, 18 Singapore; Dr Dennis Trent, UTMB, Galveston, Texas, USA; Dr Jan-Willem van der 19 Laan, RIVM, Bilthoven, The Netherlands; and Dr David Wood, WHO, Geneva, 20 Switzerland. 21 22 Taking into account comments from the April 2010, a second draft was written by Dr 23 Laurent Mallet, Sanofi Pasteur, Dr Dennis Trent, UTMB, Dr Mair Powell, MHRA, 24 Dr Diederik Bleijs, RIVM. A modified draft of the Part D of the Guidelines was further 25 developed by Dr Diederik Bleijs, RIVM, based on comments from teleconferences 26 held in January and February 2011 with an informal workgroup on environmental risk 27 assessment for dengue vaccines in which additional members consist of Dr Michael 28 Dornbusch, Office of the Gene Technology Regulator, Department of Health and Aging, 29 Canberra, Australia; Dr Anna Durbin, JHSPH ; Dr Kenneth Eckels, WRAIR; Professor 30 Robert Edelman, UMSM; Dr Lynn Morgan, Sanofi Pasteur; Dr James Robertson, 31 NIBSC; Dr Jinho Shin, WHO; and Dr Vincent Quivy, GSK. 32 33 The third draft was prepared by Dr Jinho Shin, WHO, Geneva, Switzerland. 34 35 The fourth draft of was prepared by Professor Alan Barrett, UTMB, Dr Philip Minor, 36 NIBSC, Dr Dennis Trent, UTMB, Dr Mair Powell, MHRA, Dr Diederik Bleijs, RIVM 37 and Dr Jinho Shin, WHO taking into account suggestions for modification and comments 38 by the participants of the informal consultation held on 11-12 April 2011, Geneva, 39 Switzerland attended by: Dr Beatrice Barrere, Sanofi Pasteur, Marcy l'Étoile, France; 40 Professor Alan Barrett, UTMB, Galveston, Texas, USA; Dr Diederik Bleijs, RIVM, 41 Bilthoven, Netherlands; Dr Anil Chawla, Greater Noida, UP, India; Dr Kurt Dobbelaere, 42 GSK, Rixensart, Belgium; Dr Michael Dornbusch, Office of the Gene Technology 43 Regulator, Canberra, Australia; Dr Anna Durbin, JHSPH, Baltimore, Maryland, USA; Dr 44 Kenneth Eckels, WRAIR, Silver Spring, Maryland, USA; Professor Robert Edelman, 45 UMSM, Baltimore, Maryland, USA; Mr Marcos Galves, ANVISA, Brasilia-DF, Brazil; 46

WHO/BS/11.2159

Dr Elwyn Griffiths, Biologics and Genetic Therapies Directorate (BGTD), Health Canada, 1 Ontario, Canada; Dr Joachim Hombach, WHO, Geneva, Switzerland; Dr Ivana Knezevic, 2 WHO, Geneva, Switzerland; Dr Jan-Willem van der Laan, RIVM, Bilthoven, The 3 Netherlands; Dr Laurent Mallet, Sanofi Pasteur, Ontario, Canada; Dr Lewis Markoff, 4 CBER, FDA, Bethesda, Maryland, USA; Dr Philip Minor, NIBSC, Potters Bar, 5 Hertfordshire, UK (Chair); Dr Lynn Morgan, Sanofi Pasteur, Marcy l'Étoile, France; Dr 6 Jehanara Korimbocus, AFSSAPS, Lyon, France; Dr Mair Powell, MHRA, London, UK; 7 Dr Alexander Precioso, Butantan, Sao Paulo, Brazil; Dr Vincent Quivy, GSK Biologicals, 8 Wavre, Belgium; Dr John Roehrig, CDC, Fort Collins, Collorado, USA; Dr Julia Schmitz, 9 WHO, Geneva, Switzerland; Mrs Prapassorn Thanaphollert, Thai FDA, Ministry of 10 Public Health, Nonthaburi, Thailand; and Dr Dennis Trent, UTMB, Galveston, Texas, 11 USA; and Dr Simonetta Viviani, Sanofi Pasteur, Marcy l'Étoile, France. 12 13

Acknowledgements 14

15

Acknowledgements are also due to the following experts for their useful comments: Dr 16 Mohammed Alali, Therapeutic Goods Administration, Australian Capital Territory, 17 Australia; Dr Ryoko Krause, Vaccines and Biologicals, International Federation of 18 Pharmaceutical Manufacturers & Associations (IFPMA), Geneva, Switzerland; Dr Sylvie 19 Morgeaux, AFSSAPS, Lyon, France; Dr Frédéric Mortiaux, GSK Biologicals, Rixensart, 20 Belgium; Dr Supaporn Phumiamorn, Ministry of Public Health, Nonthaburi, Thailand; Dr 21 Lucky Slamet, National Agency of Drug and Food Control Republic of Indonesia, Jakarta 22 Pusat, Indonesia; Dr Stephen Whitehead, NIAID, Bethesda, MD, USA. 23

24

Appendix 1: Model summary protocol for manufacturing and 1

control of dengue tetravalent vaccines (live, attenuated) 2

3 The following protocol is intended for guidance, and indicates the information that should 4 be provided as a minimum by the manufacturer to the national regulatory authority. 5 Information and tests may be added or deleted if necessary to be in line with the 6 marketing authorization approved by the national regulatory authority. It is thus possible 7 that a protocol for a specific product may differ in detail from the model provided. The 8 essential point is that all relevant details demonstrating compliance with the license and 9 with the relevant WHO guidelines of a particular product should be given in the protocol 10 submitted. The section concerning the final product should be accompanied by a sample 11 of the label and a copy of the leaflet that accompanies the vaccine container. If the 12 protocol is being submitted in support of a request to permit importation, it should also be 13 accompanied by a lot release certificate from the national regulatory authority of the 14 country in which the vaccine was produced and /or released stating that the product meets 15 national requirements as well as Part A guidelines of this document published by WHO. 16 17

1. Summary information on finished product (final vaccine lot) 18

International name: ____________________________ 19 Commercial name: ____________________________ 20 Product license (marketing authorization) 21

number : ____________________________ 22 Country: ____________________________ 23 24 Name and address of manufacturer: ____________________________ 25 Name and address of product license holder 26

if different: 27 Virus strains: ____________________________ 28 Origin and short history: ____________________________ 29 Batch number(s): ____________________________ 30 Finished product (final lot): ____________________________ 31 Final bulk: ____________________________ 32 Type of container: ____________________________ 33 Number of filled containers in this final lot: ____________________________ 34 Number of doses per container: ____________________________ 35 Composition (antigen concentration) / 36

volume of single human dose: ____________________________ 37 Target group: ____________________________ 38 Expiry date: ____________________________ 39 Storage conditions: ____________________________ 40

41

WHO/BS/11.2159

2. Summary Information on manufacture 1

Batch number of each monovalent bulk: ____________________________ 2 Site of manufacture of each monovalent 3

bulk: ____________________________ 4 Date of manufacture of each monovalent 5

bulk: ____________________________ 6 Batch number of final bulk: ____________________________ 7 Site of manufacture of final bulk: ____________________________ 8 Date of manufacture of final bulk: ____________________________ 9 Date of manufacture (filling or 10

lyophilizing) of finished product (final 11 vaccine lot): ____________________________ 12

Date on which last determination of virus 13 concentration was started: ____________________________ 14

Shelf-life approved (months): ____________________________ 15 Storage conditions : ____________________________ 16 Volume of single dose : ____________________________ 17 Prescribed virus concentration per human 18

dose: 19 Serotype 1: ____________________________ 20 Serotype 2: ____________________________ 21 Serotype 3: ____________________________ 22 Serotype 4: ____________________________ 23

24 Antibiotiques addenda : ____________________________ 25 Release date : ____________________________ 26 27

A genealogy of the lot numbers of all vaccine components used in the formulation of the 28 final product will be informative. 29 The following sections are intended to report the results of the tests performed during the 30 production of the vaccine 31 32

3. Control of source materials 33

3.1 Cell cultures 34

3.1.1 General information on cell banking system 35

Information and results of characterization tests on cell banking system from cell seed (if 36 applicable), master cell bank (MCB), working cell bank (WCB), end of production cells 37 (EOPC) or extended cell bank (ECB) should be provided according to the WHO 38 Recommendations for the evaluation of animal cell cultures as substrates for the 39 manufacture of biological medicinal products and for the characterization of cell banks 40

41

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Name and identification of cell substrate: ____________________________ 1 Origin and short history (attach a flowchart 2

if necessary): ____________________________ 3 Lot number & date of preparation for each 4

bank ____________________________ 5 Date established for each bank: ____________________________ 6 Date of approval by the national regulatory 7

authority: ____________________________ 8 Total number of ampoules stored for each 9

bank: _______________________________ 10 Passage/Population doubling level (PDL) 11

of each bank ____________________________ 12 Maximum passage/ population doubling 13

level approved for each bank: ____________________________ 14 Storage conditions: _______________________________ 15 Date of approval of protocols indicating 16

compliance with the requirements of the 17 relevant monographs and with the 18 marketing authorization: _______________________________ 19

3.1.2. Characterization tests on cell seed (if applicable), MCB, WCB, EOPC 20 or ECB 21

A summary table for characterization tests on each bank should be provided 22 23 Characterization tests performed on each bank 24 Methods: ____________________________ 25 Specifications: ____________________________ 26 Date tested: ____________________________ 27 Results: ____________________________ 28

3.1.3 Cell culture medium 29

30 Serum used in cell culture medium 31 32 Animal origin of serum: ____________________________ 33 Batch number: ____________________________ 34 Vendor: ____________________________ 35 Country of origin: ____________________________ 36 Certificate for TSE free: ____________________________ 37 Tests performed on serum: 38 Methods: ____________________________ 39 Specifications: ____________________________ 40 Date of test: ____________________________ 41 Results: ____________________________ 42

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1 Trypsin used for preparation of cell 2

cultures 3 4 Animal origin of trypsin: ____________________________ 5 Batch number: ____________________________ 6 Vendor: ____________________________ 7 Country of origin: ____________________________ 8 Certificate for TSE-free: ____________________________ 9 Tests performed on trypsin: 10 Methods: ____________________________ 11 Specifications: ____________________________ 12 Date of test: ____________________________ 13 Results: ____________________________ 14 15 Antibiotics 16 17 Nature and concentration of antibiotics or 18

selecting agent (s) used in production 19 cell culture maintenance medium: ____________________________ 20

21 Other source material 22 23 Identification and source of starting 24

materials used in preparing production 25 cells including excipients and 26 preservatives (particularly any materials 27 of human or animal origin e.g. albumin; 28 serum): ____________________________ 29

30

3.2 Virus seeds 31

Vaccine virus strain(s) and serotype(s): ____________________________ 32 Substrate used for preparing seed lots: ____________________________ 33 Origin and short history: ____________________________ 34 Authority that approved virus strain(s): ____________________________ 35 Date approved: ____________________________ 36

3.2.1 Information on seed lot preparation 37

38 Virus master seed (VMS) 39

Source of virus master seed lot: ____________________________ 40 Virus master seed lot number: ____________________________ 41 Name and address of manufacturer: ____________________________ 42

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Passage level: ____________________________ 1 Date of inoculation: ____________________________ 2 Date of harvest: ____________________________ 3 Number of containers: ____________________________ 4 Conditions of storage: ____________________________ 5 Date of establishment: ____________________________ 6 Maximum passage level approved for VMS:7

____________________________ 8 Date approved by the national regulatory 9

authority: ____________________________ 10 11

Virus working seed (VWS) 12 Virus working seed lot number: ____________________________ 13 Name and address of manufacturer: ____________________________ 14 Passage level from virus master seed lot: ____________________________ 15 Date of inoculation: ____________________________ 16 Date of harvest: ____________________________ 17 Number of containers: ____________________________ 18 Conditions of storage: ____________________________ 19 Date of establishment: ____________________________ 20 Date approved by the national regulatory 21

authority: ____________________________ 22

3.2.3 Tests on VMS and VWS 23

Identity test 24

Method: ____________________________ 25 Specification: ____________________________ 26 Lot number of reference reagents : ____________________________ 27 Date of test (on, off): ____________________________ 28 Result: ____________________________ 29

Genetic/phenotypic characterizations 30

Method: ____________________________ 31 Reference reagents ____________________________ 32 Specification: ____________________________ 33 Date of test (on, off): ____________________________ 34 Result: ____________________________ 35

Tests for bacteria and fungi 36

Method: ____________________________ 37 Specification: ____________________________ 38 Media: ____________________________ 39 Number of containers tested : ____________________________ 40

WHO/BS/11.2159

Volume of inoculum per container: ____________________________ 1 Volume of medium per container: ____________________________ 2 Temperatures of incubation: ____________________________ 3 Date of test (on, off): ____________________________ 4 Result: ____________________________ 5

Test for mycoplasmas 6

Method: ___________________________ 7 Specification: ____________________________ 8 Media: ___________________________ 9 Volume tested: ___________________________ 10 Temperature of incubation ____________________________ 11 Positive controls: ____________________________ 12 Date of test (on, off): ___________________________ 13 Result: ____________________________ 14

Test for mycobacteria 15

Method: ____________________________ 16 Specification: ____________________________ 17 Media: ___________________________ 18 Volume tested: ____________________________ 19 Temperature of incubation ___________________________ 20 Date of test (on, off): ___________________________ 21 Result: ___________________________ 22

Adventitious agents: 23

Volume of virus seed samples for 24 neutralization and testing ____________________________ 25

Batch number(s) of antisera/antiserum used 26 for neutralization of virus seeds ____________________________ 27

Test in tissue cultures for adventitious agents 28

Test in simian cells: 29

Type of simian cells ____________________________ 30 Quantity of neutralized sample inoculated: ____________________________ 31 Incubation conditions: ____________________________ 32 Method: ___________________________ 33 Specification: ___________________________ 34 Date of test (on, off): ____________________________ 35 Ratio of cultures viable at end of test ___________________________ 36 Result: ___________________________ 37

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Test in human cells 1


Other cell types: 10

Type of cells ____________________________ 11 Quantity of neutralized sample inoculated: ____________________________ 12 Incubation conditions: ____________________________ 13 Method: ___________________________ 14 Specification: ___________________________ 15 Date of test (on, off): ____________________________ 16 Ratio of cultures viable at end of test ___________________________ 17 Result: ___________________________ 18

Test in animals for adventitious agents 19

Method: ___________________________ 20 Specification: ___________________________ 21 Date of test (on, off): ___________________________ 22 Result: ___________________________ 23

Test by molecular methods for adventitious agents 24


Tests in non-human primates (either master or working seed lot) for 29 neurovirulence 30

(For details, please see Recommendations for yellow fever vaccine) 31 Method: ___________________________ 32 Specification: ___________________________ 33 Date of test (on, off): ___________________________ 34 Result: ___________________________ 35

Tests in suckling mice (either master or working seed lot; where necessary) for 36 neurovirulence 37

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(Detailed protocol should be developed) 1


Virus titration for infectivity 6


4. Control of vaccine production 11

4.1 Control of production cell cultures 12

4.1.1 Information on preparation 13

Lot number of MCB ___________________________ 14 Lot number of WCB ___________________________ 15 Date of thawing ampoule of WCB: ____________________________ 16 Passage number of production cells ___________________________ 17 Date of preparation of control cell cultures ___________________________ 18 Result of microscopic examination ___________________________ 19

4.1.2 Tests on control cell cultures 20

Amount or ratio of control cultures to ____________________________ 21 production cell cultures: ____________________________ 22 Incubation conditions: ____________________________ 23 Period of observation of cultures: ____________________________ 24 Date started/ended: ____________________________ 25 Ratio of cultures discarded and reason: ____________________________ 26 Results of observation: ____________________________ 27 Date supernatant fluid collected: ____________________________ 28

Test for haemadsorbing viruses 29

Quantity of cells tested: ____________________________ 30 Method: ___________________________ 31 Specification: ___________________________ 32 Date of test (on, off): ___________________________ 33 Result: ___________________________ 34

Test for adventitious agents on supernatant culture fluids 35

WHO/BS/11.2159


Type of simian cells ____________________________ 2 Quantity of pooled sample inoculated: ____________________________ 3 Incubation conditions: ____________________________ 4 Method: ___________________________ 5 Specification: ___________________________ 6 Date of test (on, off): ____________________________ 7 Ratio of cultures viable at end of test ___________________________ 8 Result: ___________________________ 9


Type of simian cells ____________________________ 11 Quantity of pooled sample inoculated: ____________________________ 12 Incubation conditions: ____________________________ 13 Method: ___________________________ 14 Specification: ___________________________ 15 Date of test (on, off): ____________________________ 16 Ratio of cultures viable at end of test ___________________________ 17 Result: ___________________________ 18


Type of cells ____________________________ 20 Quantity of pooled sample inoculated: ____________________________ 21 Incubation conditions: ____________________________ 22 Method: ___________________________ 23 Specification: ___________________________ 24 Date of test (on, off): ____________________________ 25 Ratio of cultures viable at end of test ___________________________ 26 Result: ___________________________ 27

Identity test: 28

Method: ____________________________ 29 Specification: ____________________________ 30 Date test on: ____________________________ 31 Date test off: ____________________________ 32 Result: ____________________________ 33

4.1.3 Cells used for vaccine production: 34

Observation of cells used for production 35 Specification: ____________________________ 36 Date: ____________________________ 37 Result: ____________________________ 38

WHO/BS/11.2159

1

4.2 Monovalent virus harvest pools 2

4.2.1 Information on manufacture 3

Information on each monovalent virus harvest pool should separately be provided 4 Batch number(s): _______________________________ 5 Date of inoculation: _______________________________ 6 Date of harvesting: _______________________________ 7 Lot number of Virus master seed lot _______________________________ 8 Lot number of virus working seed lot _______________________________ 9 Passage level from virus working seed lot _______________________________ 10 Methods, date of purification if relevant _______________________________ 11 Volume(s), storage temperature, storage 12

time and approved storage period: _______________________________ 13 14

4.2.2 Tests on monovalent virus harvest pools 15

Identity 16

Method: ____________________________ 17 Specification: ____________________________ 18 Lot number of reference reagents: _______________________________ 19 Specification: _______________________________ 20 Date of test _________________________ 21 Result: _______________________________ 22

Test for bacteria and fungi 23

Method: ____________________________ 24 Specification: ____________________________ 25 Media: ____________________________ 26 Number of containers tested: _______________________________ 27 Volume of inoculum per container: ____________________________ 28 Volume of medium per container: ____________________________ 29 Temperatures of incubation: _______________________________ 30 Date of test (on, off): _________________________ 31 Result: ____________________________ 32

Test for mycoplasma 33

Method: ____________________________ 34 Specification: ____________________________ 35 Media: ____________________________ 36

WHO/BS/11.2159

Volume tested: ____________________________ 1 Temperature of incubation ____________________________ 2 Positive controls: ____________________________ 3 Date of test (on, off) ____________________________ 4 Result: ____________________________ 5

Test for mycobacteria 6

Method: ____________________________ 7 Specification: ____________________________ 8 Media: ____________________________ 9 Volume tested: ____________________________ 10 Temperature of incubation ____________________________ 11 Date of test (on, off) ____________________________ 12 Result: ____________________________ 13

Test for adventitious agents 14






Type of cells ____________________________ 34 Quantity of neutralized sample inoculated: ____________________________ 35 Incubation conditions: ____________________________ 36 Method: ___________________________ 37 Specification: ___________________________ 38

WHO/BS/11.2159

Date of test (on, off): ____________________________ 1 Ratio of cultures viable at end of test ___________________________ 2 Result: ___________________________ 3

Virus titration for infectivity 4


Test for host cell proteins 9

Method: ___________________________ 10 Specification: ___________________________ 11 Date of test ___________________________ 12 Result: ___________________________ 13

Test for residual cellular DNA 14


Consistency of virus characteristics 19

Method: ___________________________ 20 Specification: ___________________________ 21 Date of test (on, off): ___________________________ 22 Result: ___________________________ 23 24

4.3. Final tetravalent vaccine bulk 25

4.3.1 Information on manufacture 26

Batch number(s): ___________________________ 27 Date of formulation: ____________________________ 28 Total volume of final bulk formulated: ____________________________ 29 Monovalent virus pools used for 30

formulation ____________________________ 31 Serotype / lot number / volume added / 32

virus concentration ___________________________ 33 Name & concentration of added substances 34

(e.g. diluent, stabilizer if relevant): ___________________________ 35

WHO/BS/11.2159

Volume(s), storage temperature, storage 1 time and approved storage period: _______________________________ 2

4.3.2 Tests on final tetravalent bulk lot 3

Residual animal serum protein 4


Test for bacteria & fungi 9

Method: ____________________________ 10 Specification: ____________________________ 11 Media: ____________________________ 12 Number of containers tested: ___________________________ 13 Volume of inoculum per container: ____________________________ 14 Volume of medium per container: ____________________________ 15 Temperatures of incubation: ___________________________ 16 Date of test (on, off): ___________________________ 17 Result: _______________________________ 18

19

5. Filling and containers 20

Lot number ___________________________ 21 Date of filling ___________________________ 22 Type of container: ___________________________ 23 Volume of final bulk filled ___________________________ 24 Filling volume per container ___________________________ 25 Number of containers filled (gross) ___________________________ 26 Date of lyophilization ___________________________ 27 Number of containers rejected during 28

inspection ___________________________ 29 Number of containers sampled ___________________________ 30 Total number of containers (net) ___________________________ 31 Maximum period of storage approved ___________________________ 32 Storage temperature and period ___________________________ 33 34

WHO/BS/11.2159

6. Control tests on final vaccine lot 1

6.1 Tests on vaccine lot 2

Inspection of final containers 3

Appearance ____________________________ 4 Specification: ____________________________ 5 Date of test ____________________________ 6 Results ____________________________ 7 Before reconstitution ____________________________ 8 After reconstitution ____________________________ 9 Diluent used ____________________________ 10 Lot number of diluent used ____________________________ 11

Test for pH 12


Identity test (each serotype) 17

Method: ____________________________ 18 Specification: ___________________________ 19 Date of test ___________________________ 20 Result: ___________________________ 21

Test for bacteria and fungi 22

Method: ____________________________ 23 Specification: ____________________________ 24 Media: ___________________________ 25 Volume tested: ____________________________ 26 Temperatures of incubation: ___________________________ 27 Date of test (on, off): ___________________________ 28 Result: ___________________________ 29

Test for potency (each serotype): 30

Method: ___________________________ 31 Batch number of reference vaccine and 32

assigned potency: ___________________________ 33 Specification: ___________________________ 34

WHO/BS/11.2159

Date of test (on, off): ___________________________ 1 Result for each serotype: ___________________________ 2

Thermal stability (each serotype) 3

Method: ___________________________ 4 Specification: ___________________________ 5 Date of test (on, off): ___________________________ 6 Result for each serotype: ___________________________ 7

General safety (unless deletion authorized) 8

Tests in mice 9

Date of inoculation ___________________________ 10 No. of animals tested ___________________________ 11 Volume and route of injection ___________________________ 12 Observation period ___________________________ 13 Specification: ____________________________ 14 Results (give details of deaths) ___________________________ 15

Tests in guinea-pigs 16

Date of inoculation ___________________________ 17 No. of animals tested ___________________________ 18 Volume and route of injection ___________________________ 19 Observation period ___________________________ 20 Specification: ____________________________ 21 Results (give details of deaths) ___________________________ 22

Residual moisture 23

Method ___________________________ 24 Specification ___________________________ 25 Date ___________________________ 26 Result ___________________________ 27

Residual antibiotics if applicable 28

Method ___________________________ 29 Specification ___________________________ 30 Date ____________________________ 31 Result ___________________________ 32 33

WHO/BS/11.2159

6.2 Diluent 1

Name and composition of diluent _______________________________ 2 Lot number _______________________________ 3 Date of filling _______________________________ 4 Type of diluent container _______________________________ 5 Filling volume per container _______________________________ 6 Maximum period of storage approved _______________________________ 7 Storage temperature and period _______________________________ 8

9 10 Name (Typed): __________________________________________________ 11 Signature: __________________________________________________ 12 Date: __________________________________________________ 13

14

WHO/BS/11.2159

Appendix 2: Model certificate for the release of dengue vaccine 1

(live, tetravalent) by national regulatory authorities 2

3 LOT RELEASE CERTIFICATE 4 5

The following lot(s) of dengue vaccine produced by _____________________(1) in 6 _______________(2), whose numbers appear on the labels of the final containers, meet all 7 national requirements(3) and Part A(4) of the WHO Guidelines to assure the quality, safety 8 and efficacy of live attenuated (recombinant) dengue virus vaccines (_____)(5), and 9 comply with Good Manufacturing Practices for Pharmaceutical Products: Main 10 Principles(6) and Good Manufacturing Practices for Biological Products(7). As a minimum, 11 this certificate is based on examination of the summary protocol of manufacturing and 12 control. 13 14 The certificate may include the following information: 15 • Name and address of manufacturer; 16 • Site(s) of manufacturing; 17 • Trade name and/common name of product; 18 • Marketing authorization number; 19 • Lot number(s) (including sub-lot numbers, packaging lot numbers if necessary); 20 • Type of container; 21 • Number of doses per container; 22 • Number of containers/lot size; 23 • Date of start of period of validity (e.g. manufacturing date) and/or expiry date; 24 • Storage condition; 25 • Signature and function of the authorized person and authorized agent to issue the certificate; 26 • Date of issue of certificate; and 27 • Certificate number. 28 29 The Director of the National Regulatory Authority (or Authority as appropriate): 30 31 Name (Typed) 32 Signature 33 Date 34 35 1 Name of manufacturer 36 2 Country of origin 37 3 If any national requirements are not met, specify which one(s) and indicate why release 38 of the lot(s) has nevertheless been authorized by the national regulatory authority 39 4 With the exception of provisions on distribution and shipping, which the national 40 regulatory authority may not be in a position to assess. 41 5 WHO Technical Report Series, No. ___, YYYY, Annex __. 42 6 WHO Technical Report Series, No. 908, 2003, Annex 4. 43 7 WHO Technical Report Series, No. 823, 1992, Annex 1. 44 45 46 47

WHO/BS/11.2159

Appendix 3: Procedure for environmental risk assessment 1

2 This appendix provides an example of the steps to be followed in conducting an 3 ERA, as exemplified by the EU Directive 2001/18/EC (Annex II) and 4 Commission Decision 2002/623/EC. 5

Step 1: 6

Identification of characteristics that may cause potential adverse effects 7 and evaluation of the potential consequences of each adverse effect 8 (hazard identification) 9 10

A hazard is defined as a potential harmful effect on human health or the 11 environment, caused by features of the GMO and its application. Effects on 12 human health or the environment are differentiated into direct and indirect effects. 13 Direct effects occur due to an interaction between the GMO itself and its direct 14 environment. Indirect effects are the result of a more extensive causal chain of 15 events. Moreover, both direct and indirect effects may be either immediate or 16 delayed. Immediate effects occur during the time period of the release, and can 17 usually be attributed to the release in a straightforward manner, whereas delayed 18 effects may become apparent at a later stage, after termination of the release, and 19 consequently they are usually less easily attributed to an effect caused by the 20 GMO. Potential adverse effects may include disease to humans (other than the 21 patient), animals or any other organism, or altered susceptibility to pathogens 22 facilitating the dissemination of infectious diseases. 23

24 25

26 Figure 1. Steps in the analysis of ERA according to the guidelines in the Commission 27 Decision 2002/623/EC and 2001/18/EC 28

WHO/BS/11.2159

Step 2: 1

Evaluation of the potential consequences of each adverse effect, if it occurs 2 3

The magnitude of the consequences is likely to be influenced by the environment 4 in which the GMO is released and the manner of release. For each adverse effect 5 that is identified the consequences for the environment need to be qualified in 6 qualitative terms ranging from high, moderate, low to negligible. 7

8

Step 3: 9

Evaluation of the likelihood 10 11

The next step in the ERA deals with the evaluation of the likelihood of the 12 occurrence of the described adverse effects and their consequences, i.e. whether 13 the potential adverse effect associated with the application of the GMO will be 14 effectuated. All characteristics that could contribute to the actual occurrence of 15 the hazard should be identified. For the determination of the likelihood, it is 16 important to consider the manner of release and the surrounding receiving 17 environment. If no quantitative data are available to determine the likelihood, the 18 likelihood should be described in qualitative terms ranging from high, moderate, 19 or low to negligible, based on an expert judgment that considers the available data. 20 In case the likelihood of a potential effect cannot be determined, a worst-case 21 scenario will be applied that assumes the potential adverse effect will occur to the 22 fullest extent. 23

Step 4: 24

Risk estimation 25 26

An estimation of the risk to human health or the environment posed by each 27 identified characteristic of the GMO which has the potential to cause adverse 28 effects should be made by multiplying the likelihood of the adverse effect 29 occurring and the consequences, were it to occur. The risk is usually characterized 30 on scale, e.g. ‘negligible’, ‘low’, ‘moderate’, or ‘high’. Usually it is concluded 31 that risk management should be applied to reduce the risk, if the risk is 32 characterized as higher than negligible. 33

34

Step 5: 35

Application of risk management strategies 36 37

WHO/BS/11.2159

Where the estimated overall risk is higher than negligible, risk management 1 strategies should be applied that are adequate to reduce the level of risk. In 2 practice, risk management measures usually reduce the likelihood instead of the 3 potential adverse effects. In most cases risk management measures aim at 4 reducing spreading of the GMO into the environment, e.g. by hospitalization of 5 the patient or bandaging of the application site. Note that risk management 6 strategies to prevent transmission of dengue virus is likely to be ineffective or 7 unfeasible in certain parts of the world. Therefore, it should be well considered to 8 demand the use of mosquito nets and repellents for an appropriate period after 9 vaccination as an effective strategy to reduce this risk. 10

11

Step 6: 12

Determination of the final risk 13 14

An evaluation of the final overall risk of the GMO should be made taking into 15 account any proposed risk management strategies. The determination of the final 16 risk should conclude an ERA based on the previous steps described under step 1 17 to 5. Steps 4 and 5 may be repeated in situations where the overall environmental 18 impact is not acceptable. In that case, the ultimate risk management tool is not to 19 perform the activity with the GMO until more scientific data are available and the 20 ERA may be repeated on the basis of these data. It is up to the competent 21 authority to decide whether risks are acceptable or not, and which risk 22 management measurements are should be installed 23 24 In the assessment of clinical trials and marketing authorization approvals, 25 environmental risk assessment and risk management strategies should be taken 26 into account, but there should always be a well balanced risk and benefit 27 consideration. 28

Abbreviations 1

2 ADE antibody-dependent enhancement 3 Ae Aedes 4 AEs adverse events 5 CCID50 cell culture infectious dose 50% 6 CDC Centers for Disease Control and Prevention 7 CMI cell-mediated immunity 8 CNS central nervous system 9 DENVs dengue viruses 10 DFI dengue febrile illness 11 DSMB data and safety monitoring board 12 E envelope 13 EIA enzyme immunoassay 14 ELISA enzyme-linked immunosorbent assay 15 EOPC end of production cell 16 ECB extended cell bank 17 ERA environmental risk assessment 18 GMO genetically modified organism 19 GMTs geometric mean titres 20 IFU immunofocus-forming unit 21 IU international unit 22 JE Japanese encephalitis 23 MCB master cell bank 24 NAAT nucleic acid amplification techniques 25 NHP non-human primate 26 NIAID National Institute of Allergy and Infectious Diseases 27 NIBSC National Institute of Biological Standards and Control 28 NS non-structural 29 ORF open reading frame 30 PBMCs peripheral blood mononuclear cells 31 PCC production cell culture 32 PDK primary dog kidney 33 PDL population doubling 34 PFU plaque-forming unit 35 prM premembrane 36 PRNT plaque reduction neutralization test 37 QPCR quantitative polymerase chain reaction 38 RCB reference cell bank 39 RT-PCR reverse transcription-polymerase chain reaction 40 TBE tick-borne encephalitis 41 TSE transmissible spongiform encephalopathies 42 UTR untranslated region 43 VE vaccine efficacy 44 VMS virus master seed 45

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VWS virus working seed 1 WCB working cell bank 2 WN West Nile 3 WRAIR Walter Reed Army Institute of Research 4 YFV yellow fever virus 5

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