guidelines on the quality, safety and efficacy of dengue ...other types 13 of dengue virus vaccines...

WHO / DRAFT / 1 May 2011 (DEN)

1 WHO / DRAFT / 1 May 2011 (DEN) 2

ENGLISH ONLY 3

4

5

Guidelines on the Quality, Safety and Efficacy of Dengue 6

Tetravalent Vaccines (Live, Attenuated) 7 8 9

Proposed replacement of TRS 932, Annex 1 10 11

NOTE: 12 13 This document has been prepared for the purpose of inviting comments and suggestions 14 on the proposals contained therein, which will then be considered by the Expert 15 Committee on Biological Standardization (ECBS). Publication of this early draft is to 16 provide information about the proposed WHO Guidelines on the Quality, Safety and 17 Efficacy of Dengue Tetravalent Vaccines (Live, Attenuated) to a broad audience and to 18 improve transparency of the consultation process. 19

20 The text in its present form does not necessarily represent an agreed formulation of 21 the Expert Committee. Written comments proposing modifications to this text MUST 22 be received by 23 June 2011 in the Comment Form available separately and should be 23 addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: 24 Quality Safety and Standards (QSS). Comments may also be submitted electronically to 25 the Responsible Officer: Dr Jinho Shin at email: [email protected]. 26 27 The outcome of the deliberations of the Expert Committee will be published in the WHO 28 Technical Report Series. The final agreed formulation of the document will be edited to be 29 in conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). 30 31 32

© World Health Organization 2011 33

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World 34 Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 35 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO 36 publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at 37 the above address (fax: +41 22 791 4806; e-mail: [email protected]). 38

The designations employed and the presentation of the material in this publication do not imply the 39 expression of any opinion whatsoever on the part of the World Health Organization concerning the legal 40 status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers 41

WHO / DRAFT/ 1 May 2011 (DEN)

or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full 1 agreement. 2 3 The mention of specific companies or of certain manufacturers’ products does not imply that they are 4 endorsed or recommended by the World Health Organization in preference to others of a similar nature that 5 are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by 6 initial capital letters. 7 8 All reasonable precautions have been taken by the World Health Organization to verify the information 9 contained in this publication. However, the published material is being distributed without warranty of any 10 kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with 11 the reader. In no event shall the World Health Organization be liable for damages arising from its use. 12

13 The named authors [or editors as appropriate] alone are responsible for the views expressed in this 14 publication. 15 16 17 18

19

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.


Table of contents 1

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

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

Part A. Guidelines on manufacturing and control of dengue tetravalent 4 vaccines (live, attenuated) ···································································································12 5

A.1 Definitions········································································································· 12 6

A.2 General manufacturing requirements······························································· 15 7

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

A.4 Control of vaccine production ·········································································· 22 9

A.5 Filling and containers······················································································· 27 10

A.6 Control tests on final lot ··················································································· 27 11

A.7 Records ············································································································· 29 12

A.8 Samples ············································································································· 29 13

A.9 Labelling ··········································································································· 29 14

A.10 Distribution and shipping ················································································· 30 15

A.11 Stability, storage and expiry date ····································································· 30 16

Part B. Nonclinical evaluation of dengue tetravalent vaccines (live, attenuated) ······32 17

B.1 General remarks ······························································································· 32 18

B.2 Product development and characterization ······················································ 34 19

B.3 Nonclinical immunogenicity and protective activity········································· 34 20

B.4 Nonclinical toxicity and safety·········································································· 35 21

B.5 Environmental risk···························································································· 38 22

Part C. Clinical evaluation of dengue tetravalent vaccines (live, attenuated) ············40 23

C.1 General considerations for clinical studies ······················································ 40 24

C.2 Immunogenicity································································································· 41 25

C.3 Clinical studies·································································································· 44 26

C.4 Post-licensure investigations ············································································ 55 27

Part D. Environmental risk assessment of dengue tetravalent vaccines (live, 28 attenuated) derived by recombinant DNA technology···················································57 29

D.1 Introduction······································································································· 57 30

D.2 Procedure for Environmental Risk Assessment ················································ 59 31


D.3 Special Considerations for Live Recombinant Dengue Virus Vaccines ··········· 59 1

Part E. Guidelines for national regulatory authorities ··················································63 2

E.1 General ············································································································· 63 3

E.2 Release and certification·················································································· 63 4

Authors···································································································································64 5

Acknowledgements···············································································································66 6

Appendix 1: Model summary protocol for manufacturing and control of 7 dengue tetravalent vaccines (live, attenuated) ·································································67 8

Appendix 2: Model certificate for the release of dengue vaccine (live, 9 tetravalent) by national regulatory authorities ·······························································81 10

Appendix 3: Procedure for environmental risk assessment ··········································82 11

Abbreviations ························································································································85 12

References ······························································································································86 13


Introduction 1

2 These Guidelines are intended to provide national regulatory authorities and 3 vaccine manufacturers with guidance on the quality, safety and efficacy of live-4 tetravalent dengue vaccines currently under clinical development to facilitate their 5 international licensure and use. 6 7 These Guidelines update the Guidelines for the production and quality control of 8 candidate tetravalent dengue virus vaccines (live) (65). 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 12 of dengue virus vaccines under development, e.g. subunit or inactivated vaccines, 13 are outside the scope of these Guidelines. However, some guiding principles 14 provided in these Guidelines, e.g. Part C clinical evaluation, may be useful for the 15 evaluation of other types of dengue vaccines. For quality control aspects, guiding 16 principles applicable to other types of vaccines such as inactivated or subunit 17 vaccines are available elsewhere if the product in development shares similar 18 manufacturing process. For example, guidelines for human papillomavirus and 19 hepatitis B vaccines may be useful for subunit vaccines for dengue as well. 20 21 These Guidelines are based on experience gained from candidate live-attenuated 22 dengue tetravalent vaccines that have been developed, as described below, and 23 will need to be updated as new data becomes available from additional studies. 24 25 Part A sets out guidelines for manufacture and quality control. Guidelines specific 26 to the nonclinical, clinical evaluation and environmental risk assessment are 27 provided in Parts B, C, and D, respectively. Part E provides guidelines for 28 national regulatory authorities. In the following section, brief overviews of 29 dengue disease and dengue vaccine development at the time of preparing this 30 document are provided as scientific basis for each part. 31 32 33

General considerations 34 35

Virology 36 Dengue is a mosquito-borne disease and represents a major public health problem 37 throughout the tropical world. The causative dengue viruses (DENVs) are 38 members of the genus Flavivirus, within the family Flaviviridae. There are four 39 serotypes (termed DENV-1 to -4) and at least 3 genetic groups (genotypes) within 40 each serotype. 41 42 All flaviviruses are lipid-enveloped, positive-sense, single-stranded RNA viruses, 43 approximately 55 nm in diameter. The genome is capped at the 5’ terminus but 44


does not have a poly A tract at the 3’ terminus, and is approximately 11,000 1 nucleotides in length. The virion RNA encodes a single open reading frame (ORF) 2 that is flanked by a 5’ untranslated region (UTR) and 3’UTR. The ORF is 3 translated into a polyprotein that is co- and post-translationally cleaved to yield at 4 least 10 proteins. Three structural proteins are derived by cleavages of the amino-5 terminal one-third of the polyprotein: the capsid or core protein forms a 6 “nucleocapsid” complex with virion RNA that lies within the lipid envelope. The 7 premembrane (prM) and envelope (E) proteins are embedded in the lipid envelope 8 via carboxy-terminal transmembrane domains and are displayed on the surface of 9 virions. Cleavage of the carboxy-terminal two-thirds of the polyprotein yields 10 seven non-structural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and 11 NS5. NS3 encodes a serine protease in the N-terminal 180 amino acids and 12 helicase, nucleotide triphosphatase, RNA 5'-triphosphatase activities in the C-13 terminal region while NS5 encodes two functions; the first one-third encodes a 14 methyltransferase that sequentially methylates the N7 and 2'-O positions of the 15 viral RNA cap using S-adenosyl-l-methionine as a methyl donor, and and the 16 remainder a RNA-dependent RNA polymerase NS1 plays various roles in the 17 virus replication cycle while NS2A, NS2B, NS4A and NS4B are all small 18 hydrophobic proteins with the central region of NS2B required for the functioning 19 of the NS3 protease. 20

21

Host range and transmission 22 DENVs are most commonly transmitted to humans by the bite of infected Aedes 23 (Ae.) aegypti mosquitoes, which are highly domesticated and the primary 24 mosquito vector; however, Ae. albopictus can also sustain human-to-human 25 transmission. The drastic increase in the incidence of DENV infection in the 26 Americas during the past 30 years is primarily due to the geographical spread of 27 Ae. aegypti following decline in vector-control efforts. The DENV that infects and 28 causes disease in humans is maintained in a human-to-mosquito-to-human cycle 29 and does not require a sylvatic cycle in non-human primates. Certain strains of 30 DENV are known to be transmitted to non-human primates in western Africa and 31 Malaysia; however, transmission to humans via mosquitoes from non-human 32 primates is believed to be very limited. 33 34

Clinical disease manifestation in humans 35 Following infection by the bite of an infected mosquito, the virus is thought to 36 replicate in local dendritic cells. Subsequent infection of macrophages and 37 lymphocytes is followed by entry into the blood stream. Haematogenous spread is 38 the likely mechanism for seeding of peripheral organs and the occasional reports 39 of CNS infections, which can lead to symptomatic illness. 40 41 Most DENV infections are either asymptomatic or only mildly symptomatic. The 42 incubation period of dengue can range from 3 to 14 days, but is generally 4 to 7 43 days. Most symptomatic DENV infections present with a sudden onset of fever 44


accompanied by headache, pain behind the eyes, generalized myalgia and 1 arthralgia, flushing of the face, anorexia, abdominal pain and nausea. Rash is 2 common in dengue and can be macular, maculopapular, morbilliform, 3 scarlatiniform or petechial in character. Rash is most often seen on the trunk, 4 insides of the arms and thighs, and plantar and palmar surfaces. Laboratory 5 abnormalities that can be observed in dengue infection include leukopenia and 6 thrombocytopenia. 7 8 Dengue illness is classified as (i) dengue with or without warning signs and (ii) 9 severe dengue. A presumptive diagnosis of dengue can be made in a patient living 10 in or traveling from a dengue-endemic area who has fever and at least two of the 11 following clinical signs or symptoms: anorexia and nausea, rash, body aches and 12 pains, warning signs, leukopenia, and a positive tourniquet test. "Warning signs" 13 include abdominal pain or tenderness, persistent vomiting, clinical fluid 14 accumulation, mucosal bleeding, lethargy or restlessness, liver enlargement of > 2 15 cm, or an increase in haematocrit concurrent with a rapid decrease in platelet 16 count. Dengue illness should be classified as severe in a patient with presumptive 17 dengue and any of the following: severe plasma leakage leading to shock or 18 respiratory compromise, clinically significant bleeding, or evidence of severe 19 organ involvement. Detailed case classification of dengue is provided in a 20 separate document (WHO/HTM/NTD/DEN/2009.1 or its update) (69). 21

22

Non-human primate dengue virus infection 23 Natural hosts for DENV infection are humans and mosquitoes. Serological 24 evidence from nonhuman primate studies indicates a sylvatic DENV cycle 25 involving several species of mosquitoes and several monkey species exists. 26 Although monkeys develop a viremia and a neutralizing antibody response to 27 DENV infection, they do not develop the hematologic abnormalities seen in 28 humans. However, in non-human primate (NHP) (and AG129 mouse, see 29 paragraph below) primary DENV infections cause a leukopenia. 30 31 Thrombocytopenia has been observed after a secondary infection (5, 8, 16, 19). 32 In the rhesus macaque, viremia typically begins 2-6 days after infection and lasts 33 for 3-6 days (5, 7, 16). Virus spreads to regional lymph nodes and can be isolated 34 from the skin, distant lymph nodes, and rarely from spleen, thymus and other 35 body organs. The NHP model for DENV is useful to measure protection from 36 viremia conferred by vaccination or passively acquired antibody. Disadvantages 37 of the NHP model include the lack of overt clinical signs of disease (8, 16, 19, 50). 38

Mouse dengue virus infection 39 Clinical isolates of DENV do not replicate well in genetically normal mice. 40 However, mouse-brain-adapted DENVs can induce fatal encephalitis after 41 intracranial inoculation of suckling mice. It has been demonstrated that 42 adaptation of a DENV2 isolate to neurovirulence in suckling mice correlated 43 positively with attenuation of virulence in humans (42). Because of this 44


ambiguity, the suckling mouse/encephalitis model is probably not useful for 1 studying candidate DEN vaccine safety or efficacy, although it has been used for 2 these purposes historically. In recent years, both chimeric mice that are 3 transplanted with human cells and severely immunocompromised strains of mice 4 have been used to elucidate the immune response to DEN infection and to study 5 pathogenesis (8, 54, 75). Interferon receptor -deficient AG129 mice support 6 replication of selected DENV strains which infect relevant cell and tissue types 7 comparable to human infection (46, 54). AG129 mice have been used to 8 investigate antibody-mediated protection. A strain of DENV2 that has been 9 adapted to AG129 mice by serial passage between mice and mosquito cells has a 10 viscerotropic phenotype, causing thrombocytopenia and vascular leakage in the 11 infected animals. The phenomenon of antibody-dependent enhancement (ADE) of 12 virus infection was observed in AG 129 mice following passive transfer of anti-13 DENV1 antibodies and challenge with the adapted strain of DENV2 (2, 46, 54). 14

Mosquito dengue virus infection 15 Vector competence refers to the efficiency with which the vector transfers 16 infection between hosts. Typically, this is a product of vector susceptibility to 17 infection, replication efficiency of the pathogen in the vector, and the sensitivity 18 of the host to infection transmitted by vector contact. Aedes aegypti mosquitoes 19 exhibit global variation in vector competence for flaviviruses. For example, in 20 sub-Saharan Africa, a black “sylvan” subspecies, Ae. formosus predominates. 21 This mosquito has a low vector competence for flaviviruses due primarily to a 22 midgut infection barrier (3). Once ingested in an infectious blood meal, DENVs 23 should replicate in the midgut and disseminate to the salivary glands to facilitate 24 transmission to a new host during feeding. In this process, the virus should 25 overcome any midgut barrier that would limit replication and prevent spread of 26 the virus to other tissues in the mosquito (19, 43, 44). 27 28 None of the live DEN vaccine preparations currently in the clinical trial phase of 29 development is effectively transmitted by mosquito vectors (29, 44),because 30 vaccine viruses replicate poorly in mosquito midgut epithelium, and/or do not 31 disseminate efficiently to the salivary glands, thereby interdicting transmission to 32 humans (19, 22, 49). In addition, the low peak titre and very short duration of 33 viremia induced by these candidates in humans has been shown to render 34 vaccinees relatively non-infectious for feeding mosquitoes. The net effect of these 35 two phenomena is a drastic reduction in vector competence. 36

At-risk populations and global health importance 37 Dengue is the most rapidly spreading mosquito-borne viral disease in the world. 38 Since 1955, the incidence of dengue and severe dengue reported to WHO has 39 increased approximately 30-fold with increasing geographic expansion to new 40 countries and from urban to rural settings. Approximately 3.5 billion people live 41 in dengue endemic countries which are located in the tropical and subtropical 42 regions of the world. An estimated 50 million dengue infections occur annually 43


and the number of cases reported annually to WHO ranged from 0.4 to 1.3 million 1 in the decade 1996 - 2005 (69). 2 3

Justification for vaccine development 4 Prevention of dengue by vector control has proven to be very difficult and costly. 5 While vector control efforts should be sustained, vaccination holds substantive 6 potential in the control of the disease. Hence, there is an urgent need to develop 7 dengue vaccines, especially to protect people from disease in endemic countries. 8

Development of candidate dengue vaccines 9 Efforts to develop a vaccine against dengue have primarily focused on live 10 attenuated viruses, derived by serial passage of virulent viruses in tissue culture or 11 via recombinant DNA technology that permits site-directed mutagenesis of the 12 genome of a virulent parent strain. Success in the development, licensure, and 13 clinical use of live attenuated flavivirus vaccines, such as yellow fever 17D 14 vaccines and Japanese encephalitis SA14-14-2, suggests that a suitably attenuated 15 live dengue vaccine could be highly efficacious. Other vaccine candidates based 16 on inactivated whole virus, subunits that include E protein, virus-like particles 17 comprised of prM and E proteins, and DNA vaccines that induce expression of 18 DENV prM/E proteins and are in preclinical or early clinical stages of 19 development. 20 21 There are no animal models that completely mimic the protean manifestations of 22 dengue. The lack of a suitable animal model makes it difficult to assess efficacy 23 of vaccine candidates and to identify/establish possible correlates of protection in 24 vivo. Therefore, the protective capacity of any vaccine candidate will be finally 25 defined by its ability to protect humans from dengue febrile illness (DFI). Results 26 of non-clinical studies using monkeys and susceptible mouse strains suggest, 27 however, that protection from dengue is best correlated with the presence of virus-28 neutralizing antibodies (1, 5, 17, 26, 27, 30, 41, 53). Studies in which vaccinated 29 volunteers were challenged with dengue viruses have been conducted in the past 30 but are not a required part of currently recommended clinical development 31 programs. 32

33 There is general agreement that DENV vaccines should ideally induce protective 34 neutralizing antibodies to each of the four serotypes simultaneously. In theory, a 35 tetravalent immune response would protect against all DFI and would also reduce 36 or eliminate the risk of a phenomenon termed antibody-dependent enhancement 37 of disease, which is thought to be one of the mechanisms that predispose to severe 38 forms of dengue. 39

40 Several strategies have been employed to derive candidate live attenuated 41 vaccines. Attenuation of DENVs by serial passage in cell culture has proven to be 42 problematic because of the difficulty in achieving long-term, balanced 43


immunogenicity against all four DENV simultaneously. Nonetheless, there are 1 four candidates that are in clinical development at the present time. 2

3 The Walter Reed Army Institute of Research (WRAIR) developed attenuated 4 DENV strains by empirical serial passage in primary dog kidney (PDK) cells and 5 produced vaccine candidates in fetal rhesus lung (FRhL) cells. Tetravalent 6 formulations of these attenuated vaccine candidates have been evaluated in Phase 7 I and Phase II clinical trials conducted by WRAIR and GlaxoSmithKline (21, 23, 8 50, 51). 9

10 The other three candidates were developed using recombinant DNA technology 11 which involves first the generation of a full-length DNA copy of the DENV 12 genome. Site-specific mutations expected to affect virulence are then introduced 13 into the DNA, and mutant full-length DNAs can then be copied in vitro to 14 produce infectious RNA transcripts that can be used to generate mutant DENVs in 15 tissue culture. The Laboratory of Infectious Diseases of the U.S. National Institute 16 of Allergy and Infectious Diseases (NIAID) has thus derived a total of five 17 candidate DEN vaccine viruses that have been tested in clinical trials. Two were 18 generated by introduction of a 30-nucleotide deletion (termed ∆30) into the 3’- 19 UTR of the DENV-4 and DENV-1 genomes (21, 23, 50, 51). These DENV-1 and 20 DENV-4 vaccine candidates were shown to be attenuated and immunogenic in 21 nonhuman primates. A DENV-2 candidate vaccine was developed by replacing 22 the gene segments encoding the prM and E proteins of the DEN4∆30 candidate 23 vaccine with those of DENV-2. An additional DENV-3 candidate vaccine was 24 developed by replacing the 3´ UTR of a DENV-3 wild-type virus with that of the 25 DEN4∆30 UTR. A third DENV-3 candidate vaccine was developed by 26 introducing a 30 nucleotide deletion into the 3´ UTR homologous to that of the 27 DEN4∆30 vaccine virus and a second non-contiguous 31 nucleotide deletion, also 28 in the 3´ UTR (6). Phase I trials have been conducted with “∆30” monovalent 29 vaccines (7, 21, 50), and Phase I trials with the tetravalent formulation were 30 initiated in 2010. 31 32 Thai scientists at Mahidol University developed a candidate DENV-2 vaccine 33 empirically by 53 serial passages of the virus in PDK cells, designated DENV-2 34 strain PDK53, which was found to be highly attenuated and immunogenic in 35 Phase I and Phase II clinical trials. The U.S. Centers for Disease Control and 36 Prevention (CDC) determined that the attenuation mutations of DENV-2 PDK53 37 virus were not located in the prM or E proteins, and in collaboration with 38 InViragen, Inc. used this genetic background to derive chimeric DENV-1, -3 and -39 4 vaccines expressing the respective prM/E genes in the context of the DENV-2 40 PDK53 genome “backbone” (21, 50). A tetravalent formulation is in Phase I 41 clinical trials. 42

43 Finally, a candidate live vaccine was developed by Acambis/Sanofi Pasteur using 44 the live attenuated yellow fever virus (YFV) vaccine, 17D, as the backbone for 45 chimeric DENV vaccine candidate. In these viral genomes, the prM and E genes 46


from each of the four DENV serotypes, respectively, are substituted for those of 1 YFV in the context of the genetic background of the 17D vaccine. A tetravalent 2 chimeric YFV-DENV vaccine has been evaluated in Phase I and II clinical trials 3 for safety and immunogenicity. Phase IIb trials to investigate protective efficacy 4 in children began late in 2009 (21, 50) and Phase III trials have been in progress 5 since late 2010. 6 7


Part A. Guidelines on manufacturing and control of 1

dengue 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 5 international name at this early stage of development will aid harmonization of 6 nomenclature if licensure is obtained. The international name should be “dengue 7 tetravalent vaccine, live attenuated”. The proper name should be the equivalent to 8 the international name in the language of the country of origin. The use of the 9 international name should be limited to vaccines that satisfy the specifications 10 formulated below. 11

A.1.2 Descriptive definition 12

A live attenuated tetravalent dengue virus vaccine defined in <A.1.1> should contain 13 live attenuated dengue viruses representing each of the four serotypes, or replication-14 competent viral vectors that express the major antigen genes of each of the four 15 dengue serotypes, that have been separately prepared in cell culture. It may be 16 presented as a sterile, aqueous suspension or as freeze-dried material. The 17 preparation should satisfy all of the specifications given below. 18

A.1.3 International reference materials 19

As the prospective vaccines are very different in type, no international reference 20 material for a candidate live dengue vaccine is available. However, an international 21 reference panel of human antisera against all four dengue serotypes is available from 22 the National Institute of Biological Standards and Control (NIBSC). The panel is 23 intended to help calibrate the response to vaccines. 24

A.1.4 Expression of dose related to vaccine potency 25

Potency of a live vaccine most often is expressed in terms of the number of 26 infectious units of virus contained in a human dose, using a specified tissue culture 27 substrate and based on results of phase I and II clinical trials. In the case of a 28 tetravalent dengue vaccine, potency will have to be assessed in terms of the 29 individual titers of each of the four serotypes of vaccine virus contained in a human 30 dose. When international reference standards for the vaccine type under production 31 become available, the dose related to vaccine potency should be calculated against 32 the international standard and expressed in international units international unit (IU) 33 to reduce variation between laboratories. Until then, the use of PFU, FFU or CCID50 34 to express the potency and doses of vaccine can be an alternative.. The dose should 35 also serve as a basis for establishing parameters for stability and for the expiry date. 36


A.1.5 Terminology 1

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

Adventitious agents 4

Contaminating microorganisms of the cell culture or source materials including 5 bacteria, fungi, mycoplasmas/spiroplasmas, mycobacteria, rickettsia, protozoa, 6 parasites, transmissible spongiform encephalopathies (TSE) agents, and viruses that 7 have been unintentionally introduced into the manufacturing process of a biological 8 product. 9

Cell bank 10 A cell bank is a collection of appropriate containers whose contents are of uniform 11 composition stored under defined conditions. Each container represents an aliquot of 12 a single pool of cells. 13

Cell culture infectious dose 50% (CCID50) 14

A measure of the number of infectious viruses per unit volume of virus suspension 15 as determined in an end-point dilution assay in monolayer cell cultures. One CCID50 16 / mL corresponds to the quantity of viruses capable of producing a cytopathic effect 17 in 50% of replicate cell cultures per mililitre of virus suspension. 18

Cell seed 19

A quantity of vials containing well-characterized cells derived from a single tissue or 20 cell of human or animal origin stored frozen in liquid nitrogen in aliquots of uniform 21 composition, one or more of which would be used for the production of a master cell 22 bank. 23

Cell substrates 24

Cells used for the production of a vaccine. Cells are derived from a master cell bank 25 or a working cell bank. 26

Dengue febrile illness (DFI) 27

DFI is defined by the detection of dengue virus in patients with two days of fever 28 irrespective of severity of illness. 29

Final lot 30

A collection of sealed final containers of finished vaccine that is homogeneous with 31 respect to the risk of contamination during filling and freeze-drying. All the final 32 containers should, therefore, have been filled from one vessel of final tetravalent 33 bulk and freeze-dried under standardized conditions in a common chamber in one 34 working session. 35


Final tetravalent bulk 1

The homogeneous finished tetravalent vaccine prepared from one or more virus 2 harvest pools in the vessel from which the final containers are filled. 3

Genetically modified organism (GMO) 4

An organism in which the genetic material has been altered in a way that does not 5 occur naturally by mating and /or natural recombination. 6

Immunofocus-forming unit (IFU) 7

A measure of the number of infectious viruses capable of forming plaques that can 8 be detected using dengue-specific antisera and a counter-stain in monolayer cell 9 cultures per unit volume of virus suspension. 10

Master cell bank (MCB) 11

A quantity of well-characterized cells of animal or other origin, derived from a cell 12 seed at a specific population doubling level (PDL) or passage level, dispensed into 13 multiple containers, cryopreserved, and stored frozen under defined conditions, such 14 as the vapour or liquid phase of liquid nitrogen in aliquots of uniform composition. 15 The master cell bank is prepared from a single homogeneously mixed pool of cells. 16 It is considered best practice that the master cell bank is used to derive working cell 17 banks. 18

Virus master seed (VMS) 19

A suspension of vaccine virus that has been aliquoted into identical vials and stored 20 at a temperature and under conditions deemed to stabilize the virus in each container. 21 The VMS is used as a source of infectious virus for generation of each virus working 22 seed lot. 23

Monovalent virus pool 24

A monovalent virus pool is a suspension of single serotype of dengue virus and may 25 be the result of one or more single harvests or multiple parallel harvests of the same 26 virus serotype collected into a single vessel before clarification. 27

Multiple parallel harvest 28

A pool of harvests coming from multiple cultures that are initiated in parallel from 29 the same ampoule of the same working cell bank infected together by the same virus 30 suspension of the same virus working seed lot 31

Plaque-forming unit (PFU) 32

A measure of the number of infectious viruses capable of forming plaques, as 33 detected by virtue of a cytopathic effect, in monolayer cell cultures per unit volume 34 of virus suspension. 35


Production cell culture 1

A collection of cell cultures used for biological production that have been prepared 2 together from one or more containers from the working cell bank or, in case of 3 primary cell cultures, from the tissues of one or more animals. 4

Single harvest 5

A quantity of virus suspension harvested from production cell cultures inoculated 6 with the same working seed lot and processed together in a single production run. 7

Working cell bank (WCB) 8

A quantity of well-characterized cells of animal or other origin, derived from the 9 master cell bank at a specific PDL or passage level, dispensed into multiple 10 containers, cryopreserved, and stored frozen under defined conditions, such as in the 11 vapour or liquid phase of liquid nitrogen in aliquots of uniform composition. The 12 working cell bank is prepared from a single homogeneously mixed pool of cells. One 13 or more of the WCB containers is used for each production culture. 14

Virus working seed (VWS) 15

A quantity of virus of uniform composition, well characterized, derived from a virus 16 master seed lot (see above) by passage, in an approved production cell line. The 17 working seed lot is used for the production of single harvest. 18 19

A.2 General manufacturing requirements 20

The general requirements for manufacturing establishments contained in the WHO 21 Good manufacturing practices for biological products (57) should be applied by 22 establishments manufacturing dengue tetravalent vaccine. 23

Separate manufacturing areas for each of the four dengue serotypes as well as 24 tetravalent vaccine formulation may be used. Alternatively, manufacturing areas may 25 be used on a campaign basis with adequate cleaning between campaigns to ensure 26 that cross-contamination does not occur. 27

Production steps and quality-control operations involving manipulations of live virus 28 should be conducted under the appropriate biosafety level as agreed with the national 29 regulatory authority and country biosafety laws. 30

31

A.3 Control of source materials 32

A.3.1 Cell cultures for virus production 33

A.3.1.1 Conformity with WHO requirements 34


Dengue viruses used in producing tetravalent dengue vaccine should be propagated 1 in cell substrates which meet the WHO Recommendations for the evaluation of 2 animal cell cultures as substrates for the manufacture of biological medicinal 3 products and for the characterization of cell banks (73) and approved by the national 4 regulatory authority. All information on the source and method of preparation of the 5 cell culture system used should be made available to the national regulatory 6 authority. 7

A.3.1.2 Types of cell culture 8

Dengue vaccine candidates have been produced in fetal rhesus lung diploid cells and 9 in continuous cell lines. For fetal rhesus lung diploid cells and continuous cells, 10 sections A.3.1.3 and A.3.1.4 apply. 11

A.3.1.3 Master cell bank and working cell bank 12

The use of a cell line such as fetal rhesus lung diploid cells or Vero cells for the 13 manufacture of dengue vaccines should be based on the cell bank system. The cell 14 seed should be approved by the national regulatory authority. The maximum number 15 of passages or population doubling (PDL) allowable between the cell seed, the WCB 16 and the production passage levels should be established by the manufacturer and 17 approved by the national regulatory authority. Additional tests may include: 18

— Propagation of the MCB or WCB cells to or beyond the maximum in vitro age for 19 production; and 20

— Examination for the presence of retroviruses and tumorigenicity in an animal test 21 system (73). 22

WHO has established a bank of Vero cells, designated as WHO Vero reference cell 23 bank (RCB) 10-87 that has been characterized in accordance with the WHO 24 Requirements for continuous cell lines used for biologicals production, WHO 25 Technical Report Series 745, 1987 (56) . The cell bank is available to manufacturers 26 as a well-characterized starting material for preparation of their own master and 27 working cell banks on application to the Coordinator, Quality, Safety and Standards, 28 WHO, Geneva, Switzerland (73). 29 30 In normal practice a master cell bank is expanded by serial subculture up to a 31 passage number (or population doubling, as appropriate) selected by the 32 manufacturer and approved by the national regulatory authority, at which point the 33 cells are combined to give a single pool distributed into ampoules and preserved 34 cryogenically to form the WCB. 35 36 The manufacturer’s WCB is used for the preparation of production cell culture, and 37 thus for production of vaccine batches 38

A.3.1.4 Test on MCB and WCB 39

The master and working cell banks should be characterized according to the WHO 40 Recommendations for the evaluation of animal cell cultures as substrates for the 41 manufacture of biological medicinal products and for the characterization of cell 42


banks (73). 1

A.3.1.5 Cell culture medium 2

Serum used for propagating cells should be tested to demonstrate freedom from 3 bacteria, fungi and mycoplasmas, according to the requirements given in Part A, 4 sections 5.2 and 5.3 of the General requirements for the sterility of biological 5 substances (Requirements for biological substances, no. 6) (55, 60), and from 6 infectious viruses. 7

Appropriate tests for detecting viruses in bovine serum are given in Appendix 1 of 8 the WHO Recommendations for the evaluation of animal cell cultures as substrates 9 for the manufacture of biological medicinal products and for the characterization of 10 cell banks (73). 11

Validated molecular tests for bovine viruses might replace the cell-culture tests of 12 bovine sera if agreed by the national regulatory authority. 13

As an additional monitor of quality, sera may be examined for freedom from 14 endotoxin. 15

Irradiation may be used to inactivate potential contaminant viruses, recognizing that 16 some viruses are relatively resistant to irradiation. 17

The sources(s) of animal components used in culture medium should be approved by 18 the national regulatory authority. These components should comply with current 19 guidelines in relation to animal transmissible spongiform encephalopathies (62, 66). 20

Human serum should not be used. If human albumin is used, it should meet the 21 revised Requirements for the collection, processing and quality control of blood, 22 blood components and plasma derivatives (Requirements for biological substances 23 no. 27) (59), as well as current guidelines in relation to human transmissible 24 encephalopathies (62, 66). 25

The use of human albumin as a component of a cell culture medium requires careful 26 consideration due to potential difficulties with the validity period of albumin (which 27 is based on the length of time for which it is suitable for use in clinical practice) in 28 relation to the potential long-term storage of monovalent bulks of each dengue 29 serotype. 30

Penicillin and other beta-lactams should not be used at any stage of the manufacture. 31 Other antibiotics may be used at any stage in the manufacture provided that the 32 quantity present in the final product is acceptable to the national regulatory authority. 33 Nontoxic pH indicators may be added, e.g. phenol red at a concentration of 0.002%. 34 Only substances that have been approved by the national regulatory authority may be 35 added. 36

If porcine or bovine trypsin is used for preparing cell cultures, it should be tested and 37 found free of cultivable bacteria, fungi, mycoplasmas and infectious viruses as 38 appropriate (73). The methods used to ensure this should be approved by the national 39 regulatory authority. 40

The source(s) of trypsin of bovine origin, if used, should be approved by the national 41


regulatory authority and should comply with current guidelines in relation to animal 1 transmissible spongiform encephalopathies (62, 66). 2

A.3.2 Virus seeds 3

A.3.2.1 Vaccine virus strains 4

The strains of DENV 1-4 viruses attenuated either by serial passage in cell cultures 5 or by recombinant DNA technology used in the production of candidate tetravalent 6 dengue vaccine should be thoroughly characterized. This will include their historical 7 records, such as information on the origin of the strain, cell culture passage history, 8 method of attenuation, results of preclinical and clinical studies demonstrating 9 attenuation, whether the strains have been biologically or molecularly cloned prior to 10 generation of the master seed; their genome sequence; and the passage level at which 11 clinical trials were performed, as well as the results of the clinical studies. 12

Only strains approved by the national regulatory authority should be used. 13

Strains of dengue recombinant viruses used for master and working seeds to produce 14 vaccine candidates should comply with the additional specifications given below 15 (section <A.3.2.2 Strains derived by molecular methods>). 16

A.3.2.2 Strains derived by molecular methods 17 If vaccine seeds derived by recombinant DNA technology are used, and because this 18 is a live attenuated vaccine, the candidate vaccine is considered a GMO in several 19 countries and should comply with the regulations of the producing and recipient 20 countries regarding GMOs. An environmental risk assessment should be undertaken 21 according to Part D of these Guidelines. 22

The nucleotide sequence of any cDNA clone used to generate vaccine virus stocks 23 should be determined prior to transfection of DNA-derived RNA into the cell 24 substrate. The cell substrate used for transfection to generate the virus should be 25 appropriate for human vaccine production and approved by the national regulatory 26 authority. 27

Pre-seed lot virus stocks derived from passaging of the primary virus stock should 28 also be sequenced as part of preclinical evaluation. 29

Viral vaccine seeds that are directly re-derived from RNA extracted from virus, to 30 reduce the risk of previous contamination by TSE or other adventitious agents, are 31 considered as new vaccine seeds and should be appropriately characterized to 32 demonstrate comparability with the starting virus seed. 33

A.3.2.3 Virus seed lot system 34

The production of vaccine should be based on the master and working seed lot 35 system to minimize the number of tissue culture passages needed for vaccine 36 production. Seed lots should be prepared in the same type of cells using the same 37 conditions for virus growth (other than scale) as those used for production of final 38 vaccine. 39


The virus working seed lot should be prepared by defined number of passages from 1 the virus master seed lot by a method and a passage level from the original virus 2 seed that is established through clinical and vaccine development studies. Once the 3 passage level of the working seed lot is established, it may not be changed without 4 approval from the national regulatory authority. 5 6 Virus seed lots should be stored in a dedicated temperature-monitored freezer at a 7 temperature that ensures stability upon storage. It is recommended that a large 8 working virus seed lot be set aside as the basic material for use by the manufacturer 9 for the preparation of each batch of vaccine. 10 11 Full in vitro and in vivo testing for detecting adventitious agents should be 12 conducted on either master or working seed lots. 13

A.3.2.4 Control cell cultures for virus seeds 14

In agreement with national regulatory authorities, tests on control cell cultures may 15 be required and performed as described in section A.4.1. 16

A.3.2.5 Tests on virus master and working seed lots 17

A.3.2.5.1 Identity 18 Each virus master and working seed lot should be identified as the appropriate 19 dengue virus serotype by immunological assay or by molecular methods. 20

A.3.2.5.2 Genetic/phenotypic characterization 21 Different live dengue vaccine viruses may have significantly different properties. 22 Such differences may influence the tests to be used to examine their genetic and 23 phenotypic stability relevant to consistency of production. The applicable tests will 24 be identified in the course of the nonclinical evaluation of the strains. Each seed 25 should be characterized by full-length nucleotide sequence determination and by 26 other relevant laboratory and animal tests, which will provide information on the 27 consistency of each virus seed. 28

Mutations introduced during derivation of each vaccine strain should be maintained 29 in the consensus sequence, unless spontaneous mutations induced during tissue 30 culture passage were shown to be innocuous in non-clinical and small-scale clinical 31 trials. Some variations in the nucleotide sequence of virus population on passage are 32 to be expected, but what is acceptable should be based on experience in production 33 and clinical use. 34

For any new virus master and working seeds, it is recommended that the first three 35 consecutive consistency vaccine lots be analysed for consensus sequence changes 36 from the virus master seed. The nucleotide sequence results should be used to 37 demonstrate the consistency of the production process. 38

Routine nucleotide sequence analysis of final bulk vaccine is not recommended. 39

A.3.2.5.3 Tests for bacteria, fungi mycoplasmas and mycobacteria 40


Each virus master and working seed lot should be shown to be free from bacterial, 1 fungal and mycoplasmal contamination by appropriate tests as specified in Part A, 2 sections 5.2 and 5.3, of the General requirements for the sterility of biological 3 substances (Requirements for biological substances, no. 6) (55, 60). Nucleic acid 4 amplification techniques (NAAT) alone or in combination with cell culture, with an 5 appropriate detection method, might be used as an alternative to one or both of the 6 compendial mycoplasma detection methods after suitable validation and agreement 7 from the national regulatory authority (73). 8

Seed lots should be shown to be free from Mycobacteria by a method approved by 9 the national regulatory authority. NAAT might be used as an alternative to 10 mycobacteria microbiological culture method and/or to the in vivo guinea-pigs test 11 for the detection of mycobacteria after suitable validation and agreement from the 12 national regulatory authority (73). 13

A.3.2.5.4 Tests for adventitious agents 14 Each virus working seed lot and/or master seed lot should be tested in cell cultures 15 for adventitious viruses relevant to the passage history of the seed virus. Where 16 antisera are used to neutralize dengue virus or the recombinant dengue virus, the 17 antigen used to generate the antisera should be produced in cell culture from a 18 species different from that used for the production of the vaccine and free from 19 extraneous agents. Simian and human cell cultures inoculated with the virus-20 antibody mixture should be observed microscopically for cytopathic changes. For 21 virus grown in simian or human cells, the neutralized virus is tested on a separate 22 culture of these cells. If other cell systems are used cells of that species, but from a 23 separate batch, are also inoculated. At the end of the observation period, the cells 24 should be tested for haemadsorbing viruses.. 25

Each virus master seed lot or working seed lot should also be tested in animals that 26 include guinea pigs, adult mice, and suckling mice. For test details, refer to the 27 section B.11 of the WHO Recommendations for the evaluation of animal cell 28 cultures as substrates for the manufacture of biological medicinal products and for 29 the characterization of cell banks (73). Additional testing for adventitious viruses 30 may be performed using validated NAAT. 31

New molecular methods with broad detection capabilities are being developed for 32 adventitious agent detection. These methods include degenerate NAAT for whole 33 virus families with analysis of the amplicons by hybridization, sequencing or mass 34 spectrometry; NAAT with random primers followed by analysis of the amplicons on 35 large oligonucleotide micro-arrays of conserved viral sequencing or digital 36 subtraction of expressed sequences; and high throughput sequencing. These methods 37 might be used in the future to supplement existing methods or as alternative methods 38 to both in vivo and in vitro test after appropriate validation and agreement from the 39 national regulatory authority. 40

A.3.2.5.5 Tests in experimental animals 41

A.3.2.5.5.1 Tests in non-human primates 42


All vaccine candidates should be evaluated at least once during pre-clinical 1 development for neurotropism in non-human primates, as detailed in Part B 2 (nonclinical evaluation). Candidate vaccines that are homogeneously “dengue” in 3 terms of genetics are not expected to be neurotropic in such a test, but where 4 attenuation has been achieved by recombination of dengue genes with those of a 5 different virus species that itself displays neurotropism (e.g. dengue/yellow fever 6 recombinants); the master seed should be tested in non-human primates. If these tests 7 were not performed at the master seed level, they should be performed at working 8 seed level. 9

National regulatory authorities may decide that such testing does not need to be 10 repeated each time a novel Working seed is derived, if results of a well conducted 11 monkey neurovirulence assay on the Master seed are negative. Recent data suggest 12 that certain small animal models for neurovirulence may serve as a surrogate for 13 non-human primates, at least where viruses expressing YF strain 17D genes are 14 concerned (37). National regulatory authorities may eventually wish to consider 15 accepting results of such studies as a surrogate for studies using non-human primates 16 to evaluate neurovirulence of novel dengue vaccines. 17

18

Test for neurotropism 19 20

To provide assurance that a candidate vaccine virus is not unexpectedly 21 neurovirulent, each vaccine strain of each serotype should be tested for 22 neurovirulence in monkeys by inoculation of Macaca mulatta (rhesus), cynomolgus 23 or other susceptible species of monkey in the course of preclinical evaluation. 24

Prior to testing for neurotropism, the neutralizing antibody test should be used to 25 assess the immune status of non-human primates to both dengue and yellow fever 26 viruses. Tests should follow the WHO Recommendations to assure the quality, safety 27 and efficacy of live attenuated yellow fever vaccines (74). 28

A.3.2.5.5.2 Tests in suckling mice 29

The virulence of different vaccine candidates in mice will depend on the strains of 30 virus and mouse. Novel vaccines that reach the clinical phase of development in 31 many cases were tested for neurovirulence in suckling and adult mice during the pre-32 clinical phase of development. 33

While mice are not considered a good model for dengue, suckling and adult mice 34 have been used to assess the neurovirulence of dengue/yellow fever recombinant 35 vaccines (17, 18). A mouse test might be considered in order to demonstrate 36 consistency of characteristics of dengue/yellow fever recombinant viruses during 37 production (see A.4.2.4.7). 38

A.3.2.5.6 Virus titration for infectivity 39 Each virus master and working seed lot should be assayed for infectivity in a 40 sensitive assay in cell culture. Depending on the results obtained in preclinical 41 studies, plaque assays, CCID50 assays, IFF assays or CCID50 with a molecular 42


readout such as quantitative polymerase chain reaction (QPCR) may be used. All 1 assays should be validated. 2

3

A.4 Control of vaccine production 4

A.4.1 Control of production cell cultures 5

Where the NRA requires the use of control cells, the following procedures should be 6 followed. From the cells used to prepare cultures for production of vaccine, a 7 fraction equivalent to at least 5 % of the total or 500 mL of cell suspension, or 100 8 million cells, should be used to prepare uninfected control cell cultures. These 9 control cultures should be observed microscopically for cytopathic and morphologic 10 changes attributable to the presence of adventitious agents for at least 14 days at a 11 temperature of 35-37 °C after the day of inoculation of the production cultures, or 12 until the time of final virus harvest, whichever comes last. At the end of the 13 observation period, supernatant fluids collected from the control culture should be 14 tested for the presence of adventitious agents as described below. Samples that are 15 not tested immediately should be stored at -60 C or lower, until such tests can be 16 conducted. 17

If adventitious agent testing of control cultures yields a positive result, the harvest of 18 virus from the parallel vaccine virus-infected cultures should not be used for 19 production. 20

For the test to be valid, not more than 20 % of the control culture flasks should have 21 been discarded, for any reason, by the end of the test period. 22

A.4.1.1 Test for haemadsorbing viruses 23

At the end of the observation period, a fraction of control cells comprising no less 24 than 25% of the total should be tested for the presence of haemadsorbing viruses, 25 using guinea-pig red blood cells. If the guinea pig red blood cells have been stored 26 prior to use in the hemadsorption assay, the duration of storage should not have 27 exceeded 7 days, and the storage temperature should have been in the range of 2–8 28 °C. 29

In some countries, the national regulatory authority requires that additional tests for 30 haemadsorbing viruses be performed using red blood cells from other species 31 including those from humans (blood group O), monkeys, and chickens (or other 32 avian species). For all tests, readings should be taken after incubation for 30 minutes 33 at 0–4 °C, and again after a further incubation for 30 minutes at 20–25 °C. The test 34 with monkey red cells should be read once more after an additional incubation for 30 35 minutes at 34–37 °C. 36

For the tests to be valid, not more than 20 % of the culture vessels should have been 37 discarded for any reason by the end of the test period. 38


A.4.1.2 Tests for cytopathic, adventitious agents in control cell 1 fluids 2

Supernatant culture fluids from each of control cell culture flasks or bottles 3 collected at the time of harvest should be tested for adventitious agents. A 10-mL 4 sample of the pool should be tested in the same cell substrate, but not the same cell 5 batch as that used for vaccine production, and an additional 10-mL sample of each 6 pool should be tested in other types of both human and monkey cells. 7

Each sample should be inoculated into cell cultures in such a way that the dilution of 8 the pooled fluid in the nutrient medium does not exceed 1:4. The surface area of the 9 flask should be at least 3 cm2 per mL of pooled fluid. At least one flask of the cells 10 should remain uninoculated, as a control. 11

The inoculated cultures should be incubated at a temperature of 35–37 °C and should 12 be examined at intervals for cytopathic effects over a period of at least 14 days. 13

Some national regulatory authorities require that, at the end of this observation 14 period, a subculture is made in the same culture system and observed for at least an 15 additional 7 days. Furthermore, some national regulatory authorities require that 16 these cells should be tested for the presence of haemadsorbing viruses. 17

For the tests to be valid, not more than 20 % of the culture vessels should have been 18 discarded for any reason by the end of the test period. 19

A.4.1.3 Identity test 20

At the production level, the cells should be identified by means of tests approved by 21 the national regulatory authority. Suitable methods are, but are not limited to, 22 biochemical tests (e.g. isoenzyme analyses), immunological tests (e.g. major 23 histocompatibility complex assays), cytogenetic tests (e.g. for chromosomal 24 markers),and tests for genetic markers (e.g. DNA fingerprinting). 25

A.4.2 Production and harvest of monovalent virus 26

A.4.2.1 Cells used for vaccine production 27

On the day of inoculation with the working seed virus, each production cell culture 28 flask (or bottle, etc) and/or cell culture control flask should be examined for 29 cytopathic effect potentially caused by infectious agents. If such examination shows 30 evidence of the presence in any flask of an adventitious agent, all cell cultures should 31 be discarded. 32

If animal serum is used in growth medium, the medium should be removed from the 33 cell culture either before or after inoculation of working virus seed. Prior to 34 beginning virus harvests, the cell cultures should be rinsed and the growth medium 35 replaced with serum-free maintenance medium. 36

Penicillin and other beta-lactam antibiotics should not be used at any stage of 37 manufacture. 38 Minimal concentrations of other suitable antibiotics may be used if approved by the 39


national regulatory authority. 1

A.4.2.2 Virus inoculation 2

Cell cultures are inoculated with dengue working seed virus at an optimal and 3 defined multiplicity of infection. After viral adsorption, cell cultures are fed with 4 maintenance medium and incubated at a temperature within a defined range and for a 5 defined period. 6

The multiplicity of infection, temperature range and duration of incubation will 7 depend on the vaccine strain and production method, and specifications should be 8 defined by each manufacturer. 9

A.4.2.3 Monovalent virus harvest pools 10

Vaccine virus is harvested within a defined period post-inoculation. A monovalent 11 harvest may be the result of one or more single harvests or multiple parallel harvests. 12 Samples of monovalent virus harvest pools should be taken for testing and stored at 13 a temperature of -60 °C or below. The sponsor should submit data to support the 14 conditions chosen for these procedures. 15

The monovalent virus harvest pool may be clarified or filtered to remove cell debris 16 and stored at a temperature that ensures stability before being used to prepare the 17 tetravalent final bulk for filling. The sponsor should provide data to support the 18 stability of the bulk over the duration of the chosen storage conditions as well as to 19 support the choice of storage temperature . 20

Harvests derived from continuous cell lines should be subjected either to further 21 purification to minimize the amount of cellular DNA and/or treatment with DNase to 22 reduce the size of the DNA. 23

A.4.2.4 Tests on monovalent virus harvest pools 24

A.4.2.4.1 Identity 25

Each monovalent virus harvest pool should be identified as the appropriate dengue 26 virus serotype by immunological assay on cell cultures using specific antibodies or 27 by molecular methods (see section A.6.1) approved by the national regulatory 28 authority. 29

A.4.2.4.2 Tests for bacteria, fungi, mycoplasmas, and mycobacteria 30 Each monovalent virus harvest pool should be shown to be free from bacterial, 31 fungal and mycoplasmal contamination by appropriate tests as specified in Part A, 32 sections 5.2 and 5.3, of the General requirements for the sterility of biological 33 substances (Requirements for biological substances, no. 6) (55, 60). 34

NAAT alone or in combination with cell culture, with an appropriate detection 35 method, might be used as an alternative to one or both of the pharmacopoeial 36 mycoplasma detection methods after suitable validation and the agreement of the 37 national regulatory authority (73). 38


Each monovalent virus harvest pool should be shown to be free from Mycobacteria 1 by an appropriate method approved by the NRA. NAAT might be used as an 2 alternative to mycobacteria microbiological culture method and/or to the in vivo 3 guinea-pig test for the detection of mycobacteria after validation and agreement of 4 the national regulatory authority (73). 5

A.4.2.4.3 Tests for adventitious agents 6

Each monovalent virus harvest pool should be tested in cell culture for adventitious 7 viruses by inoculation into continuous simian kidney cells, cell lines of human 8 origin, and the cell line used for production, but from another batch. Where antisera 9 are used to neutralize dengue virus or the recombinant virus, the antigen used to 10 generate the antisera should be produced in cell culture from a species different from 11 that used for the production of the vaccine and free from extraneous agents. The cells 12 inoculated should be observed microscopically for cytopathic changes. At the end of 13 the observation period, the cells should be tested for haemadsorbing viruses. 14

Additional testing for adventitious viruses may be performed using validated nucleic 15 acid amplification techniques. 16

New molecular methods with broad detection capabilities are being developed. 17 These methods include degenerate NAAT for whole virus families with analysis of 18 the amplicons by hybridization, sequencing or mass spectrometry; DNA 19 amplification with random primers followed by analysis of the amplicons on large 20 oligonucleotide micro-arrays of conserved viral sequencing or digital subtraction of 21 expressed sequences; and high throughput sequencing. These methods might be used 22 in the future to supplement existing methods or as alternative methods to in vitro test 23 after appropriate validation and agreement from the national regulatory authority. 24

A.4.2.4.4 Virus titration for infectivity 25 The titre for each monovalent virus harvest should be determined in a sensitive assay 26 in cell culture. Depending on the results obtained in preclinical studies, plaque 27 assays, CCID50 assays, immunofocus formation assays or CCID50 with a molecular 28 readout such as QPCR may be used. 29

A.4.2.4.5 Tests for host cell proteins 30

The host cell protein profile should be examined as part of characterization studies 31 (73). 32

A.4.2.4.6 Tests for residual cellular DNA 33 For viruses grown in continuous cell line cells, the monovalent harvest pool should 34 be tested for the amount of residual cellular DNA, and the total amount of cell DNA 35 per dose of vaccine should be not more than the upper limit agreed to by the NRA. 36 If this is technically feasible, the size distribution of the DNA should be examined as 37 a characterization test, taking into account the amount of DNA detectable using state 38 of the art methods (73) as approved by the national regulatory authority. 39

A.4.2.4.7 Test for consistency of virus characteristics 40


The dengue virus in the monovalent harvest pool should be tested to compare it with 1 the working seed virus, or suitable comparator, to ensure that the vaccine virus has 2 not undergone critical changes during its multiplication in the production culture 3 system. 4

Relevant assays should be identified in preclinical studies and may include virus 5 yield in tissue culture, plaque phenotype, or temperature sensitivity as examples. 6 Other identifying characteristics might also be applicable. 7

Assays for the attenuation of dengue/yellow fever recombinants and others as 8 appropriate include tests in suckling mice. Intracerebral inoculation of suckling mice 9 with serial dilutions of vaccine and yellow fever 17D is followed by the 10 determination of the mortality ratio and survival time. The results obtained with the 11 vaccine are compared to the yellow fever 17D control results. 12

The test may be omitted as a routine test once the consistency of the production 13 process has been demonstrated on a significant number of batches in agreement with 14 the national regulatory authority. Where there is a significant change in the 15 manufacturing process, the test should be reintroduced. 16

A.4.2.5 Storage 17

Monovalent virus harvest pools should be stored at a temperature that ensures 18 stability until tetravalent vaccine formulation. 19

A.4.3 Final tetravalent bulk lot 20

A.4.3.1 Preparation of final tetravalent bulk lot 21

The final tetravalent bulk lot should be prepared from monovalent virus pools of the 22 four dengue virus subtypes using a defined virus concentration of each component. 23

The operations necessary for preparing the final bulk lot should be conducted in such 24 a manner as to avoid contamination of the product. 25

In preparing the final bulk, any excipients (such as diluents or stabilizer) that is 26 added to the product should have been shown to the satisfaction of the national 27 regulatory authority not to impair the safety and efficacy of the vaccine in the 28 concentration used. 29

A.4.3.2 Tests on the final tetravalent bulk lot 30

A.4.3.2.1 Residual animal serum protein 31 If appropriate, a sample of the final bulk should be tested to verify that the level of 32 serum is less than 50 ng per human dose. Alternatively, if the amount of serum is too 33 low in the final bulk to be quantified, the test may be performed on the monovalent 34 bulk. 35

A.4.3.2.2 Sterility 36

Except where it is subject to in-line sterile filtration as part of the filling process, 37


each final bulk suspension should be tested for bacterial and fungal sterility 1 according to Part A, section 5.2 of the General requirements for the sterility of 2 biological substances (Requirements for biological substances, no. 6) (55), or by a 3 method approved by the national regulatory authority. 4

A.4.3.3 Storage 5

Prior to filling, the final bulk suspension should be stored under conditions shown by 6 the manufacturer to retain the desired viral potency. 7 8

A.5 Filling and containers 9

The requirements concerning good manufacturing practices for biological products 10 (57) appropriate to a vaccine should apply. 11

Care should be taken to ensure that the materials from which the container and, if 12 applicable, the closure are made do not adversely affect the infectivity (potency) of 13 the vaccine under the recommended conditions of storage. 14

A final filtration could be included during the filling operations. 15

The manufacturer should provide the national regulatory authority with adequate 16 data to prove the product is stable under appropriate conditions of storage and 17 shipping. 18 19

A.6 Control tests on final lot 20

The following tests should be carried out on the final lot. 21

A.6.1 Vaccine 22

A.6.1.1 Inspection of final containers 23

Each container in each final lot should be inspected visually, and those showing 24 abnormalities should be discarded. 25

A.6.1.1.1 Appearance 26

The appearance of the freeze-dried or liquid vaccine should be described with respect 27 to its form and colour. In the case of freeze-dried vaccines, a visual inspection should 28 be performed of the freeze-dried vaccine, the diluent, and the reconstituted vaccine. 29

A.6.1.2 pH 30

The pH of the final lot should be tested in a pool of final containers and an 31 appropriate limit set to guarantee virus stability. In the case of freeze-dried vaccines, 32 pH should be measured after reconstitution of the vaccine with the diluent. 33

A.6.1.3 Identity 34

Each monovalent component of a tetravalent dengue vaccine lot should be identified 35


as dengue or recombinant virus type-1, -2, -3 or -4 by immunological assay using 1 specific antibodies or by molecular methods. The methods used for the potency 2 assay (A.6.1.5) may serve as the identity test. 3

A.6.1.4 Sterility 4

Vaccine should be tested for bacterial and fungal sterility according to the 5 requirements in Part A, section 5.2 of the General requirements for the sterility of 6 biological substances (Requirements for biological substances, no. 6) (55) by 7 acceptable methods approved by the national regulatory authority. 8

A.6.1.5 Potency 9

At least three containers of each tetravalent vaccine lot should be assayed for 10 infectivity in a validated assay in cell culture. The titre of each individual serotype 11 should be determined. 12

A.6.1.6 Thermal stability 13

The thermal stability test is to demonstrate consistency of production. Additional 14 guidance on evaluation of vaccine stability is provided in the WHO Guideline on 15 stability evaluation of vaccines (71). At least three containers of tetravalent vaccine 16 should be incubated at the appropriate elevated temperature for appropriate time (e.g. 17 37 °C for 7 days) depending on products. The geometric mean infectious virus titre 18 of the containers for each individual virus components that have been exposed 19 should not have decreased during the period of exposure by more than an amount 20 (e.g. 1 log) justified by production data and approved by the national regulatory 21 authority. Titration of non-exposed and exposed containers should be done in 22 parallel. A validity control reagent of each of the four virus components should be 23 included in each assay to validate the assay. 24

A.6.1.7 General safety 25

Each filling lot should be tested for unexpected toxicity (sometimes called abnormal 26 toxicity) using a general safety test approved by the national regulatory authority. 27

This test may be omitted for routine lot release once consistency of production has 28 been established to the satisfaction of the national regulatory authority and when 29 good manufacturing practices are in place. Each lot, if tested, should pass a general 30 safety test. 31

A.6.1.8 Residual moisture (if appropriate) 32

The residual moisture in each freeze-dried lot should be conducive to the stability of 33 the product and the upper limit of the moisture content should be approved by the 34 national regulatory authority based on results of stability testing.. 35

A.6.1.9 Residual antibiotics (if applicable) 36

If any antibiotics are added in the vaccine production, the content of the residual 37 antibiotics should be determined and be within limits approved by the national 38 regulatory authority. 39


A.6.2 Diluent 1

The recommendations given in the Good manufacturing practices for 2 pharmaceutical products (61) should apply for the manufacturing and control of 3 diluents used to reconstitute live-attenuated dengue vaccines. An expiry date should 4 be established for the diluent based upon stability data. For lot release of the diluent, 5 tests for identity, appearance, pH, volume, sterility, and the content of key 6 components should be done. 7

A.7 Records 8

The recommendations of the Good manufacturing practices for biological products 9 (57) pp. 27–28, should apply, as appropriate for the level of development of the 10 candidate vaccine. 11

A.8 Samples 12

A sufficient number of samples should be retained for future studies and needs. 13 Vaccine lots that are to be used for clinical trials may serve as reference materials in 14 the future, and a sufficient number of vials should be reserved and appropriately 15 stored for that purpose. 16

A.9 Labelling 17

The recommendations of the Good manufacturing practices for biological products 18 (57) pp. 26–27, appropriate for a candidate vaccine should apply, with the addition 19 of the following: 20 21

The label on the carton enclosing one or more final containers, or the leaflet 22 accompanying the container, should include: 23

— a statement that the candidate vaccine fulfils Part A of these 24 Recommendations; 25

— a statement of the nature of the preparation, specifying the designation of the 26 strains of dengue or recombinant viruses contained in the live attenuated 27 tetravalent vaccine, the minimum number of infective units per human dose, 28 the nature of any cellular systems used for the production of the vaccine, and 29 whether the vaccine strains were derived by molecular methods; 30

— a statement of the nature and quantity, or upper limit, of any antibiotic present 31 in the vaccine; 32

— an indication that contact with disinfectants is to be avoided; 33

— a statement concerning the photosensitivity of the vaccine, cautioning that 34 both lyophilized and reconstituted vaccine should be protected from light; 35

— a statement indicating the volume and nature of diluent to be added to 36 reconstitute the vaccine, and specifying that the diluent to be used is that 37 supplied by the manufacturer; and 38

— a statement that after the vaccine has been reconstituted, it should be used 39


without delay, or if not used immediately, stored between 2 °C and 8 °C and 1 protected from light for a maximum period defined by stability studies. 2

3

A.10 Distribution and shipping 4

The recommendations given in the Good manufacturing practices for biological 5 products (57) appropriate for a candidate vaccine should apply. 6

Shipments should be maintained at temperatures of 8 °C or below, and packages 7 should contain cold-chain monitors (72). 8

9

A.11 Stability, storage and expiry date 10

The recommendations given in the Good manufacturing practices for biological 11 products (57) and the Guidelines on stability evaluation of vaccines (71) appropriate 12 for a candidate vaccine should apply. The statements concerning storage temperature 13 and expiry date that appear on the primary or secondary packaging should be based 14 on experimental evidence and should be submitted for approval to the national 15 regulatory authority. 16 17

A.11.1 Stability testing 18

Stability testing should be performed at different stages of production, namely on 19 single harvests, purified bulk, final bulk and final lot. Stability-indicating parameters 20 should be defined or selected appropriately according to the stage of production. It is 21 advisable to assign a shelf-life to all in-process materials during vaccine production, 22 in particular stored intermediates such as single harvests, purified bulk and final 23 bulk. 24

The stability of the vaccine in its final container and at the recommended storage 25 temperatures should be demonstrated to the satisfaction of the national regulatory 26 authorities on at least three lots of final product. Accelerated thermal stability tests 27 may be undertaken on each final lot to give additional information on the overall 28 stability of a vaccine (see section A.6.1.6). 29

The formulation of vaccine and adjuvant (if used) should be stable throughout its 30 shelf-life. Acceptable limits for stability should be agreed with national authorities 31

A.11.2 Storage conditions 32

Before being distributed by the manufacturing establishment or before being issued 33 from a storage site, the vaccine should be stored at a temperature shown by the 34 manufacturer to be compatible with a minimal loss of titre. The maximum duration 35 of storage should be fixed with the approval of the national regulatory authority and 36 should be such as to ensure that all quality specifications for final product including 37 the minimum titre specified on the label of the container (or package) will still be 38 maintained until the end of the shelf-life. 39


A.11.3 Expiry date 1

The expiry date should be defined on the basis of shelf-life and supported by the 2 stability studies with the approval of the national regulatory authority and should 3 relate to the date of the last satisfactory determination, of potency (infectious titer), 4 performed in accordance with section A.6.1.5, i.e. the date on which the cell cultures 5 were inoculated. If the vaccine is stored at a temperature lower than that used for 6 stability studies and intended for release without re-assay, the expiry date is 7 calculated from the date of removal from cold storage. The expiry dates for the 8 vaccine and the diluent may be different. 9

10


Part B. Nonclinical evaluation of dengue tetravalent 1

vaccines (live, attenuated) 2

B.1 General remarks 3

4

Nonclinical evaluation of a live dengue vaccine includes in vitro and in vivo 5 testing required prior to initiation of the clinical phase of the vaccine development 6 program. This testing should yield information suggesting the safety and 7 potential for efficacy of a dengue vaccine candidate. Testing may continue in 8 parallel with the clinical phase of product development. Tests should include 9 product characterization at each stage of manufacture (including quantification of 10 contaminants such as cellular proteins and DNA), proof of 11 concept/immunogenicity studies (including dose ranging in animals, etc.), 12 toxicology if required by the national regulatory authority, establishment of a test 13 for potency to be used throughout, and safety testing in animals (see Table B1). 14 These Guidelines should be read in conjunction with the WHO Guidelines on 15 nonclinical evaluation of vaccines (64) and are specifically aimed at nonclinical 16 evaluation of a live, attenuated dengue vaccine. 17

18 Although there is no animal model that precisely mimics dengue disease in 19 humans, animal models have been and are being used in studies on 20 immunogenicity, protective activity, toxicology, and safety. Animal models are 21 briefly reviewed at the time of preparing these Guidelines to highlight the latest 22 developments and to provide better understanding of their use in vaccine 23 development. 24

25


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


B.2 Product development and characterization 1

2 It is critical that vaccine production processes are standardized and controlled 3 to ensure consistency of manufacture in support of nonclinical data suggesting 4 potential safety and efficacy in humans. This is a pre-requisite for entering 5 the clinical trial phase. 6 7 Each of the attenuated virus candidates in the tetravalent dengue vaccine 8 formulation should be characterized to define as far as is practical the critical 9 genetic and phenotypic markers associated with attenuation. Each vaccine 10 virus should also be evaluated to determine whether the genetic basis of 11 attenuation is stable enough to reduce the risk of reversion to wild-type virus 12 (and consequently virulence), either during manufacture or during replication 13 in a vaccinee following inoculation, using available in vivo and in vitro 14 approaches. To this end, laboratory and animal studies should define genetic 15 changes in the virus genome that are associated with phenotypic attenuation 16 markers. 17 18 It is helpful when possible also to identify in vitro phenotypic markers that 19 correlate with attenuation. Both types of markers are useful to detect reversion 20 events and to differentiate vaccine strains from wild-type virus strains in 21 epidemiological surveillance following human immunization. 22 23 Qualification of each attenuated vaccine strain should include obtaining the 24 consensus nucleotide sequence of the entire genome of the vaccine candidate, 25 using the consensus nucleotide sequence of the genome of the parent virus as 26 a comparator. This is essential to map the sites of attenuating mutations. It 27 may also be possible thereby to determine the genetic basis of any in vitro 28 phenotypic markers that correlate with attenuation. Such markers include but 29 are not limited to plaque size, replication efficiency in mosquito vectors, 30 induction of viremia in non-human primates, suckling mouse neurovirulence, 31 virulence in any other animal model, and temperature sensitivity (8, 11, 12, 25, 32 26). Developers should bear in mind that consensus genome sequencing is 33 unsuitable for identifying minor or quasi-species genomes in a vaccine seed or 34 batch (19). 35 36

B.3 Nonclinical immunogenicity and protective activity 37

38 Assessment of innate and adaptive immune responses in animals can provide 39 evidence that the DEN vaccine has replicated in the host. Animals, 40 particularly mice, have also been valuable in assessing the various elements of 41 the immune response to DENV. Although there is no specific immune 42 correlate of protection, antibodies directed against the virus E protein 43 neutralize the virus and have been shown to protect animals when actively 44 induced by experimental vaccines or when passively administered, prior to 45


challenge. Based on the accumulated data, it is generally accepted that 1 protection in humans should require a DENV-specific neutralizing antibody 2 response. However, a correlation between the titre of neutralizing antibodies 3 in serum, as determined in an in vitro neutralizing antibody assay (e.g. 4 PRNT50), and protection has not been established for any of the four serotypes 5 of virus. 6 7 While protective activity in an animal model does not necessarily predict the 8 protective effect in humans, it provides useful information regarding the 9 immunological potency of the vaccine. 10 11 The immune response or protective activity to each of the four serotypes in a 12 tetravalent DENV vaccine should be assessed, including the quality of 13 response and any potential virological/immunological interference between 14 types. 15 16

B.4 Nonclinical toxicity and safety 17

18

B.4.1 Considerations 19

20 General guidance on the nonclinical safety assessment and design of 21 preclinical studies that apply to dengue vaccines is provided in the WHO 22 Guidelines on nonclinical evaluation of vaccines (64). The term “toxicity” is 23 generally associated with the untoward consequences of the administration of 24 a nonreplicating drug or biological that relate to its direct dose-dependent 25 effect in the test animal. Thus toxicity studies entail the careful analysis of all 26 major organs, as well as tissues near to and distal from the site of 27 administration, to detect unanticipated direct toxic effects typically of a drug 28 or nonreplicating biological agent, over a wide range of doses, including doses 29 sufficiently exceeding the intended clinically relevant dose amount. It is 30 generally expected that if a live attenuated vaccine does not replicate in the 31 test animal, direct toxic effects are very unlikely to be detected. Therefore, 32 vaccine developers are not expected to conduct single dose and repeated dose 33 toxicity studies, which are recommended for inactivated vaccines (sometimes 34 in combination with adjuvants). For live vaccines the emphasis is on the 35 demonstration of nonclinical safety as a consequence of vaccine virus 36 replication. 37 38 Nonclinical safety studies of live vaccines should be required for live-39 attenuated vaccines in certain stages of development. Such studies are 40 designed with the primary purpose to demonstrate the vaccine(s) are less 41 “virulent’ in the animal host than comparable wild-type viruses and that the 42 vaccine does not exhibit any unexpected harmful tissue tropism and damage 43 or the capacity to elicit a harmful immune response. There is no animal model 44


that replicates human dengue disease adequately (see B.2.1 and B.2.2). 1 However non human primates and mice may provide useful information to 2 characterize the viruses (see B.5.2 and B.5.3). The design of preclinical safety 3 studies should reflect route and frequency of administration as proposed in 4 the protocol to support clinical trials. (64) 5 6 If the live attenuated DENV vaccine is intended to be used to immunize 7 women of childbearing age, developmental/reproductive toxicity studies 8 should be performed according to WHO guidelines (64). 9

B.4.2 Assessment in the non-human primate 10

B.4.2.1 Neurotropism in non-human primates 11

12 The consensus of current opinion is that all live dengue vaccines should be 13 tested once for neurovirulence and/or neurotropism (whether vaccine viruses 14 cause diseases in the CNS or they have affinity to nerve tissues). At this time, 15 the most well-established model for vaccine neurotropism is the NHP, which 16 has been historically used to evaluate new seeds of YF vaccines (17D 17 substrains 17D204- or 17DD-derived) and live polio vaccines. Novel rodent 18 (hamster and mouse) models for YF vaccine virulence are currently under 19 development. A rodent model could eventually be considered in lieu of NHP 20 testing. 21 22 Involvement of the central nervous system in cases of dengue fever and 23 dengue hemorrhagic fever has usually been diagnosed as secondary to 24 vasculitis with resultant fluid extravasation . The rarity of reports of patients 25 with dengue encephalitis suggest that the virus does not typically cross the 26 blood-brain barrier and infect neuronal cells (32). However, since dengue 27 vaccine viruses are genetically altered compared to their wildtype parent 28 viruses, it is advisable to ensure that candidate vaccines have not acquired a 29 neurotropic phenotype as an unintended consequence of the attenuation 30 process. This is a particular issue as regards dengue vaccine viruses that 31 contain YF 17D chimeric genomes, , and it would be of similar special 32 importance in the future, if novel dengue vaccines are derived from the 33 genomes of any other known neuropathic viruses. This evaluation could be 34 done once at an early stage of development, using a master seed or working 35 seed lot of the vaccine. National regulatory authorities would need to decide 36 whether each component of the tetravalent formulation needs to be tested 37 separately for the property of neurovirulence or whether the tetravalent 38 formulation could be tested initially, in which case no further testing of the 39 individual vaccines would need to be done if results of the initial tests were 40 within predefined specifications. 41 42 Testing for neurotropism in the NHP model should follow the WHO 43 recommendations for neurotropism testing of YF vaccines (31, 74). Groups of 44


at least 10 monkeys, determined to be non-immune to DENV and YFV prior 1 to inoculation with the DENV master seed, should be inoculated 2 intracerebrally in the frontal lobe. A control group of 10 NHPs, also 3 demonstrated to be non immune to DENV and YFV, should receiveYF-17D 4 as the control group. All NHPs should be observed for 30 days for signs of 5 encephalitis, prior to necropsy. Clinical scores, and the severity of 6 histological lesions in the central nervous system, test group scores should not 7 exceed scores of the control yellow fever vaccine group. 8

B.4.2.2 Viremia in non-human primates 9

10 Non-human primates, humans and mosquitoes are the only natural hosts of 11 DENV (8, 19, 50). NHPs have been widely used to evaluate replication and 12 immunogenicity of candidate dengue vaccines (5, 16, 54). Primary infection 13 of macaques with wild type DENV results in moderate lymphadenopathy and 14 a robust immune response (8, 19). The NHP model has traditionally been 15 used as an important guide for selecting vaccine strains for further 16 development. In such studies, reduced peak titres and duration of viremia 17 induced by a candidate vaccine, compared to those induced by the non-18 attenuated parent virus, is often, but not always, a correlate of attenuation. 19 Consequently, if a dengue vaccine candidate causes viremia in NHPs 20 comparable to that caused by its wild-type parent virus the vaccine, 21 developers may wish to consider discontinuing further development. . 22

B.4.3 Assessment in mouse models 23

24 DENV infection has been studied in many different mouse models (2, 8, 46, 25 54, 75). When appropriate, a mouse model may be selected to evaluate the 26 potential of a candidate vaccine to cause disease in comparison to its wild-27 type parent virus. In such an experiment, the titres of virus in blood, spleen, 28 liver, lymph nodes, lungs, brain, and other tissues at various time points post-29 infection (8). The AG129 interferon receptor-deficient mouse will support 30 replication of DENVs of all serotypes (25, 26). A DENV2 strain adapted to 31 replicate in the AG129 induces a physiologically relevant disease in that strain 32 (54). At present, the AG129 mouse seems most suitable for safety studies, but 33 National regulatory authorities should be aware of the pitfalls of interpreting 34 results, since these animals do not possess an intact innate immune response. 35 For this same reason, as mentioned earlier, it would not be advisable to use 36 AG129 mice for classic toxicology studies. Other inbred mouse strains with 37 genes knocked-out are under investigation as models of DENV infection and 38 disease. One or more of these may have applicability to vaccine development 39 in the future. 40

B.4.4 DENV replication in vector mosquitoes 41

42


Transmission of DENV to arthropod vectors from humans is essential to 1 maintain the virus in nature. As noted previously, none of the DENV 2 attenuated candidate vaccines studied to date induce a viremia in vaccinees 3 that is sufficient in magnitude to infect feeding mosquitoes (19, 43, 49). 4 Further, if mosquitoes are infected with dengue vaccines, the viruses do not 5 replicate sufficiently to permit transmission of the virus. For these two 6 reasons, Ae. Aegypti mosquitoes are not expected to transmit dengue vaccine 7 viruses (19, 22, 49). As a measure of attenuation and safety, future novel 8 candidate vaccines should be shown to have reduced ability to replicate and 9 disseminate in Ae aegypti mosquitoes that have been infected in a controlled 10 laboratory setting, using parent strains as controls (19, 22, 35, 44). 11

12

B.5 Environmental risk 13

14 For live dengue vaccines, the primary environmental risks relate to their 15 capacity to be spread from human to human by vector mosquitoes and the risk 16 that prolonged or repeated cycles of replication in mosquitoes could permit 17 reversion to virulence. As previously noted, live vaccines currently under 18 development have been shown to replicate poorly both in vaccinees and in 19 mosquitoes, such that the risk for transmission by the mosquito vector is very 20 low, if any risk exists at all (3, 22, 28, 43, 44, 49). These factors should 21 markedly reduce the chance that any of these vaccines could revert in 22 mosquitoes to a virulent phenotype when used in a mass vaccination campaign 23 in an endemic area. In addition, genetic stability during multiple sequential 24 passages in mosquitoes has also been demonstrated for most existing live 25 dengue vaccine candidates. For future candidate novel live vaccines, similar 26 studies would need to be done. 27 28 Recently, some investigators have raised the issue, as regards live dengue 29 vaccines, that vaccine viruses could revert to virulence in mosquitoes via 30 intragenic recombination with endogenous wild-type flaviviruses. Such a 31 phenomenon would seem to be highly unlikely due to factors noted above plus 32 the controversial question of whether flaviviruses are able to undergo 33 recombination even under ideal conditions in vitro. For further details see Part 34 D guidelines for environmental risk assessment.35


Part C. Clinical evaluation of dengue tetravalent 1

vaccines (live, attenuated) 2

C.1 General considerations for clinical studies 3

4 The following should be read in conjunction with the: 5

— WHO Guidelines on clinical evaluation of vaccines: regulatory 6 expectations (TRS 924, Annex 1) (63) and 7

— Guidelines for the clinical evaluation of dengue vaccines in endemic areas 8 (WHO/IVB/08.12) (68). 9

C.1.1 Objectives of the clinical development programme 10 11

The clinical evaluation of a candidate live dengue tetravalent vaccine should aim 12 to demonstrate that it: 13

— Elicits immune responses against all four dengue serotypes 14

— Prevents DFI of any severity caused by any of the serotype 1, 2, 3 and 4 15 viruses over an appropriate minimum period of observation 16

— Has an acceptable safety profile 17 18 In addition, the program should: 19

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

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

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

30

C.1.2 Outline of the clinical development program 31 32

In the initial clinical studies (i.e. Phase 1 studies) it is expected that relatively 33 small numbers of healthy adults are vaccinated with investigational vaccine 34 formulations and that the primary focus is on assessing safety. These studies may 35 include exploration of immune responses to ascending doses of the four DENV 36 serotypes when administered alone and in combination. 37 38


The subsequent clinical studies (i.e. Phase 2 studies) should be designed to select a 1 dose of each DENV serotype for use in the tetravalent candidate vaccine 2 formulation and to identify an appropriate primary immunization schedule for 3 further study. 4 5 Since: 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 It is not currently possible to license candidate dengue vaccines based only on 11 safety and immunogenicity data. Therefore, candidate tetravalent dengue vaccines 12 should be evaluated for protective efficacy against DFI. 13 14 Sponsors may decide to conduct at least one preliminary study of protective 15 efficacy (i.e. sometimes referred to as a Phase 2b study) in order to identify a final 16 candidate vaccine and immunization schedule for further study. Alternatively, 17 depending on the data already accumulated (e.g. based on demonstration of a 18 robust neutralizing antibody response), sponsors may consider it appropriate to 19 omit such a study. 20 21 The selected candidate vaccine should be evaluated in at least one adequately 22 sized study of protective efficacy (i.e. Phase 3 study) that compares numbers of 23 cases of laboratory-confirmed DFI between groups of vaccinated and 24 unvaccinated subjects. The total DFIs counted should include those due to any of 25 the four DENV serotypes and of any degree of clinical severity that occur within a 26 defined observation period. 27 28 Section C.3 gives more details of study designs and populations to be enrolled 29 into studies conducted at each phase of development. 30 31

C.2 Immunogenicity 32

33

C.2.1 Measurement of immune responses to vaccination 34 35

Current evidence suggests that neutralizing antibody against each DENV serotype 36 is likely to be the best surrogate marker for efficacy. 37 38 It is recommended that the methodology for determination of DENV serotype-39 specific neutralizing antibody titres should follow the WHO Guidelines for the 40 plaque reduction neutralization test (PRNT) (WHO/IVB/07.07) (67). If 41 alternative methods for determining neutralizing antibody (e.g. high throughput 42


microneutralization assays) are developed these should be validated against the 1 PRNT. 2 3 Reference virus strains and cell substrates are available from the WHO and titres 4 should be expressed in IU calibrated against the WHO reference sera. In-house 5 strains may be used provided that the reference serum is employed and results 6 expressed in IU. In addition, a comparison with results obtained with the WHO 7 reference strains is advised. 8 9 An assessment of neutralizing antibody titres against a range of DENV strains, 10 including recent wild-type isolates, is encouraged. This would be valuable 11 information to obtain due to worldwide strain diversity and because neutralizing 12 antibody titres against specific isolates will be variable. Such additional assays 13 could be applied to subsets of sera collected from vaccinees that have been 14 selected randomly or based on a scientific justification (e.g. to select sera known 15 to cover a range of neutralizing antibody titres against the reference or in-house 16 strains). 17 18 The assay of DENV-specific antibody other than neutralizing antibody (e.g. IgM 19 and IgG ELISA) may be of interest but is not considered to be essential for the 20 assessment of potential vaccine efficacy. 21 22 It is considered unlikely that data on cell-mediated immunity (CMI) will provide 23 an immunological correlate of protection. However, the exploration of CMI is 24 encouraged since specific CMI assays may be useful for the assessment of 25 immunological memory and durability of protection. Assessments of cytokine 26 responses may assist in the evaluation of vaccine safety and provide some 27 indication of the potential risk that vaccination could predispose subjects to 28 develop severe DFI during subsequent natural infection (48). 29 30

C.2.2 Investigation and interpretation of immune responses to 31 vaccination 32 33

There is no established immunological correlate of protection against any DENV 34 serotype. In the initial clinical studies of safety and immunogenicity, including the 35 dose- and regimen-finding studies, it is essential to fully describe the pre- and 36 post-vaccination neutralizing antibody titres that are observed against each of the 37 four DENV serotypes (see also section C.3.1). 38 39 In a non-endemic population with no detectable pre-vaccination antibody in the 40 majority of subjects a comparison of percentages with a detectable titre post-41 vaccination (which may be defined as seroconversion in such a population) 42 against each DENV serotype should be made. The analyses should also look at 43 proportions that seroconvert (in accordance with an appropriate definition of 44


seroconversion stated in the protocol) to multiple serotypes (i.e. two, three or all 1 four serotypes). 2

3 In an endemic population in which very high proportions of subjects are already 4 seropositive with respect to at least one dengue type a comparison of pre- and 5 post-vaccination geometric mean titres (GMTs) with respect to those types will be 6 informative in addition to seroconversion rates. 7 8 In endemic and non-endemic populations detailed consideration of reverse 9 cumulative distribution curves is important. For example it may be informative to 10 compare percentages achieving a pre-defined high titre of neutralizing antibody. 11 12 In protective efficacy studies neutralizing antibody against DENV serotypes 13 should be determined and followed over time in pre-defined subsets of the study 14 population, including an assessment of antibody persistence after the protocol-15 defined period for the primary evaluation of protective efficacy. It is preferable 16 that subsets of subjects to be included in these detailed immunogenicity 17 evaluations should be identified at the time of randomization with stratification 18 for age and any other factors that might have an important impact on immune 19 responses to vaccination. In any case the data should be analysed according to 20 pre-defined subsets. Immune responses should be determined for vaccinated and 21 unvaccinated subjects so that the effects of background exposure to DENVs 22 during the study period can be assessed. 23 24 Depending on the specific vaccine construct and taking into account any pertinent 25 results of non-clinical studies, sponsors may wish to undertake some exploratory 26 investigations of antibody against other antigens (e.g. those associated with the 27 attenuated yellow fever virus backbone in a chimeric vaccine). 28 29 Long-term storage of sera is encouraged since future developments in the field 30 and/or emerging data on longer-term safety or efficacy might point to a need for 31 additional investigations that cannot be predicted at the time of conducting the 32 study. 33 34 Subsets of subjects should also be identified for collection of peripheral blood 35 mononuclear cells (PBMCs), taking into account feasibility issues, such as the 36 blood volumes required from different age groups to produce adequate cell 37 numbers for study and accessibility to adequate sample processing and storage 38 facilities. 39 40 For the analysis of the relationship between neutralizing antibody titres and 41 protection against laboratory-confirmed DFI, sera should be collected at timed 42 intervals from a substantial cohort of subjects (and preferably the entire study 43 population if feasible). Once the protocol-defined double-blind observation period 44 has been completed, the analysis of the relationship between immune response 45 and protection against DFI should follow according to: 46


1 a) A cohort study in which one or more measures of immune response are 2

related to disease in all or a large subset of immunized subjects. This 3 design is potentially the more informative if there are enough cases among 4 immunized subjects to allow for a quantitative estimation of disease risk as 5 a function of immune response(s). 6 OR 7

b) A case-control study in which immune responses are compared between 8 immunized subjects who developed disease and a subset of immunized 9 subjects who did not develop disease. This design enables establishment of 10 an association between disease and measure(s) of immune response but it 11 does not allow for a quantitative estimation of disease risk as a function of 12 immune response. 13

14 The use of serology to help identify infections with dengue viruses (whether or 15 not clinically apparent) is a quite separate issue that is discussed in section C.3.3. 16 17

C.3 Clinical studies 18 19

C.3.1 Phase 1 studies 20 21

The Phase 1 studies should be designed to provide an early indication of whether 22 severe local and/or systemic adverse events (AEs) may occur commonly after 23 vaccination. These studies may also provide preliminary data on immune 24 responses to assist in the selection of DENVs (or constructs) and doses to be 25 included in candidate tetravalent vaccine formulations for further study. 26 27 Subjects enrolled into these initial studies should be healthy adults who are naïve 28 to flaviviruses (based on medical and vaccine history and serological studies). It is 29 preferred that subjects are resident in non-endemic areas so that they are not at 30 risk of natural infection with dengue or other flaviviruses. Eligible subjects should 31 not be in need of vaccination against other flaviviruses at least throughout the 32 duration of the study. 33 34 Sponsors may choose to commence studies with monovalent (i.e. containing a 35 single live attenuated DENV serotype) before progressing to evaluate multivalent 36 versions (which may include bivalent, trivalent and then tetravalent formulations) 37 of a candidate dengue vaccine. 38 39 If a candidate tetravalent vaccine formulation elicits a much lower antibody titer 40 to one (or more than one) DENV serotype than to others it is important that 41 consideration is given to modification of the vaccine (e.g. by modifying the 42 infectious titers of serotypes) and/or the immunization schedule due to the 43 potential implications for safety and efficacy. 44


1 If a likely candidate tetravalent vaccine is identified it may be appropriate that a 2 preliminary exploration of safety and immunogenicity should be conducted in 3 healthy adult residents of an endemic area (i.e. including subjects with evidence 4 of some pre-existing immunity to dengue or other flaviviruses). Such a study 5 could provide further reassurance regarding the ability of the candidate vaccine to 6 elicit immune responses to all four DENV serotypes before progressing to studies 7 in larger numbers of subjects. 8 9


The Phase 2 studies should extend the information on safety and immunogenicity 12 of candidate tetravalent vaccine formulations. They should include studies in 13 residents of endemic areas who are therefore at risk of natural infection with 14 dengue and may have some degree of pre-existing immunity to one or more 15 DENV serotypes and to other flaviviruses. 16 17 While the first data may be obtained in adults there should be a plan to move 18 down to younger age groups in a stepwise fashion. The age range should reflect 19 that proposed for the evaluation of protective efficacy of the tetravalent vaccine 20 candidate. 21 22 Depending on the findings of the Phase 1 studies, the first Phase 2 studies may 23 further explore dose-response relationships. The data generated on safety and 24 immunogenicity should be sufficient to support the selection of one or more 25 candidate tetravalent vaccines and immunization schedules (i.e. number of doses 26 and dose intervals) for further evaluation. 27 28 If the sponsor chooses to undertake a preliminary (i.e. Phase 2b) study of safety 29 and efficacy this should be of an appropriate design and of adequate size to 30 support a robust decision regarding the vaccine formulation and schedule to be 31 further evaluated (see C.3.3). Even in a Phase 2b study it is recommended that 32 subjects should be followed up for approximately 3-5 years to collect data on 33 safety and to document antibody to DENV serotypes in subsets of each treatment 34 group. 35 36 The total number of subjects enrolled into Phase 2 studies should be sufficient to 37 describe at least common adverse reactions to vaccination with some degree of 38 confidence. Therefore it is expected that several hundred subjects should have 39 been exposed to candidate tetravalent vaccines containing the final or near final 40 doses of DENVs of each serotype. If any unusual, severe or serious adverse 41 reactions are documented it may be appropriate that further studies include the 42 assessment of safety as one of the primary objectives provided that these reactions 43 would not preclude further vaccine development. 44 45



It is not currently possible to license dengue vaccines based only on safety and 3 immunogenicity data because there is no licensed dengue vaccine and no 4 immunological correlate of protection has been established for any DENV 5 serotype. Each tetravalent candidate vaccine should be evaluated in at least one 6 study that is of an appropriate design and adequate size to estimate vaccine 7 efficacy (VE). This situation may change in the future (see section C.3.3.7). 8

9

C.3.3.1 General issues for study design 10 11

VE is estimated by comparing the total numbers of laboratory–confirmed cases of 12 DFI of any degree of severity and due to any of the four DENV serotypes between 13 the vaccinated and unvaccinated (control) groups. The primary analysis of VE 14 should be conducted at the conclusion of a protocol-defined double-blind 15 observation period. Each study should be of sufficient size and duration to provide 16 a robust estimation of VE and to provide preliminary evidence that the vaccine 17 does not predispose recipients to develop one of the severe forms of DFI 18 following natural infection. 19 20 Studies of protective efficacy should be performed in endemic areas where a 21 proportion of the population is likely to have some naturally-acquired immunity to 22 one or more of the four DENV serotypes and/or other flaviviruses. It is assumed 23 that in most, if not all, cases each study will evaluate a single tetravalent candidate 24 dengue vaccine and immunization schedule. However, the study design may be 25 adapted as necessary if more than one possible active vaccination group is to be 26 included. 27 28 Studies that involve vaccination of a large proportion of subjects at any one study 29 locality carry the potential to significantly interrupt DENV transmission during 30 the observation period. The result could be a reduced likelihood of demonstrating 31 a difference in the numbers of laboratory-confirmed cases of DFI between the 32 vaccine and control groups. Consideration should be given to this possibility 33 when designing the study. 34 35 Randomization should be performed using a centralized system. When using a 1:1 36 randomization ratio the block size should be selected with the aim of enrolling 37 approximately equal numbers in test and control groups at each of the study sites 38 so that subjects in each group are at the same risk of developing mild and severe 39 DFI throughout the observation period. It is also possible to consider the use of 40 unbalanced randomization (e.g. 3 : 2 or 2 : 1 vaccine : control) provided that care 41 is taken to ensure the desired ratio is applied at each study site (or geographically 42 localized sites) and the sample size is calculated to provide adequate power. 43 44


The decision to use unbalanced randomization should take into consideration the 1 possible advantages and disadvantages. Advantages include a larger safety 2 database and possibly easier enrolment due to the greater chance that any one 3 subject would receive the candidate dengue vaccine. Disadvantages include the 4 possibility that a larger proportion vaccinated against dengue could increase the 5 risk of achieving a reduction in DENV transmission sufficient to impact on the 6 chance of obtaining a conclusive study result. 7 8 Whenever possible, subjects randomized to the control group should receive an 9 alternative active vaccine (selected to provide an anticipated benefit to study 10 participants) rather than injections of placebo. If the active control vaccine cannot 11 be given at the same schedule as the candidate dengue vaccine, then placebo 12 injections can be used within the schedule as necessary to maintain a double-blind 13 design. If the active control vaccine has a different presentation or appearance to 14 the candidate dengue vaccine then study personnel who administer the 15 vaccinations should not have any other involvement in the conduct of the study. 16 Vaccine recipients should not be allowed to observe preparation of the vaccines 17 for injection (e.g. any reconstitution steps that may or may not be necessary) to 18 avoid the risk of them sharing this information and so identifying themselves with 19 one of the study groups. 20 21 If there is no suitable active control vaccine that can be given by the same route of 22 administration as the candidate dengue vaccine then the use of a placebo control is 23 necessary to achieve a double blind design. In such cases, the protocol could plan 24 to administer a suitable licensed vaccine to all subjects in the study (i.e. those who 25 do and do not receive the candidate dengue vaccine) at some time after 26 completion of the assigned study treatments and during the double-blind follow-27 up period. In this way all study subjects can derive some potential benefit from 28 participation in the study without compromising the study integrity. 29 30

C.3.3.2 Study location and duration 31 32

The geographic areas selected for study should have background rates of DFI that 33 are sufficient to provide enough cases in the control group during the observation 34 period to facilitate the estimation of VE. To assess background rates efficacy 35 studies should be preceded by the collection of epidemiological information to 36 document the expected incidences of DENV serotype-specific and all DFI 37 preferably over several years. The data should include information on seasonality 38 of disease to identify periods of transmission and case demographics (e.g. age and 39 gender) so that populations at highest risk of DFI can be targeted for enrolment. 40

41 There should also be an assessment of the likely extent of exposure of the 42 population to other species of flaviviruses at potential study sites because such 43 exposure may confound the interpretation of dengue-specific serological data and 44 possibly affect the clinical course of DFI. This assessment should take into 45


account any available epidemiological data, serological studies and information 1 on yellow fever vaccine coverage. 2 3 Study sites should be endemic for one or more DENV serotypes with transmission 4 expected each season during the observation period, which would likely range 5 from 1-3 years from the time of the first vaccination. Nevertheless, even if the 6 study is conducted over several seasons and at geographically dispersed study 7 sites there may not be sufficient numbers of cases of DFI to support an estimation 8 of serotype-specific VE for some or all of the four serotypes. Additional evidence 9 for protective efficacy against individual DENV serotypes should be sought from 10 post-licensure (i.e. effectiveness) studies as discussed in sections C.3.3.7 and C.4. 11 12 There should be a plan to follow-up subjects for safety and efficacy for at least 3-13 5 years from the time of completion of primary vaccination. During this period it 14 is possible that an efficacious dengue vaccine may be offered to subjects 15 originally assigned to the control group with potential implications for 16 interpretation of the data that can be collected (see C.3.3.7). 17 18

C.3.3.3 Study population 19 20

Since protective efficacy studies should be performed in endemic areas there is a 21 need to consider that the ultimate target group for vaccination may range from a 22 subgroup (e.g. infants) to the entire population. However, even in areas where a 23 substantial proportion of hospitalized cases of severe DFI are aged less than one 24 year there are likely to be concerns regarding inclusion of infants in protective 25 efficacy studies because of the possible risk of DFI that has been reported in 26 association with waning maternal antibody against one or more DENV serotypes 27 and the unknown effects of vaccination in the presence of maternal antibody. 28 29 Therefore it is expected that protective efficacy studies would likely exclude 30 subjects aged less than one year but should enrol children across a wide age range 31 subject to satisfactory results from the safety and immunogenicity studies. Section 32 C.3.3.7 considers bridging the observed VE to populations that were not included 33 in efficacy studies. 34 35

C.3.3.4 Objectives, endpoints and analyses 36 37

The primary objective of a protective efficacy is to estimate VE against DFI. The 38 primary analysis should seek to demonstrate superiority for the vaccinated group 39 versus the control group in terms of the total numbers of cases of laboratory-40 confirmed DFI in subjects who have been fully vaccinated in accordance with the 41 protocol and have been followed up for the required time with no major protocol 42 deviations. In this analysis counting of cases should commence from a designated 43 time point after the last dose of protocol-assigned doses has been administered. 44


1 VE is estimated by comparing the total numbers of laboratory–confirmed cases of 2 DFI (i.e. summation of cases due to any DENV serotype and of any degree of 3 severity) that occur in vaccinated and unvaccinated (control) groups during a 4 protocol-defined double-blind observation period. VE should be calculated using 5 the standard formula VE (%) = 100 x (1- r1/ro) [where r1 = incidence rate in the 6 vaccine group and r0 = incidence rate in the control group]. 7 8 The assessment of DENV serotype-specific VE should be a major secondary 9 objective and the subject of a planned secondary analysis. It is not expected that 10 the study would be powered to support a formal statistical analysis of DENV 11 serotype-specific efficacy. 12 13 The statistical analysis plan should explain how multiple episodes of DFI in any 14 one study participant will be handled in the analyses. 15 16 Other important secondary analyses should include the estimation of: 17

— Efficacy based on counting all DFI that occur after administration of the 18 first dose of protocol-assigned treatment; 19

— Efficacy in all vaccinated subjects regardless of protocol deviations 20 (including those with incomplete vaccination courses and missing data); 21

— Efficacy according to pre-vaccination flavivirus serological status, which 22 might be determined in a randomized subset of enrolled subjects who are 23 followed serologically; 24

— Efficacy according to severity of laboratory-confirmed DFI (with adequate 25 protocol definitions); 26

— Efficacy that includes prevention of “possible” or “probable” dengue 27 infection (e.g. applied to patients in whom serology is used as the basis for 28 dengue diagnosis without a virologically-confirmed diagnosis). The 29 justification for this secondary analysis is based on expectation that a 30 dengue vaccine may reduce the viremia so making it more difficult to 31 detect in patients who may also have abbreviated clinical signs and 32 symptoms. Thus, serological secondary end-points may help assess overall 33 efficacy assuming that serological assays are equally sensitive and specific 34 to DENV (but not to individual serotypes) in detecting dengue infection in 35 vaccine and control groups; or 36

— The effect of vaccination on the duration of hospitalization and/or need for 37 specific interventions to manage the clinical illness. 38

39 If more than one study of protective efficacy is performed with a single candidate 40 vaccine (e.g. perhaps covering different geographical regions) using the same or a 41 very similar study protocol, it may be appropriate to pre-define a pooled analysis 42 of that could provide additional insight into serotype-specific VE and the risk of 43 severe DFI in vaccine and control groups. 44


1 Each study should have in place a data and safety monitoring board (DSMB) 2 consisting of persons with no involvement in study conduct and analysis and 3 including a statistician. The DSMB charter should enable it to unblind treatment 4 assignments as necessary and to recommend that enrolment is halted or the study 5 is terminated based on pre-defined criteria designed to protect subjects from harm. 6 In addition, studies may include one or more planned interim analyses with pre-7 defined stopping rules. 8 9

C.3.3.5 Case definitions 10 11

The case definitions for the primary and for the various secondary analyses, with 12 details of the criteria to be met, should be defined in the protocol and should be in 13 accordance with the latest WHO recommendations 14 (WHO/HTM/NTD/DEN/2009.1) (69). 15

16

Clinical diagnosis 17 The most commonly diagnosed form of clinically apparent dengue virus infection 18 is characterized by the sudden onset of fever lasting at least two and up to seven 19 days. Fever is commonly accompanied by severe headache, pain behind the eyes, 20 gastrointestinal symptoms, muscle, joint and bone pain and a rash. These cases are 21 usually self-limiting and result in complete recovery. 22 23 For the purposes of classification of cases it is important to characterize the 24 severity of each DFI. The criteria used to assess severity should be those 25 described by WHO that are current when the protocol is finalized 26 (WHO/HTM/NTD/DEN/2009.1 and updates of this guidance) (69). These criteria 27 should be used to determine the features of DFI that are captured in the case 28 report form. 29

Virological diagnosis 30

All methods used for the virological component of the case definition should be 31 fully validated. Virological confirmation of the clinical diagnosis can be based on 32 direct detection of dengue viremia by isolation. However, the use of alternative 33 virological methods to confirm the diagnosis (e.g. detection of NS1 to 34 demonstrate the presence of DENV and the use of reverse transcription-35 polymerase chain reaction (RT-PCR) to determine the serotype) is acceptable. 36 The standardization of viral diagnostic methods is encouraged. Every effort 37 should be made to conduct testing in one or a small number of designated central 38 laboratories with appropriate expertise. 39 40 Obtaining specimens to attempt virological confirmation of the diagnosis should 41 be triggered by a set of clinical features laid down in the study protocol that aim 42 to identify all potential cases of DFI of any severity and as early as possible, 43


taking into account the observation that virological diagnostic methods (including 1 virus isolation and PCR-based assays) are more sensitive during the first five days 2 of infection. 3

Serological diagnosis 4

There is at least a theoretical possibility of a reduced likelihood of obtaining 5 virological confirmation of the clinical diagnosis in vaccinated subjects due to 6 levels of viremia that are below detection limits. If this were to occur in a 7 significant proportion of clinical cases there could be a bias in numbers of 8 virologically-confirmed DFI in favour of the vaccinated group. 9 10 Commercial and/or in-house serological assays (e.g. enzyme immunoassay [EIA], 11 immunofluorescence and virus neutralization tests) may be performed on paired 12 acute and convalescent sera. The results may be used to identify possible cases of 13 DFI in which a virological diagnosis was not confirmed and the numbers may be 14 compared between vaccinated and control groups in an additional secondary 15 analysis of vaccine efficacy. 16 17 For example, using the IgM capture EIA an acute primary infection may be 18 implied from a rise in IgM levels rise during the first two weeks post-infection. 19 The results need to be interpreted with some caution because the IgM response to 20 acute infection may be blunted in vaccinated subjects and in those infected 21 previously by wild type DENV or by another flavivirus. Depending on the timing 22 of the illness, the results may also be confounded by the fact that IgM and IgG 23 responses may reflect recent dengue vaccination rather than acute infection with 24 wild type dengue. In this regard, the ratio of IgM and IgG may assist in the 25 differentiation of primary and secondary infections. 26 27 The interpretation of serological data is also complicated by cross-reacting 28 antibody among flaviviruses. In those instances where cross-reaction with other 29 flaviviruses does not occur, a four-fold or greater rise in dengue neutralizing 30 antibodies makes it possible to attribute recent infection to a dengue virus 31 presumptively, but not definitively. 32 33

C.3.3.6 Case detection and description 34

35 It is essential that there is adequate surveillance to detect any possible case of DFI 36 as early as possible to optimize the chances of virological confirmation of the 37 diagnosis. The surveillance mechanisms (e.g. including arrangements for periodic 38 home visits or telephone calls and involvement of hospitals serving the study 39 catchment areas) should be tested before the study is initiated at each study site. 40 It is essential that study subjects are educated regarding the need to contact or 41 directly present to the designated study healthcare facilities whenever they 42 develop signs or symptoms that may be indicative of DFI. A check list of these 43


signs and symptoms should be provided to all study participants at the time of 1 enrolment. 2 3 In addition, measures should be in place to follow each possible case of DFI for 4 any change in disease course (e.g. progression from mild to severe DFI, onset of 5 complications) and to document the outcome, including the time to recovery or 6 death. In case of death before collection of specimens or in the absence of 7 virological confirmation of the diagnosis permission should be sought to perform 8 needle puncture of the liver for virological study and a post-mortem examination. 9 10 In some study sites that are otherwise considered suitable it may not be possible to 11 identify a local healthcare facility willing to participate in the study. These sites 12 should not be initiated unless there is at least agreement from local healthcare 13 providers to notify study staff of possible cases of DFI within a sufficient 14 timeframe to allow for specimens to be collected and transported for virological 15 diagnosis. Subjects should carry a study participant card with contact names and 16 numbers to ensure that study personnel are alerted and can arrange for the 17 collection of all the necessary clinical data and the transport of specimens for 18 virological diagnosis. 19 20 Despite taking the steps described, there will still be some cases of possible DFI 21 that are not confirmed virologically and for which serological testing is 22 inconclusive. In addition, some subjects may not comply with the study 23 requirement to present to a designated healthcare facility when they have signs 24 and symptoms indicative of a possible DFI or they may be so ill that they are 25 immediately admitted to a hospital not directly participating in the study and/or 26 may die without notification of study personnel in time to collect data and 27 specimens. It is important that as much information as possible is collected on 28 these cases whenever and however they come to light and that they are taken into 29 account in a “worse-case scenario” analysis of vaccine efficacy that counts all 30 cases (proven and unproven and regardless of protocol deviations). 31 32

C.3.3.7 Need for additional studies of efficacy 33 34

Once the efficacy of at least one candidate dengue vaccine has been satisfactorily 35 demonstrated (and perhaps already licensed and introduced into the routine 36 vaccination program in at least one country) there will be a need to reassess the 37 content of clinical development programs for other candidate dengue vaccines. In 38 particular, there will be a need to consider the appropriateness and/or feasibility of 39 conducting studies in which subjects are randomized to a control (i.e. 40 unvaccinated) group. 41 42 It is not currently possible to make a definitive recommendation regarding what 43 could or should be required in this scenario. Much will depend on the findings 44


reported from the first completed efficacy study of a candidate vaccine or from 1 ongoing studies with other candidate vaccines. 2 3 Some issues to be taken into account include the following: 4 5 i) Once a vaccine has been licensed and introduced into the routine 6

vaccination program in one or more countries the inclusion of an 7 unvaccinated group in subsequent studies in these (and possibly many 8 other) countries is likely to be considered unethical. In addition, studies 9 that include an unvaccinated group are not likely to be feasible in some 10 regions after approval of the first vaccine (with or without introduction 11 into the routine vaccination program) because subjects are unlikely to risk 12 assignment to an unvaccinated group. However, if there remains 13 considerable uncertainty about vaccine efficacy against specific DENV 14 serotype(s) then another study with an unvaccinated control group might 15 be possible in a region where such serotype(s) are predominant; 16

17 ii) The use of licensed dengue vaccine(s) in the routine vaccination program 18

in a country or region may reduce the incidence of DFI down to levels that 19 are too low to permit the estimation of VE from further studies of 20 reasonable and manageable size and duration; 21

22 iii) There should be a careful scientific evaluation of the arguments for and 23

against extrapolation of the efficacy observed for a particular vaccine to 24 other populations. For example, to other age groups, to subjects at highest 25 risk of dengue and severe dengue, to geographical areas with predominant 26 circulating DENV serotypes for which serotype-specific efficacy may not 27 have been established and to populations with high rates of immunity to 28 other flaviviruses; 29

30 iv) It is very possible that a definitive immunological correlate of protection 31

cannot be established from pre-licensure protective efficacy studies. 32 Moreover, accumulating evidence suggests that a neutralizing antibody 33 titer that seems to correlate with protection against DFI due to one DENV 34 serotype may not predict protection against DFI caused by other dengue 35 serotypes. Therefore, there needs to be a discussion of the validity of using 36 immunogenicity data to bridge the efficacy of a dengue vaccine that was 37 demonstrated in one test population to other populations, with or without 38 identification of correlates of protection; 39

40 v) There also needs to be a discussion regarding the use of bridging studies to 41

support the extrapolation of efficacy observed with one vaccine to 42 subsequent candidate vaccines that may or may not be live dengue 43 vaccines. For reasons already discussed above, there may be no alternative 44 to the use of bridging studies because the following approaches are 45 unlikely to be feasible; 46


1 vi) To continue to request evaluation of subsequent candidate dengue 2

vaccines in pre-licensure protective efficacy studies that include an 3 unvaccinated group; 4

5 vii) To request a relative efficacy study i.e. that compares rates of febrile 6

dengue illness in groups assigned to either a licensed or candidate vaccine; 7 or 8

9 viii) After consideration of all these issues it is expected that at some future 10

time point, which will depend on the safety and efficacy that is observed 11 with the first or first few vaccines to be licensed, the inclusion of 12 unvaccinated control groups in efficacy studies will no longer be possible. 13 Once this time point is reached there will have to be recommendations 14 made regarding: 15

— Additional data to be obtained after initial approval of a vaccine for 16 which VE has been estimated based on numbers of laboratory-17 confirmed DFI in vaccinated versus unvaccinated groups. 18

— Data that should be generated before and after licensure of subsequent 19 candidate vaccines. 20

21 In both instances it is important that vaccines should be evaluated in post-22 approval studies of safety and effectiveness as discussed in section C.4. 23 24

C.3.3.8 Documentation of safety during pre-licensure studies 25 26

The routine monitoring of safety during all pre-licensure clinical studies should 27 follow the usual principles taking into account issues relevant to live attenuated 28 vaccines. In addition to providing study-specific safety data there should be an 29 analysis of safety data pooled across all study groups that received the final 30 selected vaccine formulation. 31 32 In particular, there is a need to assess whether DFI (which could be of any degree 33 of severity, including a very mild illness) may be caused by vaccine strains. In 34 studies conducted in non-endemic areas there is a risk that vaccine-associated DFI, 35 which would be expected to occur only within about 30 days post-dose, could be 36 confused with an intercurrent infection while in studies in endemic areas there is a 37 need to distinguish vaccine-associated from naturally-occurring DFI. Therefore in 38 all cases it is essential that laboratory studies should be undertaken to determine 39 whether or not the clinical picture is associated with vaccine virus viremia. 40

41 The safety considerations also include the possible risk that vaccination could 42 predispose recipients to develop a severe form of DFI. The monitoring and 43 investigation of all subjects who develop signs or symptoms potentially indicative 44 of DFI during pre-licensure studies in endemic regions should provide a 45


preliminary assessment of this risk. If no undue risk is identified and the vaccine 1 is licensed it is essential that there is adequate follow-up of study subjects together 2 with further assessment of the risk in the post-licensure period (see section C.4). 3 4 The total safety database derived from all pre-licensure studies should be 5 sufficient to describe uncommon adverse reactions. Based on considerations 6 outlined in section C.3.3.7 it may be that vaccines that are developed subsequent 7 to the approval of the first vaccine(s) will not be evaluated in pre-licensure studies 8 of protective efficacy, in which case the pre-licensure safety database would 9 usually be much smaller. In these instances the minimum acceptable safety 10 database should be determined according to the nature of the vaccine and in the 11 light of experience amassed with other similar vaccines. 12 13

C.4 Post-licensure investigations 14 15

As with all medicinal products, including all vaccines, there is a need to ensure 16 that adequate surveillance is in place and is maintained to detect adverse reactions 17 during the post-licensure period in accordance with requirements of the countries 18 in which approval has been obtained. 19 20 The need for, design and extent of specific studies of safety and/or effectiveness 21 following approval of a dengue vaccine should be given careful consideration by 22 sponsors and National regulatory authorities. However, at least in the case of the 23 first dengue vaccines that reach the market there will be a clear need for well-24 designed studies, which should include long-term extension periods of pre-25 licensure efficacy studies as well as newly-initiated studies, to collect information 26 at least on the following: 27 28

— Long-term evaluation of breakthrough cases of DFI to detect any waning 29 of protective immunity against one or more DENV serotypes and the 30 possible need for booster doses; 31

32

— Antibody persistence; 33 34

— Responses to booster doses, which may be planned for pre-defined subsets 35 enrolled into studies or may be instituted as/when disease surveillance 36 indicates a possible need; 37

38

— The possible increased risk of severe DFI in vaccine recipients; and 39 40

— Vaccine effectiveness 41 42

Depending on the countries/regions in which any one licensed vaccine is 43 introduced into routine vaccination programs it is desirable that attempts should 44


be made to estimate DENV serotype-specific effectiveness and effectiveness in 1 populations that were not included in pre-licensure studies. There are several 2 possible study designs and methods for estimating vaccine effectiveness and it is 3 essential that expert advice is sought. In addition, it is likely that such studies 4 would need to be performed in close liaison with Public Health Authorities. 5

6 Sponsors and national regulatory authorities should consider the need to assess 7 immune interference and safety on co-administration of a dengue vaccine with 8 other vaccines that may be given at the same health care visits for convenience. In 9 addition, if licensure is sought in non-endemic areas with the intent to protect 10 travellers to endemic areas, sponsors and national regulatory authorities may 11 consider it necessary to obtain additional safety and immunogenicity data with the 12 final vaccine and schedule in flavivirus-naïve subjects. 13

14 Some or all post-licensure studies may be conducted as post-approval 15 commitments made to individual national regulatory authorities . In this regard, 16 both sponsors and national regulatory authorities that have approved a vaccine 17 should communicate and co-operate to ensure that studies are well-designed to 18 answer the questions posed and to avoid demands for numerous studies in 19 individual countries that are likely to be too small to provide reliable results. 20 Provisional plans for appropriate post-licensure studies should be submitted with 21 the application dossier and these should be refined during the assessment of the 22 national regulatory authority and as necessary after initial approval. 23

24


Part D. Environmental risk assessment of dengue 1

tetravalent vaccines (live, attenuated) derived by 2

recombinant DNA technology 3

D.1 Introduction 4

5

D.1.1 Scope 6

7 Some countries have legislation covering the environmental and other issues 8 related to the use of live vaccines derived by recombinant DNA technology since 9 they may be considered genetically modified organisms (GMO) including 10 vaccines. Similar issues may be raised by live vaccines derived by other methods. 11 12 This section of the guideline considers the environmental risk assessment (ERA) 13 that may be performed during DENV vaccine development. The ERA assesses 14 the risk to public health and the global environment. It does not assess the risk to 15 the intended recipient of the vaccine; this is assessed through clinical studies of 16 the vaccine. Nor does it assess the risk to laboratory workers. 17 18 The environmental impact is not usually the responsibility of the national 19 regulatory authority but of other agencies. Nonetheless the national regulatory 20 authority should receive a copy of the ERA and any associated decisions taken for 21 information and to ensure that the appropriate procedures have been followed. 22 23

D.1.2 Principles and Objectives 24

25 Live DENV vaccines in which the genetic material has been genetically modified 26 by recombinant DNA technology, should be considered to be GMO. The manu-27 facture, use and transboundary shipping of such live recombinant vaccines, for 28 research or commercial use, should comply, when applicable, with any relevant 29 legislation or regulations of the producing and recipient countries regarding 30 GMOs. In some regulatory regimes, in order to comply with environmental 31 regulations, an ERA should be undertaken if the live vaccine is being tested in a 32 clinical trial or if it is placed on the market. It should be noted that this guidance 33 on the ERA of live recombinant DENV vaccines does not intend to replace 34 existing GMO legislation when already in place in certain countries. 35 36 Generally, the objective of an ERA is to identify and evaluate, on a case-by-case 37 basis, potential adverse effects of a GMO on public health and the environment, 38 direct or indirect, immediate or delayed. This means that for each different live 39 recombinant dengue vaccine a case-by-case ERA should be performed. Direct 40 effects refer to primary effects on human health or on the environment which are 41


a result of the GMO itself and which occur through a short causal chain of events. 1 Indirect effects refers to effects occurring through a more extended causal chain 2 of events, through mechanisms such as interactions with other organisms, transfer 3 of genetic material, or changes in use or management. Immediate effects are 4 observed during the period of the release of the GMO, whereas delayed effects 5 refer to effects which may not be observed during the period of release of the 6 GMO, but become apparent as a direct or indirect effect either at a later stage or 7 after termination of the release. 8 9 The ERA should be performed in a scientifically sound and transparent manner 10 based on available scientific and technical data. Important aspects to be addressed 11 in an ERA include the characteristics of: (1) the parental organism, (2) the 12 recipient organism, (3) viral vector characteristics, (4) the donor sequence, (5) 13 genetic modification, (6) the intended use and (7) the receiving environment. The 14 data needed to evaluate the ERA do not have to derive solely from experiments 15 performed by the applicant; data available in the scientific literature can also be 16 used in the assessment. Regardless of the source of the data, it should be both 17 relevant and of an acceptable scientific quality. The ERA could be based on 18 available literature and on data of experiments previously performed for other 19 purposes, such as toxicity tests. 20 21 Ideally, the ERA is based on quantitative data and expressed in quantitative terms. 22 However, much of the information that is available for an ERA may be qualitative 23 for the reason that quantification is often hard to accomplish, and may not be 24 necessary to make a decision. The level of detail and information required in the 25 ERA is also likely to vary according to the nature and the scale of the proposed 26 release. Information requirements may differ between licensure and clinical 27 development and whether studies will be carried out in a single country or 28 multiple countries. 29 30 Uncertainty is inherent in the concept of risk. Therefore, it is important to identify 31 and analyse areas of uncertainty in the risk assessment. Since there is no 32 universally accepted approach for addressing uncertainty, risk management 33 strategies may be considered. Precise data on the environmental fate of the live 34 vaccine in early clinical trials will in most cases be insufficient or lacking. 35 However, at the market registration stage, the level of uncertainty is expected to 36 be lower as identified gaps in available data should already have been addressed. 37 38 The need for risk management measures should be based upon the estimated level 39 of risk. If new information on the GMO becomes available the ERA may need to 40 be re-performed to determine whether the estimated level of risk has changed. 41 This also holds true if the risks for the participating subjects have changed as 42 these aspects can be translated to other individuals. It should be noted that the 43 ERA will not deal with medical benefit for the subject or scientific issues such as 44 proof-of-principle. 45 46


D.2 Procedure for Environmental Risk Assessment 1

2 Risk assessment involves identification of novel characteristics of the GMO that 3 may have adverse effects (hazard), evaluating the consequences of each potential 4 adverse effect, estimation of the likelihood of adverse effects occurring, risk 5 estimation, risk management and, in some methodologies, estimation of the 6 overall risk for the environment. These processes should identify the potential 7 adverse effects by comparing the properties of the GMO with non-modified 8 organisms under the same conditions, in the same receiving environment. The 9 principles and methodology of an ERA should be applicable irrespective of the 10 geographic location of the intended environmental release of the GMO. Although 11 the ERA should take into account the specificities associated with the mosquito 12 vector being endemic or non-endemic in the region in which vaccine trials will be 13 carried out, and/or where licensure is being requested. Depending upon local 14 regulatory requirements, the ERA may be undertaken by the applicant or by the 15 local competent authority on the basis of data supplied. In all cases, the local 16 competent authority should use the ERA as a basis for deciding whether any 17 identified environmental risks are acceptable. However, the decision on whether 18 any identified risks are acceptable may vary from country to country. 19 20 There are several national and multinational documents addressing ERA issues, 21 such as: 22

— EU Directive 2001/18/EC (14) (Annex II) and Commission Decision 23 2002/623/EC (15); 24

— Risk Analysis Framework of the Australian Office of the Gene Technology 25 Regulator (39); 26

— New Substances Notification Regulations of Canadian Environmental 27 Protection Act, 1999 (13); 28

— Recombinant DNA Safety Guidelines of Genetic Engineering Approval 29 Committee in India; and 30

— Normative Resolution No.5 (12 March 2008) comprising provisions on rules 31 for commercial release of GMOs and their derivatives in Brazil. 32

33 As an example, a detailed description of a six-step ERA module of EU Directive 34 2001/18/EC is described in the Appendix 3 of these Guidelines. 35 36

D.3 Special Considerations for Live Recombinant Dengue Virus 37 Vaccines 38

39 The ERA of live recombinant DENV vaccines should be conducted according to 40 the general principles described above taking into consideration in particular the 41 vector responsible for disease transmission. The following aspects which could 42 be developed include: the genetic stability of the live recombinant virus including 43


reversion and recombination, potential transmission of vaccine virus among hosts 1 by the vector and the immune status of the population, and these are further 2 outlined below. 3 4

D.3.1 Genetic stability 5

6 DENV vaccines currently under clinical evaluation are attenuated DENV strains, 7 intertypic chimeric vaccines orDENV/YF-17D vaccine chimeras. In the intertypic 8 approach, the structural genes of an attenuated strain of DENV of a given 9 serotype are replaced by the corresponding genes of a different DENV serotype. 10 In the dengue/yellow fever chimeras, the prM/E structural genes of the dengue 11 virus are cloned into the backbone of the YF-17D vaccine, in replacement of the 12 corresponding structural YF-17D genes. 13 14

D.3.1.1 Reversion 15

16 After vaccination, there is the potential of reversion of attenuated live dengue 17 virus vaccines to a virulent form of the dengue virus, although this has not been 18 seen in clinical trials so far. The potential reversion is based on the stability of the 19 attenuating mutation(s), the number of attenuating mutations, and the nature of 20 attenuating mutation. Attenuating mutations that are dependent upon a single base 21 change may be more susceptible to reversion than a mutation that is stabilized by 22 multiple base substitutions. In addition, attenuating mutations that are derived by 23 deletions of segments of RNA are generally more stable against reversion. 24 Changes in virus genotype have the potential to influence disease transmission, 25 tropism of vector vaccine, virulence, and/or patterns of disease, resulting in a 26 virus with a previously unknown combination of properties. However, the 27 likelihood of such a reversion depends on the number of attenuation mutations 28 present and the viral genes involved in the vaccine virus (9) 29 30

D.3.1.2 Recombination 31

32 Whether or not recombination takes place among flaviviruses is controversial. 33 Theoretically, recombination between live DENV vaccines and wild type 34 flaviviruses could produce a virus with an altered phenotype, but currently there is 35 no evidence to support this (24, 33, 34, 36, 40, 45, 47) . 36 37 The potential for recombination issues within and between flaviviruses has been 38 widely discussed and challenged in the past years, based on existing literature (24, 39 33, 34, 36, 40, 47), and also from data obtained in specific experiments. In 40 particular, a "recombination trap," has been recently designed to allow the 41 products of rare recombination events to be selected and amplified, in the case of 42 West Nile (WN), tick-borne encephalitis (TBE) and Japanese encephalitis (JE) 43


viruses (47). Intergenomic but aberrant recombination was observed only in the 1 case of JE virus, and not for WN or TBE viruses. Moreover, its frequency 2 appeared to be very low and generated viruses with impaired growth properties. 3 4 While their likelihood of appearance is very low, as stated above, the potential 5 adverse effects of recombined DENVs should be evaluated in the ERA. In this 6 respect, "worst-case" scenario chimeras have been constructed to address that risk 7 (33, 34, 40). 8 9 These different studies showed that such recombinants constructed artificially 10 from a wild type flavivirus and a chimeric vaccine (40), or from two wild type 11 viruses, such as highly virulent YF Asibi virus and wild type DEN4 virus (34), 12 were highly attenuated compared to their parental viruses. Attenuation was shown 13 in culture in vitro, in mosquito vectors and in susceptible animal models, 14 including monkeys. These data provide experimental evidence that the potential 15 of recombinants, should they ever emerge, to cause disease or spread would 16 probably be very low. 17 18

D.3.2 Vector transmission 19

20 The presence of the DENV vectors such as Ae. aegypti and Ae. albopictus play a 21 key role in the transmission of flaviviruses and potentially live DENV vaccines 22 from the vaccinated subject to other individuals. Dengue does not usually spread 23 directly from person to person, except via blood transfusion. Transmission of the 24 dengue vaccine in regions in which the vector is absent is therefore highly 25 unlikely. Dengue is currently restricted to the tropical regions. Due to climate 26 change there is an increasing incidence of the detection of mosquito species not 27 normally found in regions with traditionally milder climates. This could lead to 28 the introduction of dengue in these areas and potentially change disease 29 transmission and population immune status. 30 31 Recombination between live DENV vaccines and wild type flaviviruses could 32 theoretically occur in the vaccinee (see above), but possibly also within an 33 infected mosquito although neither has been reported. A recombined DENV could 34 potentially, for instance in combination with climate change, use new vectors for 35 transmission leading to previously unknown transmission characteristics. 36 Therefore, the presence of a relevant mosquito vector and a ‘dengue favorable 37 climate’ in the vaccination region should be taken into account in the ERA of live 38 DENV vaccines. 39 40 To assess the likelihood of effective transmission of the vaccine from a vaccinated 41 individual, one has to take into consideration two parameters: the level of viremia 42 in the vaccinated hosts, and the ability of the mosquito vectors to transmit the live 43 DENV vaccine to new hosts. The blood titer required for effective transmission is 44 difficult to define. If the candidate vaccine strains replicate at low levels (1-2 logs) 45


then possible initiation of the transmission cycle (mosquito infection) is reduced. 1 For instance, in the case of the YFV17D chimeric vaccine, viremia in the hosts 2 after tetravalent vaccination is very low (38). In case the viremia titer is high 3 enough and virus is intrinsically incubated in the mosquito, then the ability to 4 transmit the virus to other hosts will be critical. For the recombinant live 5 attenuated DENV vaccines currently in clinical trial, the likelihood of occurrence 6 appears to be low to negligible. These viruses replicate to titers thousands-fold 7 lower than that which results in transmission to mosquitoes. In addition, the 8 ability of these viruses to infect, replicate, and escape the midgut is impaired (4, 6, 9 22, 28, 49, 52, 53). 10 11 The outcome of the ERA for clinical trials in regions in which the vector is absent, 12 is that the environmental risk is negligibly small. The mosquito vector is not 13 present and therefore the vaccine or possible created recombinants cannot be 14 transmitted to other people. However, in endemic areas, it is important to perform 15 an ERA as the vector is present, thus there is a possibility of transmission. 16

D.3.3 Immune status 17

18 Live DENV vaccines are able to replicate and form new viral particles in 19 vaccinated persons. The immune status of the vaccinee and the rest of the 20 population could influence the environmental risk of a live DENV vaccine. In 21 general, the presence of pre-existing immunity due to earlier exposure to DENVs 22 will reduce the extent and duration of vaccine virus replication and biodistribution. 23 The potential for transmission of the vaccine is therefore considered to be greater 24 in naïve or immunocompromised individuals. 25 26 An unvaccinated population with no pre-existing immunity will respond 27 differently upon exposure to the vaccine compared to a population in which 28 dengue is endemic. The immune status should therefore be taken into account in 29 the ERA as it can influence the environmental impact of the vaccines, and the 30 potential occurrence of adverse effects in contacts of the vaccinees. There is a 31 potential for pre-existing heterotypic antibody to cause higher levels of vaccine 32 virus. Enhanced illness in vaccine recipients who have pre-existing DENV 33 antibody (antibody-dependent enhancement) has not been observed in clinical 34 trials of live attenuated dengue vaccines to date and was not observed in a clinical 35 trial of live attenuated dengue vaccines designed to address this possibility (10, 36 20). 37

38 39


Part E. Guidelines for national regulatory authorities 1

2

E.1 General 3

4 The general recommendations for national regulatory authorities and national 5 control laboratories given in the Guidelines for national authorities on quality 6 assurance for biological products (58) should apply. In addition, the general 7 recommendations for national regulatory authorities and national control 8 laboratories provided in the Guidelines for independent lot release of vaccines by 9 regulatory authorities (70) should be followed These Guidelines specify that no 10 new biological substance should be released until consistency of manufacturing 11 and quality as demonstrated by a consistent release of batches has been 12 established. The detailed production and control procedures and any significant 13 changes in them should be discussed with and approved by the national regulatory 14 authority. The national regulatory authority should obtain the working reference 15 from manufacturers to establish a national working reference preparation until an 16 international reference reagent is available. 17

18

E.2 Release and certification 19

20 A vaccine lot should be released only if it fulfils the national requirements and/or 21 Part A of the present Guidelines. A protocol based on the model given in 22 Appendix 1, signed by the responsible official of the manufacturing establishment, 23 should be prepared and submitted to the national regulatory authority in support 24 of a request for release of vaccine for use. 25 26 A statement signed by the appropriate official of the national regulatory authority 27 should be provided if requested by a manufacturing establishment and should 28 certify whether or not the lot of vaccine in question meets all national 29 requirements, as well as Part A of these Guidelines. The certificate should also 30 state the lot number, the number under which the lot was released, and the number 31 appearing on the labels of the containers. In addition, the date of the last 32 satisfactory determination of antigen concentration as well as assigned expiry date 33 on the basis of shelf life should be stated. A copy of the official national release 34 document should be attached. The certificate should be based on the model given 35 in Appendix 2. The purpose of the certificate is to facilitate the exchange of 36 dengue virus vaccines between countries. 37 38


Authors 1

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


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


Sanofi Pasteur, Marcy l'Étoile, France; Professor Alan Barrett, UTMB, Galveston, 1 Texas, USA; Dr Diederik Bleijs, RIVM, Bilthoven, Netherlands; Dr Anil Chawla, 2 Greater Noida, UP, India; Dr Kurt Dobbelaere, GSK, Rixensart, Belgium; Dr 3 Michael Dornbusch, Office of the Gene Technology Regulator, Canberra, 4 Australia; Dr Anna Durbin, JHSPH, Baltimore, Maryland, USA; Dr Kenneth 5 Eckels, WRAID, Silver Spring, Maryland, USA; Professor Robert Edelman, 6 UMSM, Baltimore, Maryland, USA; Mr Marcos Galves, ANVISA, Brasilia-DF, 7 Brazil; Dr Elwyn Griffiths, Biologics and Genetic Therapies Directorate (BGTD), 8 Health Canada, Ontario, Canada; Dr Joachim Hombach, WHO, Geneva, 9 Switzerland; Dr Ivana Knezevic, WHO, Geneva, Switzerland; Dr Jan-Willem van 10 der Laan, RIVM, Bilthoven, The Netherlands; Dr Laurent Mallet, Sanofi Pasteur, 11 Ontario, Canada; Dr Lewis Markoff, CBER, FDA, Bethesda, Maryland, USA; Dr 12 Philip Minor, NIBSC, Potters Bar, Hertfordshire, UK (Chair); Dr Lynn Morgan, 13 Sanofi Pasteur, Marcy l'Étoile, France; Dr Jehanara Korimbocus, AFSSAPS, 14 Lyon, France; Dr Mair Powell, MHRA, London, UK; Dr Alexander Precioso, 15 Butantan, Sao Paulo, Brazil; Dr Vincent Quivy, GSK Biologicals, Wavre, 16 Belgium; Dr John Roehrig, CDC, Fort Collins, Collorado, USA; Dr Julia Schmitz, 17 WHO, Geneva, Switzerland; Mrs Prapassorn Thanaphollert, Thai FDA, Ministry 18 of Public Health, Nonthaburi, Thailand; and Dr Dennis Trent, UTMB, Galveston, 19 Texas, USA; and Dr Simonetta Viviani, Sanofi Pasteur, Marcy l'Étoile, France. 20 21

Acknowledgements 22

Acknowledgements are due to the following experts for their useful comments: Dr 23 Sylvie Morgeaux, AFSSAPS, Lyon, France; Dr Frédéric Mortiaux, GSK 24 Biologicals, Rixensart, Belgium; and Dr Ryoko Krause, Vaccines and Biologicals, 25 International Federation of Pharmaceutical Manufacturers & Associations 26 (IFPMA), Geneva, Switzerland. 27 28


Appendix 1: Model summary protocol for manufacturing 1

and control of dengue tetravalent vaccines (live, 2

attenuated) 3

4 The following protocol is intended for guidance, and indicates the information that should 5 be provided as a minimum by the manufacturer to the national regulatory authority. 6 Information and tests may be added or deleted if necessary to be in line with the 7 marketing authorization approved by the national regulatory authority. It is thus possible 8 that a protocol for a specific product may differ in detail from the model provided. The 9 essential point is that all relevant details demonstrating compliance with the license and 10 with the relevant WHO guidelines of a particular product should be given in the protocol 11 submitted. The section concerning the final product should be accompanied by a sample 12 of the label and a copy of the leaflet that accompanies the vaccine container. If the 13 protocol is being submitted in support of a request to permit importation, it should also be 14 accompanied by a lot release certificate from the national regulatory authority of the 15 country in which the vaccine was produced and /or released stating that the product meets 16 national requirements as well as Part A guidelines of this document published by WHO. 17 18

1. Summary information on finished product (final vaccine lot) 19

International name: ____________________________ 20 Commercial name: ____________________________ 21 Product license (marketing authorization) 22

number : ____________________________ 23 Country: ____________________________ 24 25 Name and address of manufacturer: ____________________________ 26 Name and address of product license holder 27

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

____________________________ 36 Number of doses per container: ____________________________ 37 Composition (antigen concentration) / 38

volume of single human dose: ____________________________ 39 Target group : ____________________________ 40 Expiry date : ____________________________ 41 Storage conditions: ____________________________ 42

43


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 Antibiotics added : ____________________________ 25 Release date : ____________________________ 26 Batch number of finished product (final 27

vaccine lot) : ____________________________ 28 Site of manufacture of finished product 29

(final vaccine lot) : ____________________________ 30 Date of manufacture of finished product 31

(final vaccine lot): ____________________________ 32 33

A genealogy of the lot numbers of all vaccine components used in the formulation of the 34 final product will be informative. 35 The following sections are intended to report the results of the tests performed during the 36 production of the vaccine 37 38

3. Control of source materials 39

3.1 Cell banks 40

3.1.1 Information on cell banking system 41

42 Name and identification of cell substrate : ____________________________ 43


Origin and short history: ____________________________ 1 Authority that approved cell bank: _______________________________ 2 Master cell bank (MCB) lot number & date 3

of preparation ____________________________ 4 Date MCB was established: ____________________________ 5 Date of approval by the national regulatory 6

authority: ____________________________ 7 Total number of ampoules stored: _______________________________ 8 Passage/Population doubling level (PDL) 9

of MCB ____________________________ 10 Maximum passage/ population doubling 11

level approved for MCB: ____________________________ 12 Storage conditions: _______________________________ 13 Date of approval of protocols indicating 14

compliance with the requirements of the 15 relevant monographs and with the 16 marketing authorization: _______________________________ 17

Manufacturer’s working cell bank (WCB) 18 lot number & date of preparation ____________________________ 19

Date WCB was established: ____________________________ 20 Total number of ampoules stored: _______________________________ 21 Passage/Population doubling level (PDL) 22

of WCB ____________________________ 23 Maximum passage /population doubling 24

level approved for WCB: ____________________________ 25 Storage conditions ____________________________ 26 Date of approval of protocols indicating 27

compliance with the requirements of the 28 relevant monographs and with the 29 marketing authorization: _______________________________ 30

Production cell lot number: _______________________________ 31 32

3.1.2 Test on MCB and WCB 33

Identification of cell substrate 34 Method: ____________________________ 35 Specification: ____________________________ 36 Date: ____________________________ 37 Result: ____________________________ 38

39 Morphological characteristics: ____________________________ 40 41 Results of tests for adventitious agents: ____________________________ 42 43 Results of tests for tumorigenicity (if 44

applicable): ____________________________ 45


1 Tests for retrovirus (if applicable): ____________________________ 2

Date of test: ____________________________ 3 Method used: ____________________________ 4 Results: ____________________________ 5

6 Nature and concentration of antibiotics or 7

selecting agent (s) used in production 8 cell culture maintenance medium: ____________________________ 9

10 Identification and source of starting 11

materials used in preparing production 12 cells including excipients and 13 preservatives (particularly any materials 14 of human or animal origin e.g. albumin; 15 serum) : ____________________________ 16

17

3.2 Virus seeds 18

Vaccine virus strain(s) and serotype(s): ____________________________ 19 Substrate used for preparing seed lots: ____________________________ 20 Origin and short history: ____________________________ 21 Authority that approved virus strain(s): ____________________________ 22 Date approved: ____________________________ 23

3.2.1 Information on seed lot preparation 24

25 Virus master seed (VMS) 26

Source of virus master seed lot: ____________________________ 27 Virus master seed lot number: ____________________________ 28 Name and address of manufacturer: ____________________________ 29 Passage level: ____________________________ 30 Date of inoculation: ____________________________ 31 Date of harvest: ____________________________ 32 Number of containers: ____________________________ 33 Conditions of storage: ____________________________ 34 Date of establishment: ____________________________ 35 Maximum passage level approved for VMS:36

____________________________ 37 Date approved by the national regulatory 38

authority: ____________________________ 39 40

Virus working seed (VWS) 41 Virus working seed lot number: ____________________________ 42 Name and address of manufacturer: ____________________________ 43 Passage level from virus master seed lot: ____________________________ 44


Date of inoculation: ____________________________ 1 Date of harvest: ____________________________ 2 Number of containers: ____________________________ 3 Conditions of storage: ____________________________ 4 Date of establishment: ____________________________ 5 Date approved by the national regulatory 6

authority: ____________________________ 7

3.2.3 Tests on VMS and VWS 8

Identity test 9

Method: ____________________________ 10 Lot number of reference reagents : ____________________________ 11 Date of test (on, off): ____________________________ 12 Result: ____________________________ 13

Genetic/phenotypic characterizations 14

Method: ____________________________ 15 Reference reagents ____________________________ 16 Specification: ____________________________ 17 Date of test (on, off): ____________________________ 18 Result: ____________________________ 19

Tests for bacteria and fungi 20 Method: ____________________________ 21 Media: ____________________________ 22 Number of containers tested : ____________________________ 23 Volume of inoculum per container : ____________________________ 24 Volume of medium per container : ____________________________ 25 Temperatures of incubation : ____________________________ 26 Date of test (on, off): ____________________________ 27 Result: ____________________________ 28

Test for mycoplasmas 29

Method: ___________________________ 30 Media: ___________________________ 31 Volume tested: ___________________________ 32 Temperature of incubation ____________________________ 33 Positive controls : ____________________________ 34 Date of test (on, off): ___________________________ 35 Result: ____________________________ 36

Test for mycobacteria 37 Method: ____________________________ 38 Media: ___________________________ 39


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

Adventitious agents: 5

Volume of virus seed samples for 6 neutralization and testing ____________________________ 7

Batch number(s) of antisera/antiserum used 8 for neutralization of virus seeds ____________________________ 9

Test in tissue cultures for adventitious agents 10

Test in simian cells : 11

Type of simian cells ____________________________ 12 Quantity of neutralized sample inoculated: ____________________________ 13 Incubation conditions: ____________________________ 14 Method: ___________________________ 15 Specification: ___________________________ 16 Date of test (on, off): ____________________________ 17 Ratio of cultures viable at end of test ___________________________ 18 Result: ___________________________ 19

Test in human cells 20


Other cell types: 29

Type of 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

Test in animals for adventitious agents 38 Method: ___________________________ 39 Specification: ___________________________ 40


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

Test by molecular methods for adventitious agents 3 Method: ___________________________ 4 Specification: ___________________________ 5 Date of test (on, off): ___________________________ 6 Result: ___________________________ 7

Tests in non-human primates (either master or working seed lot) for 8 neurovirulence 9 (For details, please see Recommendations for yellow fever vaccine) 10

Method: ___________________________ 11 Specification: ___________________________ 12 Date of test (on, off): ___________________________ 13 Result: ___________________________ 14

Tests in suckling mice (either master or working seed lot; where necessary) for 15 neurovirulence 16

(Detailed protocol should be developed) 17 Method: ___________________________ 18 Specification: ___________________________ 19 Date of test (on, off): ___________________________ 20 Result: ___________________________ 21

Virus titration for infectivity 22 Method: ___________________________ 23 Specification: ___________________________ 24 Date of test (on, off): ___________________________ 25 Result: ___________________________ 26

4. Control of vaccine production 27

4.1 Control of production cell cultures 28

4.1.1 Information on preparation 29

Lot number of MCB ___________________________ 30 Lot number of WCB ___________________________ 31 Date of thawing ampoule of WCB: ____________________________ 32 Passage number of production cells ___________________________ 33 Date of preparation of control cell cultures ___________________________ 34 Result of microscopic examination ___________________________ 35

4.1.2 Tests on control cell cultures 36

Amount or ratio of control cultures to ____________________________ 37


production cell cultures: ____________________________ 1 Incubation conditions: ____________________________ 2 Period of observation of cultures : ____________________________ 3 Date started/ended: ____________________________ 4 Ratio of cultures discarded and reason: ____________________________ 5 Results of observation: ____________________________ 6 Date supernatant fluid collected: ____________________________ 7

Test for haemadsorbing viruses 8

Quantity of cells tested: ____________________________ 9 Method: ___________________________ 10 Specification: ___________________________ 11 Date of test (on, off): ___________________________ 12 Result: ___________________________ 13

Test for adventitious agents on supernatant culture fluids 14


Type of simian cells ____________________________ 16 Quantity of pooled sample inoculated: ____________________________ 17 Incubation conditions: ____________________________ 18 Method: ___________________________ 19 Specification: ___________________________ 20 Date of test (on, off): ____________________________ 21 Ratio of cultures viable at end of test ___________________________ 22 Result: ___________________________ 23


Type of simian cells ____________________________ 25 Quantity of pooled sample inoculated: ____________________________ 26 Incubation conditions: ____________________________ 27 Method: ___________________________ 28 Specification: ___________________________ 29 Date of test (on, off): ____________________________ 30 Ratio of cultures viable at end of test ___________________________ 31 Result: ___________________________ 32


Type of cells ____________________________ 34 Quantity of pooled sample inoculated: ____________________________ 35 Incubation conditions: ____________________________ 36 Method: ___________________________ 37 Specification: ___________________________ 38 Date of test (on, off): ____________________________ 39 Ratio of cultures viable at end of test ___________________________ 40 Result: ___________________________ 41


Identity test : 1

Method : ____________________________ 2 Specification : ____________________________ 3 Date test on : ____________________________ 4 Date test off : ____________________________ 5 Result : ____________________________ 6

4.1.3 Cells used for vaccine production: 7

Observation of cells used for production 8 Date : ____________________________ 9 Result : ____________________________ 10

11

4.2 Monovalent virus harvest pools 12

4.2.1 Information on manufacture 13

Information on each monovalent virus harvest pool should separately be provided 14 Batch number(s): _______________________________ 15 Date of inoculation: _______________________________ 16 Date of harvesting: _______________________________ 17 Lot number of Virus master seed lot _______________________________ 18 Lot number of virus working seed lot _______________________________ 19 Passage level from virus working seed lot _______________________________ 20 Methods, date of purification if relevant _______________________________ 21 Volume(s), storage temperature, storage 22

time and approved storage period: _______________________________ 23 24

4.2.2 Tests on monovalent virus harvest pools 25

Identity 26 Method: ____________________________ 27 Lot number of reference reagents : _______________________________ 28 Specification: _______________________________ 29 Date of test _________________________ 30 Result: _______________________________ 31

Test for bacteria and fungi 32 Method : ____________________________ 33 Media: ____________________________ 34 Number of containers tested : _______________________________ 35 Volume of inoculum per container: ____________________________ 36 Volume of medium per container: ____________________________ 37 Temperatures of incubation: _______________________________ 38 Date of test (on, off): _________________________ 39


Result: ____________________________ 1

Test for mycoplasma 2

Method: ____________________________ 3 Media : ____________________________ 4 Volume tested: ____________________________ 5 Temperature of incubation ____________________________ 6 Positive controls : ____________________________ 7 Date of test (on, off) ____________________________ 8 Result: ____________________________ 9

Test for mycobacteria 10

Method: ____________________________ 11 Media : ____________________________ 12 Volume tested: ____________________________ 13 Temperature of incubation ____________________________ 14 Date of test (on, off) ____________________________ 15 Result: ____________________________ 16

Test for adventitious agents 17






Type of cells ____________________________ 37 Quantity of neutralized sample inoculated: ____________________________ 38 Incubation conditions: ____________________________ 39 Method: ___________________________ 40


Specification: ___________________________ 1 Date of test (on, off): ____________________________ 2 Ratio of cultures viable at end of test ___________________________ 3 Result: ___________________________ 4

Virus titration for infectivity 5


Test for host cell proteins 10 Method: ___________________________ 11 Specification: ___________________________ 12 Date of test ___________________________ 13 Result: ___________________________ 14

Test for residual cellular DNA 15

Method: ___________________________ 16 Specification: ___________________________ 17 Date of test ___________________________ 18 Result: ___________________________ 19

Consistency of virus characteristics 20

Method: ___________________________ 21 Specification: ___________________________ 22 Date of test (on, off): ___________________________ 23 Result: ___________________________ 24 25

4.3. Final tetravalent vaccine bulk 26

4.3.1 Information on manufacture 27

Batch number(s): ___________________________ 28 Date of formulation: ____________________________ 29 Total volume of final bulk formulated: ____________________________ 30 Monovalent virus pools used for 31

formulation ____________________________ 32 Serotype / lot number / volume added / 33

virus concentration ___________________________ 34 Name & concentration of added substances 35

(e.g. diluent, stabilizer if relevant): ___________________________ 36 Volume(s), storage temperature, storage 37

time and approved storage period: _______________________________ 38


4.3.2 Tests on final tetravalent bulk lot 1

Residual animal serum protein 2


Test for bacteria & fungi 7 Method: ____________________________ 8 Media: ____________________________ 9 Number of containers tested : ___________________________ 10 Volume of inoculum per container: ____________________________ 11 Volume of medium per container: ____________________________ 12 Temperatures of incubation: ___________________________ 13 Date of test (on, off): ___________________________ 14 Result: _______________________________ 15

16

5. Filling and containers 17

Lot number ___________________________ 18 Date of filling ___________________________ 19 Type of container: ___________________________ 20 Volume of final bulk filled ___________________________ 21 Filling volume per container ___________________________ 22 Number of containers filled (gross) ___________________________ 23 Date of lyophilization ___________________________ 24 Number of containers rejected during 25

inspection ___________________________ 26 Number of containers sampled ___________________________ 27 Total number of containers (net) ___________________________ 28 Maximum period of storage approved ___________________________ 29 Storage temperature and period ___________________________ 30 31

6. Control tests on final vaccine lot 32

6.1 Tests on vaccine lot 33

Inspection of final containers 34 Appearance ____________________________ 35 Date of test ____________________________ 36 Results ____________________________ 37 Before reconstitution ____________________________ 38 After reconstitution ____________________________ 39 Diluent used ____________________________ 40


Lot number of diluent used ____________________________ 1

Test for pH 2 Method: ___________________________ 3 Specification: ___________________________ 4 Date of test ___________________________ 5 Result: ___________________________ 6

Identity test (each serotype) 7 Method: ____________________________ 8 Specification: ___________________________ 9 Date of test ___________________________ 10 Result: ___________________________ 11

Test for bacteria and fungi 12 Method: ____________________________ 13 Media : ___________________________ 14 Volume tested: ____________________________ 15 Temperatures of incubation : ___________________________ 16 Date of test (on, off): ___________________________ 17 Result: ___________________________ 18

Test for potency (each serotype): 19 Method: ___________________________ 20 Batch number of reference vaccine and 21

assigned potency: ___________________________ 22 Specification: ___________________________ 23 Date of test (on, off): ___________________________ 24 Result for each serotype: ___________________________ 25

Thermal stability (each serotype) 26 Method: ___________________________ 27 Specification: ___________________________ 28 Date of test (on, off): ___________________________ 29 Result for each serotype: ___________________________ 30

General safety (unless deletion authorized) 31

Tests in mice 32

Date of inoculation ___________________________ 33 No. of animals tested ___________________________ 34 Volume and route of injection ___________________________ 35 Observation period ___________________________ 36 Results (give details of deaths) ___________________________ 37

Tests in guinea-pigs 38


Date of inoculation ___________________________ 1 No. of animals tested ___________________________ 2 Volume and route of injection ___________________________ 3 Observation period ___________________________ 4 Results (give details of deaths) ___________________________ 5

Residual moisture 6 Method ___________________________ 7 Specification ___________________________ 8 Date ___________________________ 9 Result ___________________________ 10

Residual antibiotics if applicable 11 Method ___________________________ 12 Specification ___________________________ 13 Date ____________________________ 14 Result ___________________________ 15 16

6.2 Diluent 17

Name and composition of diluent _______________________________ 18 Lot number _______________________________ 19 Date of filling _______________________________ 20 Type of diluent container _______________________________ 21 Filling volume per container _______________________________ 22 Maximum period of storage approved _______________________________ 23 Storage temperature and period _______________________________ 24

25 26 Name (Typed): __________________________________________________ 27 Signature: __________________________________________________ 28 Date: __________________________________________________ 29

30


Appendix 2: Model certificate for the release of dengue 1

vaccine (live, tetravalent) by national regulatory 2

authorities 3

4 LOT RELEASE CERTIFICATE 5 6

The following lot(s) of dengue vaccine produced by _____________________(1) in 7 _______________(2), whose numbers appear on the labels of the final containers, meet all 8 national requirements(3) and Part A(4) of the WHO Guidelines to assure the quality, safety 9 and efficacy of live attenuated (recombinant) dengue virus vaccines (_____)(5), and 10 comply with Good Manufacturing Practices for Pharmaceutical Products: Main 11 Principles(6) and Good Manufacturing Practices for Biological Products(7). As a minimum, 12 this certificate is based on examination of the summary protocol of manufacturing and 13 control. 14 15 The certificate may include the following information: 16 • Name and address of manufacturer; 17 • Site(s) of manufacturing; 18 • Trade name and/common name of product; 19 • Marketing authorization number; 20 • Lot number(s) (including sub-lot numbers, packaging lot numbers if necessary); 21 • Type of container; 22 • Number of doses per container; 23 • Number of containers/lot size; 24 • Date of start of period of validity (e.g. manufacturing date) and/or expiry date; 25 • Storage condition; 26 • Signature and function of the authorized person and authorized agent to issue the certificate; 27 • Date of issue of certificate; and 28 • Certificate number. 29 30 The Director of the National Regulatory Authority (or Authority as appropriate): 31 32 Name (Typed) 33 Signature 34 Date 35 36 1 Name of manufacturer 37 2 Country of origin 38 3 If any national requirements are not met, specify which one(s) and indicate why release 39 of the lot(s) has nevertheless been authorized by the national regulatory authority 40 4 With the exception of provisions on distribution and shipping, which the national 41 regulatory authority may not be in a position to assess. 42 5 WHO Technical Report Series, No. ___, YYYY, Annex __. 43 6 WHO Technical Report Series, No. 908, 2003, Annex 4. 44 7 WHO Technical Report Series, No. 823, 1992, Annex 1. 45 46 47 48


Appendix 3: Procedure for environmental risk 1

assessment 2

3 This appendix provides an example of the steps to be followed in conducting an 4 ERA, as exemplified by the EU Directive 2001/18/EC (Annex II) and 5 Commission Decision 2002/623/EC. 6

Step 1: 7

Identification of characteristics that may cause potential adverse effects and 8 evaluation of the potential consequences of each adverse effect (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

Step 2: 29

Evaluation of the potential consequences of each adverse effect, if it occurs 30


1

The magnitude of the consequences is likely to be influenced by the environment 2 in which the GMO is released and the manner of release. For each adverse effect 3 that is identified the consequences for the environment need to be qualified in 4 qualitative terms ranging from high, moderate, low to negligible. 5

6

Step 3: 7

Evaluation of the likelihood 8 9

The next step in the ERA deals with the evaluation of the likelihood of the 10 occurrence of the described adverse effects and their consequences, i.e. whether 11 the potential adverse effect associated with the application of the GMO will be 12 effectuated. All characteristics that could contribute to the actual occurrence of 13 the hazard should be identified. For the determination of the likelihood, it is 14 important to consider the manner of release and the surrounding receiving 15 environment. If no quantitative data are available to determine the likelihood, the 16 likelihood should be described in qualitative terms ranging from high, moderate, 17 or low to negligible, based on an expert judgment that considers the available data. 18 In case the likelihood of a potential effect cannot be determined, a worst-case 19 scenario will be applied that assumes the potential adverse effect will occur to the 20 fullest extent. 21

Step 4: 22

Risk estimation 23 24

An estimation of the risk to human health or the environment posed by each 25 identified characteristic of the GMO which has the potential to cause adverse 26 effects should be made by multiplying the likelihood of the adverse effect 27 occurring and the consequences, were it to occur. The risk is usually characterized 28 on scale, e.g. ‘negligible’, ‘low’, ‘moderate’, or ‘high’. Usually it is concluded 29 that risk management should be applied to reduce the risk, if the risk is 30 characterized as higher than negligible. 31

32

Step 5: 33

Application of risk management strategies 34 35

Where the estimated overall risk is higher than negligible, risk management 36 strategies should be applied that are adequate to reduce the level of risk. In 37 practice, risk management measures usually reduce the likelihood instead of the 38 potential adverse effects. In most cases risk management measures aim at 39 reducing spreading of the GMO into the environment, e.g. by hospitalization of 40 the patient or bandaging of the application site. Note that risk management 41 strategies to prevent transmission of dengue virus is likely to be ineffective or 42


unfeasible in certain parts of the world. Therefore, it should be well considered to 1 demand the use of mosquito nets and repellents for an appropriate period after 2 vaccination as an effective strategy to reduce this risk. 3

4

Step 6: 5

Determination of the final risk 6 7

An evaluation of the final overall risk of the GMO should be made taking into 8 account any proposed risk management strategies. The determination of the final 9 risk should conclude an ERA based on the previous steps described under step 1 10 to 5. Steps 4 and 5 may be repeated in situations where the overall environmental 11 impact is not acceptable. In that case, the ultimate risk management tool is not to 12 perform the activity with the GMO until more scientific data are available and the 13 ERA may be repeated on the basis of these data. It is up to the competent 14 authority to decide whether risks are acceptable or not, and which risk 15 management measurements are should be installed 16 17 In the assessment of clinical trials and marketing authorization approvals, 18 environmental risk assessment and risk management strategies should be taken 19 into account, but there should always be a well balanced risk and benefit 20 consideration. 21 22

23


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 DENVs dengue viruses 9 DFI dengue febrile illness 10 DSMB data and safety monitoring board 11 E envelope 12 EIA enzyme immunoassay 13 ELISA enzyme-linked immunosorbent assay 14 ERA environmental risk assessment 15 GMO genetically modified organism 16 GMTs geometric mean titres 17 IFU immunofocus-forming unit 18 IU international unit 19 JE Japanese encephalitis 20 MCB master cell bank 21 NAAT nucleic acid amplification techniques 22 NHP non-human primate 23 NIAID National Institute of Allergy and Infectious Diseases 24 NIBSC National Institute of Biological Standards and Control 25 NS non-structural 26 ORF open reading frame 27 PBMCs peripheral blood mononuclear cells 28 PDK primary dog kidney 29 PDL population doubling 30 PFU plaque-forming unit 31 prM premembrane 32 PRNT plaque reduction neutralization test 33 QPCR quantitative polymerase chain reaction 34 RCB reference cell bank 35 RT-PCR reverse transcription-polymerase chain reaction 36 TBE tick-borne encephalitis 37 TSE transmissible spongiform encephalopathies 38 UTR untranslated region 39 VE vaccine efficacy 40 VMS virus master seed 41 VWS virus working seed 42 WCB working cell bank 43 WN West Nile 44 WRAIR Walter Reed Army Institute of Research 45 YFV yellow fever virus 46


References 1

2 1. An J et al. Development of a novel mouse model for dengue virus infection. 3

Virology, 1999, 263:70-4 7.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d5 opt=Citation&list_uids=10544083] 6

2. Balsitis SJ et al. Lethal antibody enhancement of dengue disease in mice is 7 prevented by Fc modification. PLoS Pathog, 2010, 8 6:e1000790.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=P9 ubMed&dopt=Citation&list_uids=20168989] 10

3. Bennett KE et al. Quantitative trait loci that control dengue-2 virus dissemination 11 in the mosquito Aedes aegypti. Genetics, 2005, 170:185-12 94.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&13 dopt=Citation&list_uids=15781707] 14

4. Blaney JE, Jr. et al. Genetically modified, live attenuated dengue virus type 3 15 vaccine candidates. Am J Trop Med Hyg, 2004, 71:811-16 21.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&17 dopt=Citation&list_uids=15642976] 18

5. Blaney JE, Jr. et al. Recombinant, live-attenuated tetravalent dengue virus vaccine 19 formulations induce a balanced, broad, and protective neutralizing antibody 20 response against each of the four serotypes in rhesus monkeys. J Virol, 2005, 21 79:5516-22 28.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&23 dopt=Citation&list_uids=15827166] 24

6. Blaney JE, Jr. et al. Dengue virus type 3 vaccine candidates generated by 25 introduction of deletions in the 3' untranslated region (3'-UTR) or by exchange of 26 the DENV-3 3'-UTR with that of DENV-4. Vaccine, 2008, 26:817-27 28.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&28 dopt=Citation&list_uids=18191005] 29

7. Blaney JE, Jr. et al. Vaccine candidates for dengue virus type 1 (DEN1) generated 30 by replacement of the structural genes of rDEN4 and rDEN4Delta30 with those of 31 DEN1. Virol J, 2007, 4:23 32

8. Cassetti MC et al. Report of an NIAID workshop on dengue animal models. 33 Vaccine, 2010, 28:4229-34 34.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&35 dopt=Citation&list_uids=20434551] 36

9. dos Santos CN et al. Complete nucleotide sequence of yellow fever virus vaccine 37 strains 17DD and 17D-213. Virus Res, 1995, 35:35-38 41.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&39 dopt=Citation&list_uids=7754673] 40

10. Durbin AP et al. Heterotypic dengue infection with live attenuated monotypic 41 dengue virus vaccines: implications for vaccination of populations in areas where 42 dengue is endemic. J Infect Dis, 2011, 203:327-43 34.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&44 dopt=Citation&list_uids=21208923] 45

11. Durbin AP et al. rDEN4delta30, a live attenuated dengue virus type 4 vaccine 46 candidate, is safe, immunogenic, and highly infectious in healthy adult volunteers. 47


J Infect Dis, 2005, 191:710-1 8.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d2 opt=Citation&list_uids=15688284] 3

12. Edelman R et al. Phase I trial of 16 formulations of a tetravalent live-attenuated 4 dengue vaccine. Am J Trop Med Hyg, 2003, 69:48-5 60.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&6 dopt=Citation&list_uids=14740955] 7

13. Environment Canada. The New Substances Notification Regulations of the 8 Canadian Environmental Protection Act, 1999. 9

14. European Union. Directive 2001/18/EC of the European Parliament and of the 10 Council of 12 March 2001 on the deliberate release into the environment of 11 genetically modified organisms and repealing Council Directive 90/220/EEC 12 (Official Journal L 106, 17/4/2001 p. 1 - 39). 2001, 13

15. European Union. Commission Decision 2002/623/EC of 24 July 2002 14 establishing guidance notes supplementing AnnexII to Directive 2001/18/EC 15 (Official Journal L 106, 17.4.2001, p. 1). 2002, 16

16. Guirakhoo F et al. Construction, safety, and immunogenicity in nonhuman 17 primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol, 18 2001, 75:7290-19 304.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed20 &dopt=Citation&list_uids=11462001] 21

17. Guirakhoo F et al. Safety and efficacy of chimeric yellow fever-dengue virus 22 tetravalent vaccine formulations in nonhuman primates. J Virol, 2004, 78:4761-23 75.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&24 dopt=Citation&list_uids=15078958] 25

18. Guirakhoo F et al. Recombinant chimeric yellow fever-dengue type 2 virus is 26 immunogenic and protective in nonhuman primates. J Virol, 2000, 74:5477-27 85.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&28 dopt=Citation&list_uids=10823852] 29

19. Guy B et al. Preclinical and clinical development of YFV 17D-based chimeric 30 vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine, 31 2010, 28:632-32 49.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&33 dopt=Citation&list_uids=19808029] 34

20. Guy B et al. Development of Sanofi Pasteur tetravalent dengue vaccine. Hum 35 Vaccin, 2010, 36 6.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d37 opt=Citation&list_uids=20861669] 38

21. Halstead SB, Vaughn DW. Dengue vaccines. In: Plotkin, SA, Orenstein, WA et 39 al., eds. Vaccines. New York, Elsevier Inc., 2008, pp. 115-1162 40

22. Higgs S et al. Growth characteristics of ChimeriVax-Den vaccine viruses in 41 Aedes aegypti and Aedes albopictus from Thailand. Am J Trop Med Hyg, 2006, 42 75:986-43 93.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&44 dopt=Citation&list_uids=17124001] 45

23. Hombach J. Vaccines against dengue: a review of current candidate vaccines at 46 advanced development stages. Rev Panam Salud Publica, 2007, 21:254-47


60.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&1 dopt=Citation&list_uids=17612469] 2

24. Hombach J et al. Arguments for live flavivirus vaccines. Lancet, 2004, 364:498-3 9.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d4 opt=Citation&list_uids=15302184] 5

25. Huang CY et al. Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 6 virus as a potential candidate dengue type 1 virus vaccine. J Virol, 2000, 74:3020-7 8.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d8 opt=Citation&list_uids=10708416] 9

26. Huang CY et al. Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent 10 dengue vaccine development. J Virol, 2003, 77:11436-11 47.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&12 dopt=Citation&list_uids=14557629] 13

27. Johnson AJ, Roehrig JT. New mouse model for dengue virus vaccine testing. J 14 Virol, 1999, 73:783-15 6.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d16 opt=Citation&list_uids=9847388] 17

28. Johnson BW et al. Analysis of the replication kinetics of the ChimeriVax-DEN 1, 18 2, 3, 4 tetravalent virus mixture in Aedes aegypti by real-time reverse 19 transcriptase-polymerase chain reaction. Am J Trop Med Hyg, 2004, 70:89-20 97.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&21 dopt=Citation&list_uids=14971704] 22

29. Khin MM et al. Infection, dissemination, transmission, and biological attributes of 23 dengue-2 PDK53 candidate vaccine virus after oral infection in Aedes aegypti. 24 Am J Trop Med Hyg, 1994, 51:864-25 9.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d26 opt=Citation&list_uids=7810824] 27

30. Kinney RM et al. Construction of infectious cDNA clones for dengue 2 virus: 28 strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology, 1997, 29 230:300-30 8.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d31 opt=Citation&list_uids=9143286] 32

31. Levenbook IS et al. The monkey safety test for neurovirulence of yellow fever 33 vaccines: the utility of quantitative clinical evaluation and histological 34 examination. J Biol Stand, 1987, 15:305-35 13.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&36 dopt=Citation&list_uids=3680299] 37

32. Lum LC et al. Dengue encephalitis: a true entity? Am J Trop Med Hyg, 1996, 38 54:256-39 9.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d40 opt=Citation&list_uids=8600761] 41

33. McGee CE et al. Recombinant chimeric virus with wild-type dengue 4 virus 42 premembrane and envelope and virulent yellow fever virus Asibi backbone 43 sequences is dramatically attenuated in nonhuman primates. J Infect Dis, 2008, 44 197:693-45 7.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d46 opt=Citation&list_uids=18266603] 47


34. McGee CE et al. Substitution of wild-type yellow fever Asibi sequences for 17D 1

vaccine sequences in ChimeriVax-dengue 4 does not enhance infection of Aedes 2 aegypti mosquitoes. J Infect Dis, 2008, 197:686-3 92.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&4 dopt=Citation&list_uids=18266608] 5

35. Miller BR et al. Dengue-2 vaccine: oral infection, transmission, and lack of 6 evidence for reversion in the mosquito, Aedes aegypti. Am J Trop Med Hyg, 1982, 7 31:1232-8 7.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d9 opt=Citation&list_uids=7149108] 10

36. Monath TP et al. Recombination and flavivirus vaccines: a commentary. Vaccine, 11 2005, 23:2956-12 8.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d13 opt=Citation&list_uids=15811640] 14

37. Monath TP et al. Safety testing for neurovirulence of novel live, attenuated 15 flavivirus vaccines: infant mice provide an accurate surrogate for the test in 16 monkeys. Biologicals, 2005, 33:131-17 44.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&18 dopt=Citation&list_uids=15975826] 19

38. Morrison D et al. A novel tetravalent dengue vaccine is well tolerated and 20 immunogenic against all 4 serotypes in flavivirus-naive adults. J Infect Dis, 2010, 21 201:370-22 7.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d23 opt=Citation&list_uids=20059357] 24

39. Office of Gene Technology Regulator. Risk Analysis Framework (RAF) for The 25 Gene Technology Regulator. 2009, 26 [http://www.health.gov.au/internet/ogtr/publishing.nsf/Content/riskassessments-1] 27

40. Pugachev KV et al. Construction and biological characterization of artificial 28 recombinants between a wild type flavivirus (Kunjin) and a live chimeric 29 flavivirus vaccine (ChimeriVax-JE). Vaccine, 2007, 25:6661-30 71.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&31 dopt=Citation&list_uids=17693000] 32

41. Putnak RJ et al. An evaluation of dengue type-2 inactivated, recombinant subunit, 33 and live-attenuated vaccine candidates in the rhesus macaque model. Vaccine, 34 2005, 23:4442-35 52.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&36 dopt=Citation&list_uids=16005749] 37

42. Sabin AB, Schlesinger RW. Production of Immunity to Dengue with Virus 38 Modified by Propagation in Mice. Science, 1945, 101:640-39 2.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d40 opt=Citation&list_uids=17844088] 41

43. Salazar MI et al. Dengue virus type 2: replication and tropisms in orally infected 42 Aedes aegypti mosquitoes. BMC Microbiol, 2007, 43 7:9.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed44 &dopt=Citation&list_uids=17263893] 45

44. Schoepp RJ et al. Infection of Aedes albopictus and Aedes aegypti mosquitoes 46 with dengue parent and progeny candidate vaccine viruses: a possible marker of 47 human attenuation. Am J Trop Med Hyg, 1991, 45:202-48


10.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&1 dopt=Citation&list_uids=1877715] 2

45. Seligman SJ, Gould EA. Safety concerns with regard to live attenuated flavivirus 3 vaccines. J Infect Dis, 2008, 198:794-4 5.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d5 opt=Citation&list_uids=18694340] 6

46. Shresta S et al. Murine model for dengue virus-induced lethal disease with 7 increased vascular permeability. J Virol, 2006, 80:10208-8 17.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&9 dopt=Citation&list_uids=17005698] 10

47. Taucher C et al. A trans-complementing recombination trap demonstrates a low 11 propensity of flaviviruses for intermolecular recombination. J Virol, 2010, 12 84:599-13 611.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed14 &dopt=Citation&list_uids=19864381] 15

48. Thomas SJ et al. Scientific consultation on cell mediated immunity (CMI) in 16 dengue and dengue vaccine development. Vaccine, 2009, 27:355-17 68.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&18 dopt=Citation&list_uids=19022321] 19

49. Troyer JM et al. A live attenuated recombinant dengue-4 virus vaccine candidate 20 with restricted capacity for dissemination in mosquitoes and lack of transmission 21 from vaccinees to mosquitoes. Am J Trop Med Hyg, 2001, 65:414-22 9.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d23 opt=Citation&list_uids=11716092] 24

50. Vaughn DW et al. Dengue. In: Barrett, AD & Stanberry, L, eds. Vaccines for 25 Biodefense and Emerging and Neglected Diseases. New York, Elsevier Inc, 2009, 26 pp. 287-324 27

51. Whitehead SS et al. Prospects for a dengue virus vaccine. Nat Rev Microbiol, 28 2007, 5:518-29 28.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&30 dopt=Citation&list_uids=17558424] 31

52. Whitehead SS et al. A live, attenuated dengue virus type 1 vaccine candidate with 32 a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and 33 immunogenic in monkeys. J Virol, 2003, 77:1653-34 7.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&d35 opt=Citation&list_uids=12502885] 36

53. Whitehead SS et al. Substitution of the structural genes of dengue virus type 4 37 with those of type 2 results in chimeric vaccine candidates which are attenuated 38 for mosquitoes, mice, and rhesus monkeys. Vaccine, 2003, 21:4307-39 16.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&40 dopt=Citation&list_uids=14505913] 41

54. Williams KL et al. A mouse model for studying dengue virus pathogenesis and 42 immune response. Ann N Y Acad Sci, 2009, 1171 Suppl 1:E12-43 23.[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&44 dopt=Citation&list_uids=19751398] 45

55. World Health Organization. General requirements for the sterility of biological 46 substances (revised 1973). In: WHO Expert Committee on Biological 47 Standardization. Twenty-fifth report. Geneva, World Health Organization, 1973, 48


Annex 4 (WHO Technical Report Series, No. 530) 1 [http://www.who.int/biologicals/publications/trs/areas/vaccines/sterility/WHO_T2 RS_530_A4.pdf] 3

56. World Health Organization. Requirements for continuous cell lines used for 4 biologicals production. In: WHO Expert Committee on Biological 5 Standardization. Thirty-sixth report. Geneva, World Health Organization, 1987, 6 Annex 3 (WHO Technical Report Series, No. 745) 7 [http://whqlibdoc.who.int/trs/WHO_TRS_745.pdf] 8

57. World Health Organization. Good manufacturing practices for biological products. 9 In: WHO Expert Committee on Biological Standardization. Forty-second report. 10 Geneva, World Health Organization, 1992, Annex 1 (WHO Technical Report 11 Series, No. 822) 12 [http://www.who.int/biologicals/publications/trs/areas/vaccines/gmp/WHO_TRS_13 822_A1.pdf] 14

58. World Health Organization. Guidelines for national authorities on quality 15 assurance for biological products. In: WHO Expert Committee on Biological 16 Standardization. Forty-second report. Geneva, World Health Organization, 1992, 17 Annex 2 (WHO Technical Report Series No. 822) 18 [http://www.who.int/biologicals/publications/trs/areas/vaccines/regulatory/WHO_19 TRS_822_A2.pdf] 20

59. World Health Organization. Requirements for the collection, processing and 21 quality control of blood, blood components and plasma derivatives (requirements 22 for biological substances no. 27). In: WHO Expert Committee on Biological 23 Standardization. Forty-third report. Geneva, World Health Organization, 1994, 24 Annex 2 (WHO Technical Report Series, No. 840) 25 [http://www.who.int/bloodproducts/publications/WHO_TRS_840_A2.pdf] 26

60. World Health Organization. General requirements for the sterility of biological 27 substances (requirements for biological substances no. 6, revised 1973, 28 amendment 1995). In: WHO Expert Committee on Biological Standardization. 29 Forty-sixth report. Geneva, World Health Organization, 1998, Annex 3 (WHO 30 Technical Report Series, No. 872) 31 [http://www.who.int/biologicals/publications/trs/areas/vaccines/sterility/WHO_T32 RS_872_A3.pdf] 33

61. World Health Organization. Good manufacturing practices for pharmaceutical 34 products: main principles. In: WHO Expert Committee on Specifications for 35 Pharmaceutical Preparations. Thirty-seventh report. Geneva, World Health 36 Organization, 2003, Annex 4 (WHO Technical Report Series, No. 908) 37 [http://whqlibdoc.who.int/trs/WHO_TRS_908.pdf; or 38 http://whqlibdoc.who.int/publications/2004/9241546190_part1.pdf] 39

62. World Health Organization. Guidelines on transmissible spongiform 40 encephalopathies in relation to biological and pharmaceutical products. Geneva, 41 World Health Organization, 2003, 42 [http://www.who.int/biologicals/publications/en/whotse2003.pdf] 43

63. World Health Organization. WHO guidelines on clinical evaluation of vaccines: 44 regulatory expectations. In: WHO Expert Committee on Biological 45 Standardization. Fifty-second report. Geneva, World Health Organization, 2004, 46 Annex 1 (WHO Technical Report Series, No. 924) 47 [http://www.who.int/biologicals/publications/trs/areas/vaccines/clinical_evaluatio48 n/035-101.pdf] 49


64. World Health Organization. WHO guidelines on nonclinical evaluation of 1 vaccines. In: WHO Expert Committee on Biological Standardization. Fifty-fourth 2 report. Geneva, World Health Organization, 2005, Annex 1 (WHO Technical 3 Report Series, No. 927) 4 [http://www.who.int/biologicals/publications/trs/areas/vaccines/nonclinical_evalu5 ation/ANNEX%201Nonclinical.P31-63.pdf] 6

65. World Health Organization. Guidelines for the production and quality control of 7 candidate tetravalent dengue virus vaccines (live). In: WHO Expert Committee on 8 Biological Standardization. Fifty-fifth report. Geneva, World Health Organization, 9 2006, Annex 1 (WHO Technical Report Series No. 932) 10 [http://www.who.int/biologicals/publications/trs/areas/vaccines/dengue/TRS932A11 nnex%201_Dengue%20virus%20vacc%20live.pdf] 12

66. World Health Organization. WHO guidelines on tissue infectivity distribution in 13 transmissible spongiform encephalopathies. Geneva, Switzerland, World Health 14 Organization, 2006, 15 [http://www.who.int/bloodproducts/TSEPUBLISHEDREPORT.pdf] 16

67. World Health Organization. Guidelines for plaque-reduction neutralization testing 17 of human antibodies to dengue viruses.21. 2007, 18 [http://whqlibdoc.who.int/hq/2007/WHO_IVB_07.07_eng.pdf] 19

68. World Health Organization. Guidelines for the clinical evaluation of dengue 20 vaccines in endemic areas. In: WHO/IVB/08.12. Geneva, World Health 21 Organization, 2008, 22 [http://whqlibdoc.who.int/hq/2008/WHO_IVB_08.12_eng.pdf] 23

69. World Health Organization. Dengue: guidelines for diagnosis, treatment, 24 prevention and control. Geneva, World Health Organization, 2009, 25 WHO/HTM/NTD/DEN/2009.1 26 [http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf] 27

70. World Health Organization. Guidelines for independent lot release of vaccines by 28 regulatory authorities (Adopted, 2010). In: WHO Expert Committee on Biological 29 Standardization. Geneva, World Health Organization, In press, 30

71. World Health Organization. Guidelines on stability evaluation of vaccines 31 (Adopted, 2006). In: WHO Expert Committee on Biological Standardization. 32 Geneva, World Health Organization, In press, 33 [http://www.who.int/biologicals/publications/trs/areas/vaccines/stability/Microsof34 t%20Word%20-%20BS%202049.Stability.final.09_Nov_06.pdf] 35

72. World Health Organization. Model guidance for the storage and transport of time 36 and temperature-sensitive pharmaceutical products (Adopted, 2010). In: WHO 37 Expert Committee on Biological Standardization. Geneva, World Health 38 Organization, In press, 39

73. World Health Organization. Recommendations for the evaluation of animal cell 40 cultures as substrates for the manufacture of biological medicinal products and for 41 the characterization of cell banks (Adopted, 2010). In: WHO Expert Committee 42 on Biological Standardization. Geneva, World Health Organization, In press, 43

74. World Health Organization. Recommendations to assure the quality, safety and 44 efficacy of live attenuated yellow fever vaccines (Adopted, 2010). In: WHO 45 Expert Committee on Biological Standardization. Geneva, World Health 46 Organization, In press, 47


75. Yauch LE, Shresta S. Mouse models of dengue virus infection and disease. 1

Antiviral Res, 2008, 80:87-93. 2 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&do3 pt=Citation&list_uids=18619493] 4

5

guidelines on the quality, safety and efficacy of dengue ...other types 13 of dengue virus vaccines...

Documents