recommendations to assure the quality, safety and efficacy ......14 paper on hepatitis e in 2015...

1

WHO/BS/2018.2348 2

ENGLISH ONLY 3

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION 4

Geneva, 29 October to 2 November 2018 5

Recommendations to assure the quality, safety and efficacy of 6

recombinant hepatitis E vaccines 7

Proposed new recommendations 8

NOTE: 9

This document has been prepared for the purpose of inviting comments and suggestions on the proposals 10

contained therein, which will then be considered by the Expert Committee on Biological Standardization 11

(ECBS). Publication of this draft is to provide information about the Recommendations to assure the 12

quality, safety and efficacy of recombinant hepatitis E vaccines to a broad audience and to improve 13

transparency of the consultation process. 14

These Recommendations were developed based on the outcomes and consensus of the WHO working 15

group meeting and informal consultation convened in 2017 and 2018 with participants from national 16

regulatory authorities, national control laboratories, vaccine manufacturers and academia researchers. 17

The text in its present form does not necessarily represent an agreed formulation of the Expert 18 Committee on Biological Standardization. Written comments proposing modifications to this text 19 MUST be received by 28 September 2018 in the Comment Form available separately and should be 20

addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Department of 21 Essential Medicines and Health Products (EMP). Comments may also be submitted electronically to the 22

Responsible Officer: Dr Dianliang Lei at email: [email protected]. 23

24

The outcome of the deliberations of the Expert Committee will be published in the WHO Technical Report 25

Series. The final agreed formulation of the document will be edited to be in conformity with the "WHO 26

style guide" (WHO/IMD/PUB/04.1). 27

© World Health Organization 2018 28

All rights reserved. 29 30 This draft is intended for a restricted audience only, i.e. the individuals and organizations having received this draft. The 31 draft may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in part or in whole, 32 in any form or by any means outside these individuals and organizations (including the organizations' concerned staff and 33 member organizations) without the permission of the World Health Organization. The draft should not be displayed on any 34 website. 35 36 Please send any request for permission to: 37 38

WHO/BS/2018.2348 of 60 Dr Ivana Knezevic, Technologies Standards and Norms, Department of Essential Medicines and Health Products, World 1 Health Organization, CH-1211 Geneva 27, Switzerland. Email: [email protected]. 2 3 The designations employed and the presentation of the material in this draft do not imply the expression of any opinion 4 whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or 5 of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate 6 border lines for which there may not yet be full agreement. 7 8 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 9 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors 10 and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 11 12 All reasonable precautions have been taken by the World Health Organization to verify the information contained in this 13 draft. However, the printed material is being distributed without warranty of any kind, either expressed or implied. The 14 responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health 15 Organization be liable for damages arising from its use. 16 17 This draft does not necessarily represent the decisions or the stated policy of the World Health Organization. 18 19

WHO/BS/2018.2348 of 60

Contents 1

Introduction ............................................................................................................................................. 5 2

Scope ....................................................................................................................................................... 5 3

General considerations ............................................................................................................................ 6 4

Terminology .......................................................................................................................................... 10 5

Part A. Manufacturing recommendations ............................................................................................. 12 6

A.1 Definitions ............................................................................................................................. 12 7

A.2 General manufacturing recommendations ............................................................................ 12 8

A.3 Control of source materials ................................................................................................... 12 9

A.4 Control of HEV protein production ...................................................................................... 13 10

A.5 Control of purified antigen bulk ............................................................................................ 15 11

A.6 Control of adsorbed antigen bulk .......................................................................................... 17 12

A.7 Control of final bulk .............................................................................................................. 19 13

A.8 Filling and containers ............................................................................................................ 20 14

A.9 Control tests on the final filled lot ........................................................................................ 21 15

A.10 Records ................................................................................................................................. 22 16

A.11 Retained samples .................................................................................................................. 23 17

A.12 Labelling ............................................................................................................................... 23 18

A.13 Distribution and transport ..................................................................................................... 23 19

A.14 Stability testing, storage and expiry date .............................................................................. 23 20

Part B. Nonclinical evaluation of recombinant hepatitis E vaccines .................................................... 25 21

B.1 Strategy for cloning and expressing the gene product .......................................................... 25 22

B.2 Product characterization and process development .............................................................. 25 23

B.3 Pharmacodynamic studies ..................................................................................................... 26 24

B.4 Biodistribution studies .......................................................................................................... 28 25

B.5 Toxicology studies ................................................................................................................ 28 26

Part C. Clinical evaluation of recombinant hepatitis E vaccines .......................................................... 29 27

C.1 Introduction ........................................................................................................................... 29 28

C.2 Assays .................................................................................................................................. 29 29

C.3 Immunogenicity ................................................................................................................... 30 30

C.4 Efficacy ................................................................................................................................ 32 31

C.5 Safety ................................................................................................................................... 37 32

Part D. Recommendations for NRAs .................................................................................................... 37 33

D.1 General recommendations..................................................................................................... 37 34

D.2 Official release and certification ........................................................................................... 38 35

Authors and acknowledgements ........................................................................................................... 38 36

References ............................................................................................................................................. 40 37

Appendix 1. Model summary protocol for the manufacturing and control of recombinant hepatitis E 38

vaccines ................................................................................................................................................. 49 39

Appendix 2. Model NRA lot release certificate for recombinant hepatitis E vaccines......................... 60 40

41

WHO/BS/2018.2348 of 60 1

Recommendations published by WHO are intended to be scientific and advisory in nature.

Each of the following sections constitutes recommendations for national regulatory

authorities (NRAs) and for manufacturers of biological products. If an NRA so desires, these

WHO Recommendations may be adopted as definitive national requirements, or

modifications may be justified and made by the NRA. It is recommended that modifications

to these WHO Recommendations are made only on condition that such modifications ensure

that the product is at least as safe and efficacious as that prepared in accordance with the

recommendations set out below. The parts of each section printed in small type are comments

or examples intended to provide additional guidance to manufacturers and NRAs.

2

Abbreviations 3

ALT Alanine aminotransferase 4

DNA Deoxyribonucleic acid 5

ECBS Expert Committee on Biological Standardization 6

ELISA Enzyme-linked immunosorbent assay 7

GACVS Global Advisory Committee on Vaccine Safety 8

GLP Good laboratory practice 9

HEV Hepatitis E virus 10

HIV Human immunodeficiency virus 11

HPLC High-performance liquid chromatography 12

HPSEC High-performance size exclusion chromatography 13

IFA Immunofluorescence foci assay 14

IgG Immunoglobulin G 15

IgM Immunoglobulin M 16

LMICs Low- and middle-income countries 17

MALDI-TOF Matrix assisted laser desorption/Ionisation time-of-flight mass spectrometry 18

MCB Master cell bank 19

NAT Nucleic acid amplification technique 20

NCL National control laboratory 21

NIBSC National Institute for Biological Standards and Control 22

NRA National regulatory authority 23

ORF Open reading frame 24

PCR Polymerase chain reaction 25

PEI Paul Ehrlich Institut 26

WHO/BS/2018.2348 of 60

RNA Ribonucleic acid 1

SAGE Strategic Advisory Group of Experts on Immunization 2

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis 3

TEM Transmission electron microscopy 4

ULN Upper limit of normal 5

WCB Working cell bank 6

WHO World Health Organization 7

8

Introduction 9

Hepatitis E virus (HEV) is a major cause of sporadic and epidemic hepatitis that is found 10

worldwide. The highest seroprevalence rates are observed in regions where low standards of 11

sanitation increase the risk for transmission of the virus (1). 12

The WHO Strategic Advisory Group of Experts on Immunization (SAGE) issued a position 13

paper on hepatitis E in 2015 which reviewed existing evidence on the burden of hepatitis E 14

and on the safety, immunogenicity, efficacy and cost-effectiveness of a hepatitis E vaccine 15

that was first licensed in China (1). This vaccine contains the HEV open reading frame 2 16

(ORF2) capsid protein manufactured in E. coli using recombinant technology. The WHO 17

Global Advisory Committee on Vaccine Safety (GACVS) reviewed this same hepatitis E 18

vaccine in 2014 and concluded that the vaccine had an acceptable safety profile (2). In 2016, 19

WHO published its Global Health Sector Strategy on Viral Hepatitis 20162021 (3), which 20

addresses hepatitis A, B, C and E. Hepatitis E is probably the most neglected of the four. This 21

document highlights the need for some urgency in addressing all viral hepatitis, and 22

particularly hepatitis E for which only one vaccine is approved anywhere in the world and no 23

good therapies exist. 24

Manufacturers and other stakeholders have requested WHO to develop recommendations to 25

ensure the quality, safety and efficacy of hepatitis E vaccines. In response, a series of 26

meetings was convened by WHO to review the current status of development and likely time 27

to licensure of recombinant hepatitis E vaccines (4). These meetings were attended by experts 28

from around the world involved in the research, manufacture, regulatory assessment and 29

approval, control-testing and release of hepatitis E vaccines. Participants were drawn from 30

academia, national regulatory authorities (NRAs), national control laboratories (NCLs) and 31

industry. 32

Scope 33

These WHO Recommendations provide guidance to NRAs and manufacturers on the 34

manufacturing process, and on nonclinical and clinical aspects, to assure the quality, safety 35

and efficacy of recombinant hepatitis E vaccines. 36

37

WHO/BS/2018.2348 of 60

The present document encompasses recombinant hepatitis E vaccines for prophylactic use 1

based on the ORF2 capsid protein. 2

3

This document should be read in conjunction with other relevant WHO guidance, especially 4

on the nonclinical (5) and clinical (6) evaluation of vaccines. Other WHO guidance should be 5

considered, including – as appropriate to the vaccine – guidance on the evaluation of animal 6

cell cultures as substrates for the manufacture of biological medicinal products, on the 7

characterization of cell banks (7) and on the nonclinical evaluation of vaccine adjuvants and 8

adjuvanted vaccines (8). 9

10

General considerations 11

Hepatitis E virus 12

13

HEV is a non-enveloped positive-sense RNA virus of the Hepeviridae family. The single-14

stranded viral genome is 7.2 kb in length and contains three open reading frames. Of these, 15

ORF2 codes for the viral capsid protein which is the target of neutralizing antibodies against 16

HEV (9). HEV isolates were classified into four human genotypes (genotypes 14) such that 17

the nucleic acid variation of ORF2 between different genotypes is more than 20%; however, 18

the four genotypes form a single serotype based on their immune reactivity. Different 19

genotypes differ by more than 8.8% in highly conserved ORF1 and ORF2 amino acid 20

sequences (10). These genotypes have been subdivided further into numerous subtypes, 21

although the underlying criteria are controversial (11, 12). Nevertheless, HEV strains that 22

infect humans belong to one currently identifiable serotype, with marked serological cross-23

reactivity as well as evidence for cross-protection in non-human primates and in humans (13). 24

25

Epidemiology 26

27

Almost all of the information available on the epidemiology of HEV concerns genotypes 14. 28

HEV genotypes 1 and 2 primarily infect humans, whereas genotypes 3 and 4 mainly infect 29

mammalian animals with occasional cross-species transmission to humans. 30

31

The epidemiology and clinical presentation of HEV infection vary greatly by geographical 32

location, based primarily on differences in circulating HEV genotypes (1416). A Global 33

Burden of Disease study estimated that HEV genotypes 1 and 2 account for approximately 34

20.1 million HEV infections, 3.4 million symptomatic cases, 70 000 deaths, and 3000 35

stillbirths annually (17). 36

37

Hepatitis E infection due to genotypes 1 and 2 has been identified in at least 63 countries, of 38

which about half have reported large outbreaks (17). The overall burden of disease due to 39

hepatitis E is greatest in low- and middle-income countries (LMICs), especially where clean 40

drinking water is scarce, as in Africa and Asia, as faecal contamination of drinking-water is a 41

major route of HEV transmission (17). There is no evidence of large outbreaks of hepatitis E 42

WHO/BS/2018.2348 of 60

in developed countries although small clusters of cases associated with foodborne 1

transmission have occurred in Europe and Japan (18). There are also countries with no 2

recorded sporadic disease or outbreak but where serological evidence of past HEV infection 3

has been reported, suggesting that HEV infection may be endemic. 4

5

Waterborne hepatitis E outbreaks have been reported from at least 30 countries on three 6

continents (Africa, Asia and North America [Mexico]); these outbreaks were caused chiefly 7

by HEV genotype 1. Large waterborne hepatitis E outbreaks frequently occur in the Indian 8

subcontinent (19). In Australia, Europe and North America, cases due to genotype 1 have 9

been reported in returning travellers. The distribution of HEV genotype 2 has been elusive, 10

with most cases reported from Mexico, Namibia, Nigeria some other West African countries, 11

but it seems rare with few cases to date (2022). 12

13

In recent years, there have been numerous outbreaks caused by HEV genotype 1 in camps for 14

internally displaced persons and refugees in Africa. There is some evidence that other modes 15

of transmission, including from person to person, may contribute to the prolongation of 16

outbreaks, particularly in displaced populations (23). Recent large outbreaks have occurred 17

among displaced persons in Chad, Niger, Sudan and Uganda (20, 2426). The first 18

serologically confirmed outbreak documented in Africa occurred among Angolan refugees in 19

Namibia in 1983. During a recent outbreak in northern Uganda, a high mortality rate was 20

recorded among children under 2 years of age (24); however, the cause of death in these 21

children was not verified. As in the outbreak in northern Uganda in 2007, the South Sudan 22

outbreak also started during the rainy season with high disease attack rates (7.4%) among 23

camp residents and high mortality among pregnant women (10.4%) (25). A sero-survey 24

conducted during this outbreak showed that more than half of residents had no evidence of 25

recent or past HEV infection, suggesting that these persons remained uninfected. Both the 26

Ugandan and South Sudanese outbreaks lasted well over a year, indicating that prevention 27

and control efforts in such outbreaks can be challenging. 28

29

Although waterborne HEV outbreaks can result in large numbers of cases over a short period 30

of time, most hepatitis E cases in LMICs probably occur within smaller clusters or result 31

from sporadic transmission (27). The risk factors for sporadic hepatitis E are less well 32

understood, although water contamination may play a role. 33

34

In industrialized countries, where the disease burden is much lower, zoonotic transmission, 35

mainly through consumption of uncooked or undercooked meat, is a possible mode of 36

transmission and HEV genotype 3 is the predominant genotype (15). Despite the ubiquity of 37

HEV genotype 3 in the domestic pig population, clinically apparent human infections with 38

this genotype have been reported almost entirely in developed countries. 39

40

In recent years, HEV genotype 4 has been found to circulate in animals in China, India and 41

several European countries; most human cases of hepatitis due to HEV genotype 4 have been 42

WHO/BS/2018.2348 of 60

reported in China. The main mode of transmission of HEV genotype 4 is also believed to be 1

through consuming of infected pork and having contact with domestic pigs. 2

3

There is no evidence of sexual transmission of HEV (15). Transfusion transmission of HEV 4

occurs and is well documented; however, its contribution to the overall disease burden is 5

limited (15, 28, 29). 6

7

Disease and diagnosis 8

9

HEV-infected persons exhibit a wide clinical spectrum, ranging from asymptomatic infection 10

through acute icteric hepatitis to fulminant hepatitis. In immunocompetent persons, 11

asymptomatic infection is common and the disease presentation is often mild. The ratio of 12

symptomatic to asymptomatic infection has been estimated to range from 1:2 to 1:10 or more 13

in outbreak settings and increases with the age at the time of infection (30). The incubation 14

period ranges from 15 to 60 days, with a mean of 40 days (31). Infection with HEV genotype 15

1 is associated with serious disease more often than infection with other genotypes, though 16

the extent to which such severe disease occurs with genotypes 2 and 4 is not well documented. 17

Studies in non-human primates have shown a relationship between the dose of viral inoculum 18

and the host’s immunological response and degree of liver injury (32). 19

20

In LMICs, where HEV genotypes 1 and, to a lesser extent, 2 are the most commonly 21

identified causes of hepatitis E, the disease mainly affects young adults (e.g. aged 15–39 22

years), with preponderance in males. During waterborne outbreaks, children may develop 23

severe hepatitis E due to coinfection with hepatitis A virus (33). 24

25

During epidemics, fulminant hepatitis E occurs at a disproportionately high rate among 26

pregnant women (3436). The disease is typically most severe during the third trimester of 27

pregnancy (37, 38). While mortality from hepatitis E ranges from 0.1% to 4% in the general 28

population, it can range from 10% to 50% among women in the third trimester of pregnancy 29

(1, 37, 39, 40). The mechanism for the high mortality among pregnant women is unclear (30). 30

The causes of death include fulminant liver failure and obstetric complications, including 31

excessive bleeding (35). HEV can be transmitted from mother to fetus during pregnancy, 32

resulting in poor fetal outcomes, including miscarriage, premature delivery and stillbirths (19, 33

34). 34

35

HEV genotypes 3 and 4 have been repeatedly reported to cause severe disease as well as 36

chronic hepatitis E in immunocompromised persons in China and Europe. Chronic infections 37

do not occur in otherwise healthy individuals. HEV infection in persons who receive 38

immunosuppressive treatment following solid organ or bone marrow transplantation, and in 39

persons with severe immunodeficiency of other origins, is associated with risk of progression 40

to chronic hepatitis E (41). HIV-infected patients are not at higher risk for HEV infection; the 41

number of acute infections reported in these populations is low and very few chronic cases 42

have been reported (4244). The clinical manifestation and progression of chronic hepatitis E 43

WHO/BS/2018.2348 of 60

(lasting > 6 months) are variable; some cases progress to significant fibrosis in a relatively 1

short period of time. 2

3

Persons with pre-existing chronic liver disease are prone to develop severe hepatitis 4

following HEV infection. Those with advanced liver disease, including cirrhosis, may 5

develop acute hepatic failure when infected with HEV (19). The burden of HEV-induced 6

acute liver failure in patients with pre-existing chronic liver disease is unknown. 7

8

Laboratory diagnosis of recent HEV infection is based on detection of HEV-specific IgM 9

antibodies, the recent appearance or several-fold increase in titres of specific IgG antibodies 10

or detection of HEV RNA in blood samples (45). Specific detection of HEV antigen can also 11

be a marker for diagnosis of hepatitis E (46). However, the performance characteristics 12

(sensitivity and specificity) of some of the currently-available commercial assays for anti-13

HEV antibodies are suboptimal (4753). In one study that compared six different assays, 14

sensitivity of the individual assays ranged from 72% to 98%, and specificity from 78% to 15

96%; further, the kappa coefficients for agreement between results of various pairs of tests 16

varied from 0.42 to 0.80 (54). This has implications for clinical trials based on serological 17

outcomes and for studies that rely on these serological tests to estimate the burden of disease 18

and previous history of infection. One recent study compared results from a newer diagnostic 19

assay to the results from the assay used in the original study of seroprevalence in rural 20

Bangladesh and found that the newer assay showed much higher seroprevalence in the 21

population (55). 22

23

Immune response to natural HEV infection 24

25

Past HEV infection is characterized by the presence of serum IgG antibodies directed against 26

the viral capsid protein, which may confer protection against reinfection; however, the 27

protective IgG antibody concentration is not known and the duration of protection following 28

natural infection is uncertain. In Kashmir, serological follow-up of 45 persons known to have 29

had hepatitis E during the 1978 outbreak found that 47% had detectable anti-HEV IgG 14 30

years after infection (56), though the difficulties with interpreting serological assay results 31

has already been mentioned. A recent study based on 67 months’ serological follow-up data 32

and mathematical modelling suggested that naturally-acquired anti-HEV IgG will remain 33

detectable in half of the seropositive individuals for 14.5 years (57). In another follow-up 34

study, 100% of persons had measurable anti-HEV IgG 5 years after infection (58). However, 35

the subjects studied were living in hyperendemic areas where the possibility of multiple re-36

exposures and natural boosting cannot be ruled out. 37

38

There is other evidence that naturally-acquired infection does not confer lifelong immunity. 39

For instance, even in endemic areas, the prevalence of anti-HEV IgG in the population does 40

not reach the very high levels observed for hepatitis A which does confer lifelong protection, 41

and attack rates are highest among young-to-middle-aged adults, suggesting that infection 42

during early life may not confer lifetime protection, or that infections usually occur later in 43

WHO/BS/2018.2348 of 60

life. In addition, outbreaks recur in countries where previous epidemics would be expected to 1

have resulted in a level of population immunity sufficient to prevent future outbreaks. The 2

duration of protection conferred by naturally-acquired antibodies has important implications 3

for long-term vaccine efficacy. 4

5

Vaccines against HEV 6

7

Although many experimental HEV vaccines have been evaluated in virus challenge studies in 8

non-human primates, up to mid-2017 there had been only one vaccine licensed for human use 9

in any country. This vaccine was licensed in China in December 2011 for use in persons aged 10

16 years and over. It is based on a 239-amino acid recombinant HEV peptide, corresponding 11

to amino acids 368–606 of ORF2 which encodes the capsid protein of genotype 1 HEV 12

(5962). Vaccine efficacy after three doses was 100% over a 12-month period after the last 13

dose and was 95.5% over 19 months in all subjects who had received at least one dose. Other 14

vaccines based on HEV capsid protein are currently in nonclinical or clinical development. 15

16

Terminology 17

The definitions given below apply to the terms as used in these WHO Recommendations. 18

They may have different meanings in other contexts. 19

Adventitious agents: contaminating microorganisms that may include bacteria, fungi, 20

mycoplasmas, and endogenous and exogenous viruses, that have been unintentionally 21

introduced into the manufacturing process. 22

Cell bank: a collection of containers containing aliquots of a suspension of cells from a 23

single pool of uniform composition, stored frozen under defined conditions (typically −60 °C 24

or below for yeast or bacteria and in liquid nitrogen for insect or mammalian cell lines). The 25

terms master cell bank (MCB) and working cell bank (WCB) are used in these 26

recommendations. An MCB is a bank of a cell substrate from which all subsequent cell banks 27

used for vaccine production will be derived; an MCB represents a well characterized 28

collection of cells derived from a single tissue or cell. A WCB is a cell bank derived by 29

propagation of cells from an MCB under defined conditions and is used to initiate production 30

of cell cultures on a lot-by-lot basis. The WCB is also referred to as “manufacturer’s working 31

cell bank” in other documents. 32

The individual containers (e.g. ampoules or vials) should be representative of the pool 33

of cells from which they are taken and should be frozen on the same day following the 34

same procedure and using the same equipment and reagents. 35

Cell substrate: cells used to manufacture a biological product. 36

Final bulk: the formulated vaccine present in a container from which the final containers are 37

filled. The final bulk may be prepared from one or more antigen bulks. If prepared from one 38

WHO/BS/2018.2348 of 60

or more antigen bulks, mixing should result in a uniform preparation to ensure that the final 1

containers are homogenous. 2

Final filled lot: a collection of sealed final containers of finished vaccine that is 3

homogeneous with respect to the risk of contamination during the filling process. All of the 4

final containers must therefore have been filled from a single vessel of final bulk in one 5

working session. Also referred to as final lot or final product in other documents. 6

Single harvest pool: a pool of a number of single harvests of the same virus type processed 7

at the same time. 8

Seed lot (master, working seed lot): a quantity of bacterial, viral or cell suspension that has 9

been derived from one strain, has been processed as a single lot, and has a uniform 10

composition. It is used to prepare the inoculum for the production medium. 11

Single harvest: the biological material prepared from a single production run before further 12

downstream processing. 13

14

International reference materials 15

Subsequent sections of this document refer to WHO reference materials that may be used in 16

laboratory or clinical evaluations. Key standards used in the control of hepatitis E vaccines 17

include the following: 18

An International Standard for antibodies to hepatitis E virus is available for 19

standardization of diagnostic tests for use in seroprevalence studies and for assessing 20

immunity. The first International Reference Reagent for antibodies to hepatitis E virus 21

(95/584), which was established by the Expert Committee on Biological 22

Standardization (ECBS) in 1997, contains 50 units per ampoule (63). This preparation 23

is in the custody of the National Institute for Biological Standards and Control 24

(NIBSC) Potters Bar, England. 25

International Standards for hepatitis E RNA are also available. These standards are 26

suitable for the calibration of in-house or working standards for the amplification and 27

detection of hepatitis E virus RNA. The first International Standards for hepatitis E 28

virus RNA for nucleic acid amplification technique (NAT)-based assays was 29

established by ECBS in 2011 and contains 250 000 IU per ampoule (64). The first 30

International Reference Panel for hepatitis E virus NRA genotypes for NAT-based 31

assays (8578/13) contains 11 members and was established by the ECBS in 2015 (65). 32

These two preparations are in the custody of the Paul Ehrlich Institut (PEI), Langen 33

Germany. 34

WHO’s Catalogue of International Reference Preparations should be consulted for the 35

latest list of appropriate WHO International Standards and reference materials. See: 36

http://www.who.int/bloodproducts/catalogue/en/. 37

http://www.who.int/bloodproducts/catalogue/en/

WHO/BS/2018.2348 of 60

Product-specific national reference for potency assay is under development by the 1

National Institutes for Food and Drug Control, China. 2

3

4

Part A. Manufacturing recommendations 5

A.1 Definitions 6

7

A.1.1 International name and proper name 8

The international name should be “recombinant hepatitis E vaccine”. The proper name should 9

be the equivalent of the international name in the language of the country of origin. 10

The use of the international name should be limited to vaccines that meet the specifications 11

elaborated below. 12

13

A.1.2 Descriptive definition 14

The recombinant hepatitis E vaccine is a sterile liquid vaccine preparation that contains 15

purified recombinant capsid protein of hepatitis E virus. The protein may be formulated with 16 a suitable adjuvant. Such vaccines are for prophylactic use. 17

18

A.2 General manufacturing recommendations 19

20

The general manufacturing recommendations contained in WHO good manufacturing 21

practices for pharmaceutical products: main principles (66) and Good manufacturing 22

practices for biological products (67) should apply to the design, establishment, operation, 23

control and maintenance of manufacturing facilities for recombinant hepatitis E vaccines. 24

A.3 Control of source materials 25

26

A.3.1 Cells for antigen production 27

The use of any type of cell should be based on a cell bank system (68, 69) and should be 28

approved by and registered with the NRA. The maximum allowable number of passages or 29

population doublings from the MCB to production level should be approved by the NRA. 30

A.3.1.1 Recombinant cells for production 31

The history and characteristics of the parental cells, including bacteria or eukaryotic cells if 32

relevant, should be fully described. The recombinant production strain (parental cell 33

transformed with the recombinant expression construct) should be fully described and 34

information should be given on the results of any adventitious agent testing required and on 35

homogeneity and accuracy of the inserted sequence (including copy number per cell) for the 36

WHO/BS/2018.2348 of 60

MCB and WCB. Plasmid retention should be demonstrated as part of process validation. A 1

full description of the biological characteristics of the host cell and expression strategy should 2

be given. This should include genetic markers of the host cell, the construction, genetics and 3

structure of the expression system, and the origin and identification of the HEV sequence that 4

is being cloned. The complete sequence of the entire construct should be determined, 5

including control elements, and should be provided as part of the validation of the production 6

process. The molecular and physiological measures used to promote and control the 7

expression of the cloned HEV sequence in the host cell should be described in detail (70). 8

Cells must be maintained in a state that allows for recovery of viable cells without alteration 9

of genotype (e.g. frozen in liquid nitrogen). The cells should be recovered if necessary in 10

selective media so that the genotype and phenotype are maintained and clearly identifiable. 11

Cell banks should be identified and fully characterized by appropriate tests. 12

Data (e.g. on plasmid restriction enzyme mapping, nutritional requirements or antibiotic 13

resistance, if applicable) that demonstrate the genetic stability of the expression system 14

during passage of the recombinant WCB up to and beyond the passage level used for 15

production should be provided to and approved by the NRA as part of the validation of the 16

production process. Any instability of the expression system occurring in the seed culture or 17

after a production-scale run should be documented. Stability studies should also be performed 18

to confirm cell viability, maintenance of the expression system etc. after retrieval from 19

storage. These studies may be performed as part of their routine use in production or may 20

involve samples being taken specifically for this purpose. 21

A.3.1.1.1 Tests on recombinant bacteria MCB and WCB 22

MCBs and WCBs should be tested for the absence of bacterial and fungal contamination by 23

appropriate tests, as specified in Part A, section 5.2 of WHO’s General requirements for the 24

sterility of biological substances (71), or by a method approved by the NRA, to demonstrate 25

that only the bacterial production strain is present in the MCB and WCB. 26

A.3.1.2 Other expression systems 27

If other expression systems are used, characterization may be based on WHO’s 28

Recommendations to assure the quality, safety and efficacy of recombinant human 29

papillomavirus virus-like particle vaccines (72) and Recommendations for the evaluation of 30

animal cell culture as substrates for the manufacture of biological medicinal products and 31

for the characterization of cell banks (7). 32

A.4 Control of HEV protein production 33

34

A.4.1 Microbial purity 35

Microbial purity of recombinant bacterial cultures should be monitored in each fermentation 36

vessel at the end of the production run by methods approved by the NRA. 37

WHO/BS/2018.2348 of 60

Any agent added to the fermenter or bioreactor with the intention of feeding cells or of 1

inducing production or increasing cell density should be approved by the NRA. No 2

antibiotics should be added at any stage of manufacturing unless approved by the NRA. 3

A.4.2 Control of single harvests 4

A.4.2.1 Storage and intermediate hold times 5

After the production run, the cell suspension or the product partially purified from it (e.g. by 6

preparation of inclusion bodies) should be maintained under conditions shown by the 7

manufacturer to retain the desired biological activity. Hold times should be approved by the 8

NRA. 9

A.4.2.2 Tests on single harvest or single harvest pool 10

If appropriate, tests may be conducted on a single antigen harvest or on a pool of single 11

antigen harvests depending on the production strategy. The protocol should be approved by 12

the NRA. 13

A.4.2.2.1 Sampling 14

Samples required for the testing of antigen harvests should be taken immediately on 15

harvesting or pooling and before further processing. Tests for sterility and adventitious agents, 16

as described below in sections A.4.2.2.2 and A.4.2.2.4, should preferably be performed 17

within 24 hours. If these tests are not performed within 24 hours, the samples taken for these 18

tests should be kept at an appropriate temperature. Where mammalian or insect cell 19

expression systems are used, samples should be stored at 60°C and subjected to no more 20

than one freeze–thaw cycle. For other systems in which the infectivity of adventitious agents 21

will not need to be preserved, an appropriate temperature should be chosen. Moreover, 22

evidence should be provided that the freezing process does not affect the viability of the 23

adventitious agents putatively present in the sample. 24

A.4.2.2.2 Tests for bacteria, fungi and mycoplasmas 25

Harvests from bacterial expression systems could have bacterial contamination. Therefore, a 26

method such as the microbial limits test may be appropriate for addressing microbial purity. 27

Such testing should be approved by the NRA. 28

For non-bacterial production systems, each single antigen harvest or single harvest pool 29

should be shown to be free from bacterial and fungal contamination by appropriate tests, as 30

specified in Part A, section 5.2, of WHO’s General requirements for the sterility of biological 31

substances (71) and mycoplasma (73). 32

A.4.2.2.3 Test for identity 33

Each harvest should be identified as HEV antigen by a suitable assay such as SDS-PAGE, 34

ELISA or other methods. The tests should be approved by the NRA. Alternatively, the 35

identity can be confirmed as part of testing of the purified antigen. 36

WHO/BS/2018.2348 of 60

A.4.2.2.4 Tests for adventitious agents if insect or mammalian cells are used in production 1

Each single harvest or single harvest pool should be tested for adventitious viruses in cell 2

cultures selected for their appropriateness to the origin and passage history of the insect cell 3

substrate and recombinant baculovirus or the mammalian cell substrate. These cell cultures 4

should include, as a minimum, a monkey kidney cell line and a human cell line. Antisera 5

used for the purpose of neutralizing the recombinant baculovirus should be free from 6

antibodies that may neutralize adventitious viruses and should preferably be generated by the 7

immunization of specific-pathogen-free animals with an antigen made from a source (other 8

than the production cell line) which has itself been tested for freedom from adventitious 9

agents. The inoculated indicator cells should be examined microscopically for cytopathic 10

changes. At the end of the examination period, the cells should also be tested for 11

haemadsorbing viruses. 12

Additional testing for specific adventitious viruses may be performed (e.g. by PCR 13

amplification techniques). 14

A.5 Control of purified antigen bulk 15

16

The purification process can be applied to a single antigen harvest, part of a single antigen 17

harvest or a pool of single antigen harvests and should be approved by the NRA. The 18

maximum number of harvests that may be pooled should also be approved by the NRA. 19

Adequate purification may require several purification steps based on different biophysical 20

and/or biochemical principles and may involve disassembly and reassembly of particles. The 21

entire process (sequence of process steps) used for the purification of the final antigen bulk 22

should be appropriately validated and should be approved by the NRA. Any reagents added 23

during the purification processes (such as DNase) should be documented. 24

The purified antigen bulk can be stored under conditions shown by the manufacturer to allow 25

it to retain the desired biological activity. Intermediate hold times should be approved by the 26

NRA. 27

A.5.1 Tests on the purified antigen bulk 28

Purified antigen bulks should be subjected to the tests listed below. Some tests may be 29

omitted if performed on the adsorbed antigen bulk. All quality control release tests and 30

specifications for purified antigen bulk, unless otherwise specified, should be validated by the 31

manufacturer and approved by the NRA. 32

A.5.1.1 Purity 33

The degree of purity of the purified antigen bulk, and levels of residual host-cell protein and 34

DNA, should be assessed by suitable methods. One suitable method for analysing the 35

proportion of potential contaminating proteins is SDS-PAGE under reducing denaturing 36

conditions. The protein bands within the gel should be identified by sensitive staining 37

WHO/BS/2018.2348 of 60

techniques and quantified by densitometric analysis. Other suitable methods such as HPLC 1

may also be used for purity analysis. 2

A.5.1.2 Protein content 3

Each purified antigen bulk should be tested for the total protein content using a suitable 4

method. 5

Alternatively, the total protein content may be calculated from measurement of an earlier 6

purified process intermediate. 7

A.5.1.3 Antigen content 8

The antigen content should be measured on the purified antigen bulk or the adsorbed antigen 9

bulk (see section A.6.3.7) by an appropriate method. 10

The ratio of antigen content to protein content may be calculated and monitored for each 11

purified antigen bulk. 12

International Standards and reference reagents are not available for the control of HEV 13

vaccine antigen content. Therefore, product-specific reference preparations should be 14

developed and used. 15

A.5.1.4 Sterility tests for bacteria and fungi 16

Each purified antigen bulk should be tested for bacterial and fungal sterility, as specified in 17

Part A, section 5.2, of WHO’s General requirements for the sterility of biological substances 18

(71), or by a method approved by the NRA. Alternatively, this test can be performed on the 19

related adsorbed antigen bulks if the purified bulk is not stored prior to adsorption. 20

A.5.1.5 Percentage of intact monomer 21

The integrity of the HEV protein should be carefully monitored at least in the early stages of 22

process validation and should be assessed by suitable methods. The purity assay (see section 23

A.5.1.1) may also serve to assess the integrity of the HEV monomer. This test could be 24

eliminated with the approval of the NRA. 25

A.5.1.6 Particle size and structure 26

The protein is expected to aggregate to form particles; the size and structure of the particles 27

should be established and monitored. The distribution of particle sizes should be determined. 28

This test may be omitted for routine lot release once consistency of production has been 29

established, with the agreement of the NRA. 30

Suitable methods for assessing particle size include dynamic light scattering, size-exclusion 31

chromatography – high-performance liquid chromatography (SEC–HPLC) and transmission 32

electron microscopy (TEM). A reference preparation should be included for comparison. 33

A.5.1.7 Tests for reagents used during purification or other phases of manufacture 34

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A test should be carried out to detect the presence of any potentially hazardous reagents used 1

during manufacture, using a method(s) approved by the NRA. This test may be omitted for 2

routine lot release upon demonstration that the process consistently eliminates the reagent 3

from the purified antigen bulks, with the approval of the NRA. 4

A.5.1.8 Tests for residual host derived material 5

Where a eukaryotic expression system is used, the amount of residual host-cell DNA derived 6

from the expression system should be determined in each purified antigen bulk by suitably 7

sensitive methods. The level of host-cell DNA should not exceed the maximum level agreed 8

with the NRA, taking into consideration issues such as those discussed in WHO’s 9

Recommendations for the evaluation of animal cell cultures as substrates for the manufacture 10

of biological medicinal products and for the characterization of cell banks (68). 11

These tests may be omitted for routine lot release upon demonstration that the process 12

consistently inactivates the biological activity of the residual DNA or reduces the amount and 13

size of the contaminating residual DNA present in the purified antigen bulks, subject to the 14

agreement of the NRA. 15

Levels of residual protein from the host cell should be determined for all systems. 16

A.5.1.9 Test for viral clearance 17

When an insect or mammalian cell substrate is used for the production of antigens, the 18

production process should be validated in terms of its capacity to remove and/or inactivate 19

adventitious viruses – as described in the Q5A guidelines (74) of the International 20

Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals 21

for Human Use. This testing is performed during vaccine manufacturing development or as 22

part of process validation and is not intended as an assessment for lot release. 23

If a replicating viral vector such as a baculovirus is used, the production process should be 24

validated for its capacity to eliminate (by removal and/or inactivation) residual recombinant 25

virus. 26

A.6 Control of adsorbed antigen bulk 27

28

In some cases, the adsorbed antigen bulk may be further modified by dilution or addition of 29

excipients to generate the final bulk, in which case the considerations described below apply. 30

Where the adsorbed bulk is filled directly without further modification it is the final bulk and 31

this section does not apply (instead, see section A.7). 32

A.6.1 Addition of adjuvant 33

The purified HEV antigen may be adsorbed onto an adjuvant such as an aluminium salt or 34

other substance. The adjuvant and the concentration used should be approved by the NRA. 35

A.6.2 Storage 36

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Until the adjuvanted antigen bulk is formulated into the final bulk, the suspension should be 1

stored under conditions shown by the manufacturer to allow it to retain the desired biological 2

activity. Hold times should be approved by the NRA. 3

A.6.3 Tests on adsorbed antigen bulk 4

All tests and specifications for adsorbed antigen bulk, unless otherwise specified, should be 5

approved by the NRA. 6


Each adsorbed antigen bulk should be tested for bacterial and fungal sterility, as specified in 8

Part A, section 5.2, of WHO’s General requirements for the sterility of biological substances 9

(71), or by an alternative method approved by the NRA. 10

A.6.3.2 Bacterial endotoxins 11

Each adsorbed antigen bulk should be tested for bacterial endotoxins using a method 12

approved by the NRA. 13

If it is inappropriate to test the adsorbed antigen bulk, the test should be performed on the 14

purified antigen bulk prior to adsorption, subject to the approval of the NRA. 15

A.6.3.3 Identity 16

Each adsorbed antigen bulk should be identified as the appropriate HEV antigen by a suitable 17

method. The test for antigen content may also serve as the identity test. This test may be 18

omitted if it is performed on the finished product. 19

A.6.3.4 Adjuvant concentration 20

Adsorbed antigen bulk may be assayed for adjuvant content. 21

A.6.3.5 Degree of adsorption 22

The degree of adsorption (completeness of adsorption) of antigen to the adjuvant should be 23

assessed, if applicable. This test may be omitted upon demonstration of process consistency, 24

subject to the agreement of the NRA. 25

A.6.3.6 pH 26

The pH value of the adsorbed antigen bulk may be monitored until production consistency is 27

demonstrated, subject to the agreement of the NRA. 28

A.6.3.7 Antigen content 29

The antigen content of the adsorbed antigen bulk should be measured using appropriate 30

methods. If this test is conducted on the purified antigen bulk, it may be omitted from the 31

testing of the adsorbed antigen bulk. 32

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International Standards and reference reagents are not available for the control of HEV 1

vaccine antigen content. Therefore, product-specific reference preparations may be used. 2

A.7 Control of final bulk 3

4

The antigen concentration in the final formulation should be sufficient to ensure that the dose 5

is consistent with that shown to be safe and effective in human clinical trials. Should an 6

adjuvant be added to the vaccine formulation, this adjuvant and the concentration used should 7

be approved by the NRA. 8

The operations necessary for preparing the final bulk should be conducted in such a manner 9

as to avoid contamination of the product. In preparing the final bulk vaccine, any substances 10

such as diluents, stabilizers or adjuvants that are added to the product should have been 11

shown to the satisfaction of the NRA not to impair the safety and efficacy of the vaccine at 12

the concentration used. The final bulk should be stored under conditions shown by the 13

manufacturer to allow it to retain the desired biological activity until it is filled into 14

containers. 15

A.7.1 Tests on the final bulk 16

All tests and specifications for final bulk should be approved by the NRA, unless otherwise 17

specified. Where the antigen bulk is the final formulation, the tests below will be performed 18

in section A.6 at the level of the adsorbed antigen bulk and not repeated here. 19


Each final bulk should be tested for bacterial and fungal sterility, as specified in Part A, 21

section 5.2, of WHO’s General requirements for the sterility of biological substances (71), or 22

by a method approved by the NRA. 23

A.7.1.2 Adjuvant content 24

Each final bulk should be assayed for adjuvant content. 25

Where aluminium compounds are used, the aluminium content should not exceed 1.25 mg 26

per single human dose. 27

Tests for adjuvant content on the final bulk may be omitted if conducted on each final lot 28

derived from the final bulk. 29

A.7.1.3 Degree of adsorption 30

The degree of adsorption of the antigen to the adjuvant in each final bulk should be assessed, 31

if applicable (e.g. if the adjuvant is aluminium salts). 32

This test may be omitted upon demonstration of process consistency or if performed on the 33

final lot. 34

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A.7.1.4 Preservative content 1

The final bulk may be tested for the presence of preservative, if added. The method used and 2

the permitted concentration should be approved by the NRA. 3

A.7.1.5 Potency 4

The potency of each formulated final bulk before filling should be assessed by an appropriate 5

in vivo method. If an in vivo potency test is used to test final fill lots, this test may be omitted 6

on the formulated final bulk before filling. The methods for detection of antibodies in in vivo 7

tests and the analysis of data should be approved by the NRA. The vaccine potency should be 8

compared with that of a reference preparation; the NRA should determine the limits of 9

potency and approve the reference preparation used. 10

In vitro methods such as ELISA may be developed to assess potency. With the approval of 11

the NRA, the in vitro assay may replace the in vivo assay when appropriately validated and 12

when consistency of production is demonstrated. 13

Manufacturers should establish a product-specific reference preparation that is traceable to a 14

specific lot of vaccine, or bulks used in the production of a specific lot, which has been 15

shown to be efficacious in clinical trials. The performance of this reference vaccine should be 16

monitored by trend analysis using relevant test parameters and the reference vaccine should 17

be replaced when necessary. An acceptable procedure for replacing reference vaccines should 18

be in place (75, 76). 19

A.7.1.6 Osmolality 20

The osmolality of the final bulk may be tested. The osmolality test may be omitted if 21

performed on the final lot. 22

An alternative test (e.g. freezing point) may be used as a surrogate measure for ionic 23

strength/osmolality. 24

A.8 Filling and containers 25

26

The requirements concerning filling and containers given in WHO good manufacturing 27

practices for pharmaceutical products: main principles and WHO good manufacturing 28

practices for biological products (66, 67) should apply to vaccine filled in the final form. 29

Care should be taken to ensure that the materials of which the container – and if applicable 30

the transference devices and closure – are made do not adversely affect the quality of the 31

vaccine. 32

Manufacturers should provide the NRA with adequate data to prove the stability of the 33

product under appropriate conditions of storage and shipping. 34

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A.9 Control tests on the final filled lot 1

2

The following tests should be performed on each final filled lot (i.e. in the final containers). 3

Unless otherwise justified and authorized, the tests should be performed on labelled 4

containers from each final filled lot by means of validated methods approved by the NRA. 5

All tests and specifications, including methods used and permitted concentrations, should be 6

approved by the NRA, unless otherwise specified. 7

A.9.1 Inspection of containers 8

Every container in each final lot should be inspected visually or mechanically, and those 9

showing abnormalities (e.g. improper sealing, clumping, presence of particles) should be 10

discarded and recorded for each relevant abnormality. A limit should be established for the 11

percentage of containers rejected. 12

A.9.2 Appearance 13

The appearance of the vaccine should be described with respect to its form and colour. 14

A.9.3 Identity 15

An identity test should be performed on at least one container from each final lot, using a 16

validated method approved by the NRA. The potency test may serve as the identity test. 17

A.9.4 Sterility tests for bacteria and fungi 18

Each final lot should be tested for bacterial and fungal sterility, as specified in Part A, section 19

5.2, of WHO’s General requirements for the sterility of biological substances (71), or by a 20

method approved by the NRA. 21

A.9.5 pH and osmolality 22

The pH value and osmolality of the final lot should be tested. The osmolality test may be 23

omitted if performed on the final bulk. The osmolality test may also be omitted for routine lot 24

release upon demonstration of product consistency, subject to the approval of the NRA. 25

An alternative test (e.g. freezing point) may be used as a surrogate measure for ionic 26

strength/osmolality. 27

A.9.6 Preservatives 28

Each final lot should be tested for the presence of preservative, if added. 29

A.9.7 Test for pyrogenic substances 30

Each final lot should be tested for pyrogenic substances. Where appropriate, tests for 31

endotoxin (e.g. the limulus amebocyte lysate [LAL] test) should be performed. However, 32

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where there is interference in the test (e.g. from the adjuvant) a test for pyrogens in rabbits 1

should be performed. 2

A suitably validated monocyte-activation test may also be considered as an alternative to the 3

rabbit pyrogen test. 4

The test is conducted until consistency of production is demonstrated, subject to the 5

agreement of the NRA. 6

A.9.8 Adjuvant content 7

Each final lot should be assayed for adjuvant content, if applicable. Where aluminium 8

compounds are used, the aluminium content should not exceed 1.25 mg per single human 9

vaccine dose. 10

A.9.9 Extractable volume 11

For vaccines filled into single-dose containers, the extractable content should be checked and 12

shown to be not less than the intended dose. 13

For vaccines filled into multi-dose containers, the extractable content should be checked and 14

should be shown to be sufficient for the intended number of doses. 15

A.9.10 Degree of adsorption 16

The degree of adsorption to the adjuvant (completeness of adsorption) of each antigen present 17

in each final vaccine lot should be assessed, if applicable, and the limit should be approved 18

by the NRA. 19

This test may be omitted for routine lot release upon demonstration of product consistency, 20

subject to the approval of the NRA. 21

A.9.11 Potency 22

A potency test should be carried out on each final lot as outlined in Part A, section A.7.1.5, if 23

such a test has not been performed on the final bulk. 24

A.9.12 General safety (innocuity) test 25

The need to test the final lots of the HEV vaccine for unexpected toxicity (also known as 26

abnormal toxicity) should be agreed with the NRA. 27

Some countries no longer require this test (77, 78). 28

A.10 Records 29

30

The requirements given in WHO good manufacturing practices for pharmaceutical products: 31

main principles and WHO good manufacturing practices for biological products (66, 67) 32

should apply. 33

WHO/BS/2018.2348 of 60

A.11 Retained samples 1

2



should apply. 5

A.12 Labelling 6

7



should apply, with the addition of the following information: 10

The label on the carton, the container or the leaflet accompanying the container should state: 11

that the vaccine has been prepared from recombinant bacterial cells, or another 12

expression system; 13

the genotype of the HEV antigen present in the preparation; 14

protein/antigen content and potency per dose; 15

number of doses, if the product is issued in a multiple-dose container; 16

name and maximum quantity of any residual antibiotic present in the vaccine; 17

name and concentration of any preservative added; 18

name and concentration of any adjuvant added; 19

name and concentration of any other excipient added; 20

temperature recommended during storage and transport; 21

expiry date; 22

name of the manufacturer; 23

lot/batch number; 24

any special dosing schedules. 25

A.13 Distribution and transport 26

27



should apply. Further guidance is provided in WHO’s Model guidance for the storage and 30

transport of time- and temperature-sensitive pharmaceutical products (79). 31

A.14 Stability testing, storage and expiry date 32

33

A.14.1 Stability testing 34

Adequate stability studies form an essential part of vaccine development. Current guidance 35

on the evaluation of vaccine stability is provided in WHO’s Guidelines on stability 36

WHO/BS/2018.2348 of 60

evaluation of vaccines (80). Stability testing should be performed at different stages of 1

production, namely on single antigen harvests or single harvest pools, purified antigen bulk, 2

final bulk (whenever materials are stored before further processing) and final lot. Stability-3

indicating parameters appropriate to the stage of production should be defined or selected. A 4

shelf-life should be assigned to all in-process materials during vaccine production – 5

particularly intermediates such as single antigen harvests, purified antigen bulk and final bulk. 6

The stability and expiry date of the vaccine in its final container, maintained at the 7

recommended storage temperature up to the expiry date, should be demonstrated to the 8

satisfaction of the NRA using final containers from at least three final lots made from 9

different adsorbed antigen bulks. 10

Accelerated stability tests may be undertaken to give additional information on the overall 11

characteristics of a vaccine and may also aid in assessing comparability when the 12

manufacturer plans to change aspects of manufacturing. 13

The formulation of vaccine antigens and adjuvant (if used) must be stable throughout the 14

shelf-life of the vaccine. Acceptable limits for stability should be agreed with the NRA. 15

Following licensure, ongoing monitoring of vaccine stability is recommended to support 16

shelf-life specifications and to refine the stability profile (80). Data should be provided to the 17

NRA in accordance with local regulatory requirements. 18

The final stability-testing programme should be approved by the NRA and should include an 19

agreed set of stability-indicating parameters, procedures for the ongoing collection and 20

sharing of stability data, and criteria for rejecting vaccines. 21

A.14.2 Storage conditions 22

The final lot should be kept at 2–8 °C. If other storage conditions are used, they should be 23

fully validated and approved by the NRA. The vaccine should be ensured that the minimum 24

potency specified on the label of the container or package will be maintained after release and 25

until the end of the shelf-life, provided that the vaccine is stored under the recommended 26

conditions. During storage, adsorbed vaccines should not be frozen. 27

If a vaccine has been shown to be stable at temperature ranges higher than the approved 2–28

8 °C range, it may be stored in an extended controlled temperature conditions for a defined 29

period, subject to approval by the NRA (81). 30

A.14.3 Expiry date 31

The expiry date should be based on the shelf-life supported by stability studies and should be 32

approved by the NRA. The expiry date should be based on the date of blending of final bulk, 33

the date of filling or the date of the first valid potency test on the final lot. 34

Where an in vivo potency test is used, the date of the potency test is the date on which the test 35

animals are inoculated. 36

WHO/BS/2018.2348 of 60

1

Part B. Nonclinical evaluation of recombinant hepatitis E 2

vaccines 3

Nonclinical evaluation of hepatitis E vaccines should be based on the WHO guidelines on 4

nonclinical evaluation of vaccines (5). Details of the design, conduct, analysis and evaluation 5

of nonclinical studies are available in the WHO guidelines (5). Further guidance on the 6

general principles for nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines 7

can be found in a separate WHO guidelines document (8). 8

Prior to clinical testing of any new hepatitis E vaccine in humans there should be extensive 9

product characterization, proof-of-concept studies, immunogenicity studies and safety testing 10

in animals. The extent of nonclinical evaluation will depend on the complexity of the vaccine 11

formulation on a case-by-case basis. The following specific issues should be considered in 12

the context of the development of recombinant hepatitis E vaccines based on the ORF2-13

encoded viral capsid protein. 14

B.1 Strategy for cloning and expressing the gene product 15

16

The viral genome contains three open reading frames. Of these, ORF2 codes for the viral 17

capsid protein which is the target of neutralizing antibodies against HEV (8284). 18

A full description should be given of the biological characteristics of the host cell and 19

expression vectors used in production. This should include details of: 1) the construction, 20

genetics and structure of the expression vector; 2) the origin and identification of the gene 21

that is being cloned; and 3) potential retrovirus-like particles in, and genetic markers for, 22

mammalian cell-based expression systems. The physiological measures used to promote and 23

control the expression of the cloned gene in the host cell should be described in detail. 24

25

Data should be provided to demonstrate the genetic stability of the expression system beyond 26

the passage level used for production. Any instability of the expression system occurring in 27

the seed culture or after a production-scale run (for instance, involving rearrangements, 28

deletions or insertions of nucleotides) must be documented. The NRA should approve the 29

system used. 30

B.2 Product characterization and process development 31

32

Rigorous identification and characterization of recombinant DNA-derived vaccines is 33

required as part of the application for marketing authorization. The ways in which these 34

products differ chemically, structurally, biologically or immunologically from the naturally-35

occurring antigen must be fully documented. Such differences could arise during processing 36

at the genetic or post-translational level, or during purification. 37

WHO/BS/2018.2348 of 60

1

The expressed protein should be characterized by biochemical, biophysical and 2

immunological methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis 3

(SDS-PAGE), MALDI-TOF, high-performance size exclusion chromatography (HPSEC) and 4

binding activity to monoclonal antibodies. Immunogenicity of the protein should be analysed 5

in an appropriate animal. 6

7

It is crucially important that vaccine production processes are appropriately standardized and 8

controlled to ensure consistency in manufacturing, and in the collection of nonclinical data 9

that may indicate potency and safety in humans. The extent of product characterization may 10

vary according to the stage of development. The vaccine lots used in nonclinical studies 11

should be adequately representative of the formulation intended for use in clinical 12

investigation and, ideally, should be the same lots as those used in clinical trials. If this is not 13

feasible, the lots used in nonclinical studies should be comparable to clinical lots with respect 14

to physicochemical characteristics, stability and formulation. 15

16

B.3 Pharmacodynamic studies 17

18

B.3.1 Immunogenicity studies 19

Immunogenicity of the vaccine should be evaluated in relevant animal species that respond 20

well to vaccine antigen (e.g. rodent, rabbit, swine, non-human primates) (85). 21

Immunogenicity data can provide initial insights into immunological characteristics of the 22

vaccine antigen and are useful for evaluation of the vaccine formulation and underlying 23

protective mechanisms and as justification for including an adjuvant in a vaccine. 24

The nonclinical passive immunization studies in non-human primates and human 25

epidemiology studies indicate that the humoral immunity is probably the primary effector 26

mechanism that directly mediates protection against HEV. Clear correlation between the 27

serum IgG responses to vaccine antigen and protection has indeed been demonstrated in 28

clinical (86) and nonclinical (87) studies conducted with recombinant hepatitis E capsid-29

based vaccines. On this basis, it is recommended that evaluation of vaccine immunogenicity 30

should include assessment of serum anti-HEV IgG antibodies. 31

Immunogenicity studies should establish a dose-response relationship by testing different 32

doses of vaccine antigen. Ideally, immune responses are assessed after each dose of vaccine 33

in line with intended posology. For an adjuvanted vaccine, the advantage of adjuvant 34

included in vaccine should be demonstrated by serological data, with or without additional 35

elucidation of cellular immune response, depending upon the adjuvant used. 36

B.3.2 Challenge studies 37

The protective effect of vaccine antigen should be evaluated in an appropriate animal model. 38

Several animal models are known to be experimentally permissive to infection by human 39

WHO/BS/2018.2348 of 60

HEV (85), including swine (genotypes 3 and 4), rabbit (genotype 4), and different species of 1

non-human primates such as cynomolgus macaques (genotypes 1 and 2) and rhesus macaques 2

(genotypes 14). The challenge studies conducted in non-human primates demonstrated 3

protective immunity of the hepatitis E vaccines which are subsequently shown to be 4

efficacious in humans (60, 86). 5

The animals should be HEV-naïve. The naïve status of animals at baseline should be 6

confirmed by the absence of detectable anti-HEV total IgG antibody in sera and HEV RNA 7

in faeces and in sera. The virus used for animal challenge should correspond to the wild type 8

virus strain from which the vaccine antigen is derived. 9

The design of challenge studies may vary, depending on platforms with which the vaccine is 10

produced. Vaccination of animals is usually conducted in accordance with the intended 11

posology, and challenge of animals is pursued at the time when vaccinated animals develop 12

peak protective responses. In general, challenge via the intravenous route is acceptable 13

because transmission through the oral route is less efficacious. The challenge dose should be 14

sufficiently high to ensure the establishment of reliable infection and/or histopathological 15

hepatitis. Important endpoints used to define protection should be specified in the study 16

protocol and should include: 17

infection marker such as HEV RNA in stool and serum at serial time points, and/or 18

histopathological evidence for hepatitis using liver biopsy, and 19

biochemical parameter of ALT change at serial time points. 20

In addition, passive immunization studies in animal models that involve passive transfer of 21

human vaccinees’ antisera to naïve animals, followed by HEV virus challenge, might be 22

useful for estimating a specific IgG titre associated with protection. 23

B.3.3 Cross-neutralization protection of different genotypes 24

The genetic differentiation of HEV strains is determined by whether the nucleic acid 25

variation of ORF2 between two viruses is more than 20%. According to this criterion, human 26

HEV isolates are classified into four genotypes (genotypes 14). These four genotypes share 27

a single serotype based on their immunoreactivity and cross-neutralization (88, 89). 28

Therefore, HEV vaccine with recombinant pORF2s derived from a given genotype is 29

expected to provide cross-genotype protection against all four genotypes. Results from 30

preclinical and clinical studies have substantiated this notion. In preclinical animal models, 31

the same protection was observed in animals challenged with different genotypes of HEV 32

after immunization of recombinant pORF2 derived from distinct genotypes. Purcell et al. (90) 33

and Li et al. (59) demonstrated that immunization with recombinant pORF2 derived from 34

genotype 1 HEV was able to protect against genotype 1, 2, 3 and 4 HEV infection in HEV 35

non-human primate models. The recombinant pORF2 derived from genotype 4 HEV 36

provided cross-protective effects in genotype 1 and 4 HEV infection on a non-human primate 37

model (91). More recently, results from an HEV rabbit model suggested that recombinant 38

WHO/BS/2018.2348 of 60

capsid proteins derived from genotype 1 cross-protects against genotype 4 HEV infection (92, 1

93). 2

Furthermore, in clinical trials conducted in China and Nepal, recombinant pORF2 protein 3

vaccine derived from genotype 1 HEV sequences showed protection for acute hepatitis 4

caused by genotype 1 and 4 HEV infection (61, 86, 93). 5

From the biochemical analysis, a cross-genotype and neutralizing epitope, as recognized by 6

mAb 8G12, was identified between genotypes 1 and 4 HEV using the recombinant capsid 7

proteins (94). The mAb 8G12 was shown to block the binding of naturally-acquired 8

antibodies in human and animal sera. The presence of these “8G12-like” antibodies or the 9

epitopes recognized by these antibodies could partially rationalize the cross-genotype 10

protection of vaccines with antigens derived from a given type (such as those based on 11

genotype 1). 12

To date, clinical data on cross-protection from completed clinical trials with recombinant 13

HEV capsid-based vaccines are still limited. If cross-protection is claimed, challenge studies 14

should be conducted in appropriate animal models to evaluate the potential for cross-15

protection against heterologous viruses. 16

B.4 Biodistribution studies 17

18

The classical pharmacokinetic studies are not required for recombinant human hepatitis E 19

vaccines. If a novel excipient, including a novel adjuvant, is included in vaccine, 20

biodistribution study should be considered (8). 21

B.5 Toxicology studies 22

23

Toxicology studies should be undertaken in accordance with the WHO guidelines on 24

nonclinical evaluation of vaccines (5). Such studies should be performed with final vaccine 25

formulation in relevant animal species and should reflect the intended clinical use of the 26

vaccine (5). Repeated dose toxicity and local tolerance should be evaluated in relevant 27

species following good laboratory practice (GLP) principles, prior to initiation of early 28

human clinical trials. Because the target population for the hepatitis E vaccines includes 29

women of childbearing age, GLP-compliant reproductive and developmental toxicity studies 30

are also required. 31

32

In general, toxicity evaluation in one relevant species is justified. The route and dosing 33

regimen should reflect the intended clinical use. For evaluation of developmental toxicity, the 34

dosing regimen should consider one or two doses prior to mating so that pregnant animals 35

and embryo and fetus are exposed to a maximal vaccine response during the critical window 36

of organogenesis. 37

38

WHO/BS/2018.2348 of 60

If the vaccine is formulated with a novel adjuvant, nonclinical toxicology studies should be 1

conducted as appropriate for the adjuvant concerned and the recommendations in WHO’s 2

Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines 3

should be followed (8). 4

5

If a novel cell substrate (i.e. a substrate that has not been previously licensed or used in 6

humans) is used for the production of a hepatitis E vaccine, safety aspects, such as potential 7

immune responses elicited by residual host-cell proteins, should be investigated in a suitable 8

animal model. Such studies should be undertaken particularly if the final product contains an 9

adjuvant that might enhance responses to low levels of residual proteins. 10

11

Part C. Clinical evaluation of recombinant hepatitis E vaccines 12

C.1 Introduction 13

14

Clinical trials should adhere to the principles described in WHO’s Guidelines for good 15

clinical practice (GCP) for trials on pharmaceutical products (95). General guidance on 16

vaccine clinical development programmes is provided in the WHO Guidelines on clinical 17

evaluation of vaccines: regulatory expectations (96) and is not repeated here. This section 18

addresses issues for clinical development programmes that are specific to, or of special 19

concern for, vaccines intended to prevent clinically apparent infections with HEV. 20

21

C.2 Assays 22

23

This section considers: 24

serological assays for establishing the baseline sero-status of trial subjects and evaluating 25

the humoral immune response to vaccination (see section C.3); and 26

serological and virus detection assays for laboratory confirmation of acute hepatitis 27

caused by HEV infection in vaccine efficacy trials (see section C.4). 28

Sponsors should also consult section 5.3.3 of the Guidelines on clinical evaluation of 29

vaccines: regulatory expectations (96). 30

31

C.2.1 Serological assays 32

C.2.1.1 Functional antibody 33

Currently there is no well-established assay for measuring anti-HEV virus neutralizing 34

antibody. Since there is no efficient HEV cell infection model, a direct measurement of anti-35

HEV neutralizing antibody is not feasible. Neutralizing antibody has been estimated using 36

methods such as real-time PCR (97) or an immunofluorescence foci assay (IFA) to detect 37

virus, but these methods are not standardized or suitable for processing large numbers of sera 38

and each has its drawbacks. Sponsors are encouraged to develop high-throughput assays for 39

anti-HEV neutralizing antibody. For instance, a potential high-throughput neutralization 40

WHO/BS/2018.2348 of 60

assay based on recombinant HEV capsid particles has been compared with the IFA and with 1

anti-HEV IgG using sera from HEV-infected and vaccinated macaques (97). 2

3

C.2.1.2 Total binding antibody 4

For the purposes of estimating the immune response to vaccination, sponsors may choose to 5

develop in-house anti-HEV IgG assays in which the antigen used to coat the wells is at least 6

the same as, or is a truncated version of, that in the vaccine. It is recommended that the 7

quantitation of anti-HEV IgG should be referenced to the WHO standard (98) sera as part of 8

the validation of the assay. Using the selected assay methodology, a cut-off value should be 9

identified and justified for distinguishing seronegative and seropositive sera. 10

11

For the detection of acute infection with HEV, commercial assays are available to detect 12

HEV-specific IgG, IgM, IgA and total immunoglobulin. These commercial assays vary 13

considerably in their use of synthetic or recombinant antigen, viral strain origin and genotype, 14

viral gene product(s) and detection method (e.g. anti-HEV IgM antibody detection commonly 15

uses a μ chain capture ELISA whereas IgG antibody detection usually involves a direct 16

antigen-coating ELISA with secondary enzyme conjugated antibody for detection). 17

Comparative studies have shown considerable differences in the sensitivity and specificity of 18

commercially available assays, with even more variability for IgM compared to IgG assays 19

(50, 51). The assays used to detect and quantify anti-HEV antibody in suspected cases of 20

hepatitis E in efficacy trials must be adequately justified, taking into account what is known 21

about their performance characteristics. 22

23

C.2.2 Virus detection assays 24

Appropriate HEV RNA or antigen detection assays are required to confirm the presence of 25

virus in blood and/or stools of suspected cases of hepatitis E (see section C.4). Commercial 26

quantitative PCR assays are available along with WHO HEV RNA International Standards of 27

genotype 3a and 3b strains (64, 65, 99). Assays with different targets (e.g. assays that target 28

ORF2 or the ORF2/3 overlapping region) have been shown to have different performance 29

characteristics. The ability of assays to detect and quantify HEV RNA from specific 30

genotypes should be taken into account when selecting the method to be used in trials. 31

32

In vaccine efficacy trials it is recommended that HEV should be identified at least to 33

genotype level for all PCR-positive cases. The fragments that are amplified by real-time PCR 34

are usually less than 100 nucleotides in length and are located on conserved parts of the 35

genome. Therefore, additional genomic sequencing, which published data suggest may be 36

targeted to a specific region, is currently required to determine the HEV (sub-)genotype. 37

Sponsors should provide full details of the methodology applied and appropriate controls 38

should be used. 39

C.3 Immunogenicity 40

41

C.3.1 Formulation, dose and regimen 42

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C.3.1.1 Primary series 1

HEV vaccines will be used mainly or exclusively in regions with relatively high rates of 2

clinically apparent infections, but pre-vaccination testing for HEV serostatus will not be 3

feasible in routine use. In naturally primed persons, not all of whom may have detectable pre-4

vaccination anti-HEV IgG, the first dose of hepatitis E vaccine may elicit large increments in 5

antibody due to an anamnestic response. In contrast, multiple doses of the same vaccine may 6

be required to achieve similar antibody levels in HEV-naïve subjects. Consequently, it is 7

important that the primary series should be selected on the basis of the immune responses 8

observed in subjects who are seronegative, including seronegative subjects who are unlikely 9

to have been naturally primed. 10

11

In the absence of an established immune correlate of protection for HEV, selection of the 12

vaccine dose and regimen may be based on reaching an antibody plateau response unless this 13

is precluded by concerns over reactogenicity. It is desirable that immunogenicity studies 14

should explore the minimum number of doses and the shortest dose interval(s) required to 15

achieve a plateau immune response. 16

17

C.3.1.2 Need for re-vaccination 18

In the absence of an immune correlate of protection, it is recommended that the possible need 19

for revaccination is not based solely on waning antibody levels. There should be planned 20

long-term follow-up for hepatitis E cases in vaccine efficacy trials, and/or data should be 21

collected from vaccine effectiveness studies to determine waning protection against clinically 22

apparent HEV infection (see section C.4.2.7). 23

24

In anticipation that revaccination may be necessary to maintain protection, it is recommended 25

that the immune response to additional doses of vaccine is assessed. For example, subjects 26

enrolled into an immunogenicity trial could be sub-randomized to receive further doses at 27

predefined intervals after completion of a primary series. The immune responses to additional 28

doses could be compared with the post-primary responses of the same persons and/or 29

compared with the response to a single dose administered to previously unvaccinated and 30

seronegative control subjects. 31

32

C.3.1.3 Cross-protection 33

The ability of a candidate hepatitis E vaccine to protect against a range of wild-type strains 34

covering the four main genotypes of HEV may vary according to the vaccine construct; it is 35

important that this should be investigated in nonclinical studies (see section B.3.3). 36

37

In clinical trials in which vaccine-elicited antibody is determined against the antigen in the 38

vaccine (see section C.2.1.2), it is recommended that IgG is also measured using antigens 39

derived from a range of wild-type hepatitis E viruses. If marked differences are observed in 40

IgG antibody when measured using vaccine versus non-vaccine antigens and/or by HEV 41

genotype, it would be of particular interest to assess whether a similar effect is observed for 42

functional antibody levels. In addition, depending on the range of investigations already 43

WHO/BS/2018.2348 of 60

completed, it may be appropriate to conduct additional nonclinical studies to evaluate the 1

possible implications of the findings for protection before proceeding to conduct efficacy 2

trials. 3

4

C.3.2 Special populations 5

Thus far, the efficacy of hepatitis E vaccination has been demonstrated in healthy subjects 6

aged 16 years and above, most of them under 45 years of age. There may be interest in the 7

use of hepatitis E vaccines in younger age groups and/or subjects at particular risk of 8

developing severe or fulminant hepatitis (e.g. during pregnancy and in persons with pre-9

existing liver disease) and/or immunodeficient subjects who are at risk of developing chronic 10

HEV infection. If a vaccine has already been shown to be efficacious in healthy adults it may 11

be possible, on the basis of safety and immunogenicity data, to extend its use to various 12

special populations. For instance: 13

There may be interest in completing primary vaccination before the period of greatest 14

risk, in which case safety and immunogenicity data should be generated to support use 15

of appropriate regimen(s) in specific paediatric age subgroups. 16

Section 5.6.4 of the WHO Guidelines on clinical evaluation of vaccines: regulatory 17

expectations (96) discusses the evaluation of the safety and immunogenicity of vaccines 18

during pregnancy. Protection against hepatitis E disease by vaccination during 19

pregnancy, as opposed to vaccination of women before or between pregnancies, would 20

require the development of a vaccine that can elicit antibody levels that are likely to be 21

protective (e.g. similar to those observed in adults enrolled in vaccine efficacy trials) 22

after a single dose or after two doses administered within a short interval. 23

Subjects with pre-existing liver disease and immunodeficient subjects may have very 24

variable immune responses to vaccination depending on the underlying cause and 25

specific nature of their condition. Vaccine regimens should be supported by immune 26

responses documented in specific subgroups that are representative of the intended 27

target populations. 28

29

C.4 Efficacy 30

31

C.4.1 Requirement for a demonstration of vaccine efficacy 32

It is currently recommended that the protective efficacy of a candidate vaccine against 33

clinically apparent HEV infection should be evaluated in a pre-licensure vaccine efficacy trial. 34

The following considerations apply: 35

At the time of preparing this guidance there is one vaccine against hepatitis E that is 36

licensed in one country (See General considerations) (60, 61). 37

The licensed vaccine is not widely used and it is not included in national immunization 38

programmes, so the use of a control group that does not receive vaccination against 39

hepatitis E is possible. 40

In jurisdictions in which a licensed vaccine is available, it is possible that individual 41

NRAs may consider that licensure can be based on a trial that evaluates the efficacy of 42

WHO/BS/2018.2348 of 60

the candidate relative to that of the licensed vaccine in a population similar to that in 1

which the efficacy of the licensed vaccine was established. 2

The lack of an immune correlate of protection against hepatitis E does not rule out 3

immunobridging a candidate vaccine to a licensed vaccine that has been shown to be 4

efficacious. However, this approach is possible only if both vaccines contain the same 5

antigen(s) so that immune responses can be compared directly. In addition, the 6

demonstration of efficacy of the first licensed vaccine was confined to HEV genotypes 7

1 and 4 and it is not known whether the protective immune response may vary between 8

genotypes. Furthermore, the baseline seropositivity rate of the population in which 9

efficacy was demonstrated was estimated at 47% (based on data from less than one 10

tenth of the total subjects randomized into the trial) (61). It cannot be assumed that the 11

point estimate of vaccine efficacy would be applicable to populations with very 12

different pre-vaccination seropositivity rates. 13

14

Taking these considerations into account, the focus of this section is on clinical development 15

programmes that include vaccine efficacy trials in which the control group does not receive 16

vaccination against hepatitis E. Most of the recommendations are also applicable to trials in 17

which the control group receives a licensed vaccine against hepatitis E. Clinical programmes 18

leading to licensure based on immunobridging are not addressed in this guidance. The general 19

principles to consider are discussed in sections 5.6.2 and 6.3.3 of Guidelines on clinical 20

evaluation of vaccines: regulatory expectations (96). 21

22

C.4.2 Considerations for efficacy trial design 23

C.4.2.1 Primary objective 24

The primary objective will be to demonstrate that the candidate vaccine protects against 25

clinically apparent (i.e. symptomatic) HEV infection of any genotype (see section C.4.2.4). 26

It is not required for efficacy to be shown against asymptomatic HEV infection. With 27

the exception of immunodeficient subjects, who may develop chronic infection with 28

possible sequelae, asymptomatic infection is of no clinical significance. 29

It is not required for vaccine efficacy trials to be powered to demonstrate genotype-30

specific efficacy (see section C.4.2.2). 31

32

C.4.2.2 Trial sites 33

Efficacy trials will be conducted in endemic areas in which the estimated attack rate for 34

clinically apparent HEV infection is sufficient to complete enrolment into an adequately 35

powered vaccine efficacy trial within a reasonable time frame. Sites may be chosen on the 36

basis of available public health disease surveillance data and/or pre-trial evaluations of 37

epidemiology conducted by the sponsor. In two prior efficacy trials (6062, 86), HEV 38

genotypes that caused clinically apparent infections in the control (placebo) groups were 39

limited in accordance with strains circulating at trial sites in the years in which they were 40

conducted. Sponsors are encouraged to consider selecting sites in a range of geographical 41

areas in which different genotypes are circulating and/or to conduct separate vaccine efficacy 42

trials in regions with different genotype distributions. 43

WHO/BS/2018.2348 of 60

1

C.4.2.3 Subject selection criteria 2

Because of the peak age incidence of hepatitis E it is likely that vaccine efficacy trials will 3

target adolescents and adults. An upper age limit may be set depending on the age-specific 4

attack rates. 5

6

In endemic areas the adult population will include a variable proportion of subjects who are 7

seropositive for IgG against HEV. Options for subject selection include the following: 8

Adults could be enrolled without knowledge of their baseline serostatus for HEV, 9

which is the usual approach in vaccine efficacy trials conducted in endemic regions. 10

This provides an assessment of the benefit of vaccination over and above the level of 11

pre-existing protection against HEV infection due to natural exposure. The proportion 12

of subjects enrolled who are seropositive at the time of enrolment should be estimated 13

retrospectively by determining anti-HEV IgG in samples obtained from all, or a 14

randomly selected subset of, subjects prior to vaccination. This information is helpful 15

when considering extrapolation of the estimate of vaccine efficacy observed to other 16

regions not included in the trial. 17

A possible alternative approach would be to pre-screen subjects for anti-HEV IgG and 18

to enrol only those who are considered to be seronegative on the basis of a threshold 19

determined by the assay used. This may allow for a smaller sample size to be 20

randomized into the trial due to an expected higher attack rate; however, it is also 21

possible that seronegative adults are less likely to encounter HEV compared to 22

seropositive adults of a similar age range and resident in the same region due to 23

differences in their living conditions. Therefore, detailed knowledge of the local 24

epidemiology of hepatitis E should be taken into account before choosing this 25

approach. 26

27

C.4.2.4 Primary endpoint 28

In accordance with the recommended primary objective, the primary endpoint should be 29

clinically apparent acute hepatitis that is confirmed to be due to HEV. Sponsors could 30

consider appointing an independent data adjudication committee to review the data and 31

determine which subjects meet the case definition to be counted in the primary analysis. 32

33

Clinical features for the case definition 34

The clinical features that trigger subjects to present to study site staff or to a local designated 35

health-care facility for laboratory investigations for acute hepatitis E should be selected with 36

the aim of capturing as many cases as possible while limiting unnecessary investigations. On 37

this basis it is reasonable to define a possible case of acute viral hepatitis requiring laboratory 38

investigation as an illness presenting with any or a minimum number of signs and symptoms 39

– including malaise, fatigue, anorexia, right upper quadrant tenderness for longer than 3 days 40

or any duration of jaundice or dark urine. Additional symptoms that could be considered 41

include any abdominal pain, nausea or vomiting that persists for at least 3 days and for which 42

there is no known likely explanation. 43

WHO/BS/2018.2348 of 60

1

Laboratory confirmation of acute hepatitis E infection 2

It is recommended that the laboratory confirmation of acute hepatitis E cases should be 3

conducted in a designated central laboratory. If more than one central laboratory is necessary 4

for practical reasons, it is essential that the laboratories use identical methodologies and 5

consideration should be given to testing a randomly selected subset of samples at each 6

laboratory to assess concordance. 7

8

There is variability in the onset and duration of elevated alanine aminotransferase (ALT) 9

levels in serum, detectable HEV RNA in serum or stool, the appearance of anti-HEV IgM 10

and the appearance of or detectable increase in anti-HEV IgG in relation to the first 11

appearance of symptoms. An increase in ALT to at least 2.5-fold the upper limit of normal 12

(ULN) based on the local or central laboratory normal range should lead to investigations to 13

determine whether HEV is the causative agent. If the first sample does not show a > 2.5-fold 14

elevation in ALT, the test should be repeated after approximately 12 weeks for any subject 15

with jaundice, persistent symptoms or an elevated total serum bilirubin in the first sample. 16

17

The confirmation of HEV as causative of the clinical picture should be based on any two or 18

more of: 19

IgM against HEV, which is often detectable at the time of onset of clinical symptoms 20

but may peak after 12 weeks and in some cases remains detectable for several months; 21

at least a 4-fold rise in anti-HEV IgG between the first sample and a convalescent 22

sample; 23

detection of HEV RNA in blood or in stool, which is usually shortlived but may persist 24

for longer in stool than in blood. 25

To avoid missing cases, protocols should plan for appropriately timed repeat specimens (e.g. 26

at 26 weeks after the first sample) in persons with elevated ALT. Since HEV RNA is most 27

likely to be detectable early in the course of a clinical illness, it is recommended that samples 28

are obtained as early as possible and, if negative, repeat testing is conducted after a short 29

interval. 30

31

Samples obtained at first presentation should also be tested to detect acute infection or to 32

establish coinfection with other hepatitis viruses that can cause the same clinical picture, 33

including: 34

IgM against hepatitis A virus; 35

hepatitis B surface antigen and anti-core IgM; 36

antibody against hepatitis C and/or HCV RNA. 37

If the first sample is negative for evidence of acute infection with hepatitis A, B, C or E 38

viruses and further samples are indicated to rule out hepatitis E, it is recommended that these 39

should also be retested for evidence of hepatitis A, B and C to document any possible 40

coinfections. 41

42

C.4.2.5 Primary, secondary and other analyses 43

WHO/BS/2018.2348 of 60

In a vaccine efficacy trial, it may be permissible that the primary analysis includes only 1

confirmed acute hepatitis E cases, whether there is evidence of coinfection with other 2

hepatitis viruses or not, as follows: 3

in subjects who completed the vaccination series within predetermined visit windows, 4

if more than one dose is required; and 5

with symptom onset more than a defined period after the only or final dose of the series 6

that takes into account what is known about the timing of the post-dose anti-HEV IgG 7

peak. 8

This approach gives the most optimistic estimation of vaccine efficacy. 9

10

If the primary analysis is confined to cases counted as described above it is essential that 11

there are predefined secondary analyses to estimate vaccine efficacy based on confirmed 12

cases of clinically apparent HEV infection defined and counted as follows: 13

all cases in subjects who received at least one assigned dose as randomized and 14

regardless of adherence to study visit windows; 15

cases that occurred at any time after the last dose received (i.e. counted from the day of 16

dosing) in those who completed the assigned number of doses; 17

cases that occurred after each sequential dose, depending on the number of doses in the 18

series and counted from the day of dosing. 19

20

Vaccine efficacy should be explored according to HEV genotype if this is feasible, depending 21

on the numbers of cases that occur due to individual genotypes. 22

23

It is recommended that an additional analysis should explore any differences in clinical or 24

laboratory features, including severity, between cases that occur in the candidate vaccine 25

group and the control group (whether the control group receives placebo or a licensed vaccine 26

against hepatitis E). The analysis should take into account whether the severity observed in 27

individual subjects could reflect coinfection with other hepatitis viruses, whether acute (most 28

likely coinfection with hepatitis A) or chronic (i.e. acute hepatitis E occurring in subjects who 29

have chronic hepatitis B or C infection). 30

31

C.4.2.6 Case ascertainment 32

It is recommended that an active case ascertainment strategy is used throughout the time 33

frame of a vaccine efficacy trial. This is essential at least up to the time of the primary 34

analysis, which may be conducted after a specific number of total cases has been 35

accumulated or after a predefined period in which a sufficient number of cases are expected 36

to occur to estimate vaccine efficacy. 37

38

C.4.2.7 Duration of protection 39

While the primary analysis may lead to licensure, it is recommended that trials continue to 40

use active case ascertainment to follow up subjects for several years to provide data on 41

waning vaccine protection without unblinding of treatment assignment at the level of the 42

WHO/BS/2018.2348 of 60

individual. These data can be reported at some time after licensure of the vaccine and may 1

point to the need for further doses to be administered at intervals to maintain protection. 2

3

C.4.2.8 Vaccine effectiveness 4

The need for vaccine effectiveness studies should be established at the time of licensure. 5

6

If longer-term follow-up within a pre-licensure trial is not considered to be feasible, the 7

duration of vaccine protection should be investigated within a vaccine effectiveness study 8

and/or as part of routine disease surveillance conducted by public health authorities. 9

Furthermore, the efficacy of the vaccine against individual genotypes should be explored 10

either as part of a vaccine effectiveness study and/or during routine disease surveillance. 11

12

C.5 Safety 13

The evaluation of the safety of candidate hepatitis E vaccines should be in accordance with 14

the recommendations made in section 7 of WHO’s Guidelines on clinical evaluation of 15

vaccines: regulatory expectations (96). If the primary series consists of several doses it is 16

important to document whether reactogenicity increases with sequential doses. Additionally, 17

the safety of post-primary doses should be evaluated. There may be some special 18

considerations for vaccine safety, depending on the vaccine construct and the intended target 19

population (e.g. if the vaccine is proposed for administration during pregnancy). 20

21

If a candidate vaccine is evaluated in a large pre-licensure trial, and if the safety profile 22

documented in immunogenicity trials did not give rise to any major concerns, it may be 23

acceptable that a full assessment of safety (i.e. including detailed documentation of local and 24

systemic reactogenicity as well as all unsolicited adverse events) could be confined to a 25

randomized subset of the total subjects. Serious adverse events should be documented in all 26

subjects enrolled at all trial sites. 27

28

Part D. Recommendations for NRAs 29

D.1 General recommendations 30

31

The general recommendations for NRAs and NCLs given in WHO’s Guidelines for national 32

authorities on quality assurance for biological products (100) and Guidelines for 33

independent lot release of vaccines by regulatory authorities (101) should apply. These 34

recommendations specify that no new biological substance should be released until 35

consistency of lot manufacturing and quality has been demonstrated. 36

37

The detailed production and control procedures – as well as any significant changes in them 38

that may affect the quality, safety and efficacy of recombinant hepatitis E vaccines – should 39

be discussed with and approved by the NRA (102). For control purposes, the relevant 40

International Standards currently in force should be obtained for the purpose of calibrating 41

WHO/BS/2018.2348 of 60

national, regional and working standards (103). The NRA may obtain from the manufacturer 1

the product-specific or working reference to be used for lot release. 2

3

Consistency of production has been recognized as an essential component in the quality 4

assurance of recombinant hepatitis E vaccines. In particular, the NRA should carefully 5

monitor production records and quality control test results for clinical lots, as well as for a 6

series of consecutive lots of the vaccine. 7

D.2 Official release and certification 8

9

A vaccine lot should be released only if it fulfils all national requirements and/or satisfies 10

Part A of these WHO Recommendations (101). 11

12

A protocol for the manufacture and control of recombinant hepatitis E vaccines, based on the 13

model protocol provided in Appendix 1 and signed by the responsible official of the 14

manufacturing establishment, should be prepared and submitted to the NRA in support of a 15

request for the release of a vaccine for use. 16

17

A lot release certificate signed by the appropriate NRA official should then be provided if 18

requested by a manufacturing establishment and should certify whether or not the lot of 19

vaccine in question meets all national requirements and/or Part A of these WHO 20

Recommendations. The certificate should provide sufficient information on the vaccine lot. 21

The purpose of this official national release certificate is to facilitate the exchange of vaccines 22

between countries and should be provided to importers of the vaccines. A model NRA Lot 23

Release Certificate is provided in Appendix 2. 24

25

26

Authors and acknowledgements 27

The first draft of this document was prepared by a WHO drafting group: Dr P Minor, 28

National Institute for Biological Standards and Control, United Kingdom; Dr Y Wang, 29

National Institutes of Food and Drug Control, China; Dr R Wagner, Paul-Ehrlich-Institut , 30

Germany; Dr E Gurley, Center for Communicable Diseases, International Centre for 31

Diarrheal Diseases Research, Bangladesh and Johns Hopkins University, USA; Dr M Powell, 32

Medicines and Healthcare Products Regulatory Agency, United Kingdom; Dr Q Zhao, 33

Xiamen University, China; Dr D Lei, World Health Organization, Switzerland and Dr Y Sun, 34

Paul-Ehrlich-Institut, Germany; taking into consideration the discussions of a WHO working 35

group meeting to develop WHO recommendations to assure the quality, safety and efficacy 36

of recombinant hepatitis E vaccines, held in Geneva Switzerland, 1112 May 2017 with 37

members of the drafting group and participants and representatives from regulators, vaccine 38

developers and academia: Dr I Ciglenecki, Médecins Sans Frontières International, 39

Switzerland; Dr N Kamar, Toulouse University Hospital, France; Mrs D Kusmiaty, National 40

Quality Control Laboratory of Drug and Food, Indonesia; Dr M Li, Center for Drug 41

WHO/BS/2018.2348 of 60

Evaluation of China Food and Drug Administration, China; Dr A Lommel, Paul-Ehrlich-1

Institut, Germany; Dr C Ngabéré, Ministry of Public Health, Chad; Dr S Phumiamorn, 2

Institute of Biological Products, Thailand; Dr H Iftikhar Qureshi, Pakistan Health Research 3

Council, Pakistan; Mr B Shrestha, Walter Reed/ AFRIMS Research Unit Nepal (WARUN), 4

Nepal; Dr D Steele, Bill & Melinda Gates Foundation, USA; Dr E Teshale, Centers for 5

Disease Control and Prevention, USA; Dr X Wu, National Institutes of Food and Drug 6

Control, China; Dr C Young, Health Canada, Canada; Dr J Zhang, Xiamen University, China; 7

Mr S Gao, Innovax Biotech Co. Ltd, China; Mr B Huang, Xiamen Innovax Biotech Co. Ltd, 8

China; Dr J Lynch, International Vaccine Institute, Republic of Korea; Dr K Maithal, Zydus 9

Cadila Healthcare Ltd., India; Dr JWK Shih, Xiamen Innovax Biotech Co. Ltd, China; Dr I 10

Knezevic, World Health Organization, Switzerland; Dr O Lapujade, World Health 11

Organization, Switzerland; Dr G Enwere, World Health Organization, Switzerland; Ms C 12

Rodriguez Hermandez, World Health Organization, Switzerland and Dr P Duclos, World 13

Health Organization, Switzerland. 14

The second draft was prepared by the Dr P Minor, Consultant, United Kingdom; Dr Y Sun, 15

Paul-Ehrlich-Institut, Germany; Dr M Powell, Medicines and Healthcare Products Regulatory 16

Agency, United Kingdom; Dr Q Zhao, Xiamen University, China; Dr R Wagner, Paul-17

Ehrlich-Institut, Germany; Dr E Gurley, Johns Hopkins Bloomberg School of Public Health, 18

USA; Dr Y Wang, National Institutes of Food and Drug Control, China and Dr D Lei, World 19

Health Organization, Switzerland after public consultation with incorporated comments 20

received from regulators, manufacturers and academia into the text as appropriate. The third 21

draft was then prepared by Dr P Minor, Consultant, United Kingdom; Dr D Lei World 22

Health Organization, Switzerland; Dr Y Sun, Paul-Ehrlich-Institut, Germany; Dr M Powell, 23

Medicines and Healthcare Products Regulatory Agency, United Kingdom; Dr Y Wang, 24

National Institutes of Food and Drug Control, China; Dr E Gurley, Johns Hopkins Bloomberg 25

School of Public Health, USA and Dr Q Zhao, Xiamen University, China following an 26

informal consultation held in Beijing, China, 1819 April 2018 and attended by Dr E Gurley, 27

Johns Hopkins Bloomberg School of Public Health, USA; Mr D Minh Hung, Ministry of 28

Health, Viet Nam; Dr M Li, Center for Drug Evaluation of the China Food and Drug 29

Administration, China; Dr Z Liang, National Institutes of Food and Drug Control, China; Dr 30

J Martin, National Institute for Biological Standards and Control, United Kingdom; Dr P 31

Minor, Consultant, United Kingdom; Dr C Ngabéré, Ministry of Public Health, Chad; Dr S 32

Phumiamorn, Ministry of Public Health, Thailand; Dr H Iftikhar Qureshi, Pakistan Health 33

Research Council, Pakistan; Dr J Shin, Regional Office for the Western Pacific, World 34

Health Organization, Philippines; Mr Y Tang, WHO China Country Office, China; Dr D Lei, 35

World Health Organization, Switzerland; Mr B Shrestha, Walter Reed/AFRIMS Research 36

Unit Nepal, Nepal; Dr R Simalango, National Agency of Drug and Food Control, Indonesia; 37

Dr D Steele, Bill & Melinda Gates Foundation, USA; Dr Y Sun, Paul-Ehrlich-Institut, 38

Germany; Dr E Teshale, Centers for Disease Control and Prevention, USA; Dr Y Wang, 39

National Institutes of Food and Drug Control, China; Dr H Wahu Triestano Wibowo, 40

National Agency of Drug and Food Control, Indonesia; Dr X Wu, National Institutes of Food 41

and Drug Control, China; Dr M Xu, National Institutes of Food and Drug Control, China; Dr 42

WHO/BS/2018.2348 of 60

X Yao, Center for Drug Evaluation of China Food and Drug Administration, China; Dr J 1

Zhang, Xiamen University, China; Dr Q, Zhao, Xiamen University, China. 2

Representatives from industry: Dr H Chandra, Cadila Healthcare Limited, Vaccine 3

Technology Center, India; Ms W Weidan Huang, Innovax Biotech Co. Ltd, China; Dr B 4

Huang, Innovax Biotech Co. Ltd, China; Dr JWK Shih, Xiamen Innovax Biotech Co. Ltd, 5

China. 6

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diagnosis, and protective immunity, Rev Med Virol. 2012;22(5):339–49. 8

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hepatitis E virus infection. ILAR J. 2014:55(1):187–99. 10

86. Shrestha MP, Scott RM, Joshi DM, Mammen MP Jr, Thapa GB, Thapa N et al. Safety 11

and efficacy of a recombinant hepatitis E vaccine. N Engl J Med. 2007;356(9):895–12

903. 13

87. Tsarev SA, Tsareva TS, Emerson SU, Govindarajan S, Shapiro M, Gerin JL et al. 14

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89. Meng J, Pillot J, Dai X, Fields HA, Khudyakov YE. Neutralization of different 20

geographic strains of the hepatitis E virus with anti-hepatitis E virus-positive serum 21

samples obtained from different sources. Virology. 1998;249:316–24. 22

90. Purcell RH, Nguyen H, Shapiro M, Engle RE, Govindarajan S, Blackwelder WC et al. 23

Pre-clinical immunogenicity and efficacy trial of a recombinant hepatitis E vaccine. 24

Vaccine. 2003;21:2607–15. 25

91. Huang WJ, Zhang H, Harrison TJ, Lan HY, Huang GY, Wang YC. Immunogenicity 26

and protective efficacy in rhesus monkeys of a recombinant ORF2 protein from 27

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neutralization of hepatitis E virus by a cross-genotype antibody. Cell Res. 36

2015;24(5):604–20. 37

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international and other biological reference standards (revised 2004). In: WHO Expert 32

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Appendix 1. Model summary protocol for the manufacturing and 1

control of recombinant hepatitis E vaccines 2

3

The following protocol is intended for guidance. It indicates the information that should be 4

provided as a minimum by the manufacturer to the NRA. Information and tests may be added 5

or deleted/omitted as necessary with the approval of the NRA. 6

It is possible that a protocol for a specific product may differ in detail from the model 7

provided. The essential point is that all relevant details demonstrating compliance with the 8

licence and with the relevant WHO recommendations for a particular product should be 9

provided in the protocol submitted. 10

The section concerning the final product must be accompanied by a sample of the label and a 11

copy of the leaflet (package insert) that accompanies the vaccine container. If the protocol is 12

being submitted in support of a request to permit importation, it must also be accompanied by 13

a lot release certificate from the NRA of the country in which the vaccine was produced or 14

released, stating that the product meets national requirements as well as the recommendations 15

in Part A of this document. 16

Summary information on final lot 17

18

International name: _________________ 19

Trade name/commercial name: _________________ 20

Product licence (marketing authorization) number: _________________ 21

Country: _________________ 22

Name and address of manufacturer: _________________ 23

Name and address of licence holder, if different: _________________ 24

Final packaging lot number: _________________ 25

Type of container: _________________ 26

Number of containers in this final lot: _________________ 27

Final container lot number: _________________ 28

Date of manufacture: _________________ 29

Nature of final product (adsorbed): _________________ 30

Preservative and nominal concentration: _________________ 31

Volume of each single human dose: _________________ 32

Number of doses per final container: _________________ 33

34

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Summary of the composition (include a summary of the qualitative and quantitative 1

composition of the vaccine per human dose, including any adjuvant used and other 2

excipients): 3

___________________________________________________________________________4

___________________________________________________________________________ 5

6

Shelf-life approved (months): _________________ 7

Expiry date: _________________ 8

Storage condition: _________________ 9

10

The following sections are intended for recording the results of the tests performed during the 11

production of the vaccine, so that the complete document will provide evidence of 12

consistency of production. If any test has to be repeated, this must be indicated. Any 13

abnormal result must be recorded on a separate sheet. 14

15

16

Detailed information on manufacture and control 17

18

Starting materials 19

The information requested below is to be presented on each submission. Full details on 20

master and working seed lots and cell banks are requested upon first submission only and 21

whenever a change has been introduced. 22

23

Identity of seed lot strain used for vaccine production: ______________________ 24

25

26

Reference number of seed lot: ______________________ 27

Date(s) of reconstitution (or opening) of seed lot 28

ampoule(s): ______________________ 29

30

Single harvests used for preparing the bulk 31

32

Lot number(s): ______________________ 33

Volume(s) of fermentation paste, storage temperature, 34

storage time and approved storage period: ______________________ 35

Name of the culture medium: ______________________ 36

Date of inoculation: ______________________ 37

Temperature of incubation: ______________________ 38

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Control of bacterial purity 1

Method: ______________________ 2

Specification: ______________________ 3

Date: ______________________ 4

Result: ______________________ 5

6

Date of harvest: ______________________ 7

Volume of harvest: ______________________ 8

Yield (mg/ml): ______________________ 9

Volume after filtration: ______________________ 10

11

Identity test 12

Method: ______________________ 13

Specification: ______________________ 14

Date: ______________________ 15

Result: ______________________ 16

17

Test for bacteria and fungi 18

Method: ______________________ 19

Media: ______________________ 20

Volume inoculated: ______________________ 21

Date of start of test: ______________________ 22

Date of end of test: ______________________ 23

Result: ______________________ 24

25

Test for Mycoplasmas (if applicable) 26

Method: ______________________ 27




Result: ______________________ 31

32

Test for adventitious agents (if applicable) 33

Method: ______________________ 34

Specification: ______________________ 35

Date: ______________________ 36

Result: ______________________ 37

38

Control of purified antigen bulk 39

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Lot number of purified bulk: ______________________ 1

Date of purification: ______________________ 2

Volume(s), storage temperature, storage time 3

and approved storage period: ______________________ 4

Purity 5

Method: ______________________ 6

Specification: ______________________ 7

Date: ______________________ 8

Result: ______________________ 9

10

Protein content 11

Method: ______________________ 12

Specification: ______________________ 13

Date: ______________________ 14

Result: ______________________ 15

16

Antigen content 17

Method: ______________________ 18

Specification: ______________________ 19

Date: ______________________ 20

Result: ______________________ 21

22

Sterility test for bacteria and fungi 23

Method: ______________________ 24

Media: ______________________ 25




Result: ______________________ 29

30

Percentage of intact monomer 31

Method: ______________________ 32

Specification: ______________________ 33

Date: ______________________ 34

Result: ______________________ 35

36

Particle size and structure 37

Method: ______________________ 38

WHO/BS/2018.2348 of 60

Specification: ______________________ 1

Date: ______________________ 2

Result: ______________________ 3

4

Test for reagents used during purification or other phases of manufacture (if relevant) 5

Method: ______________________ 6

Specification: ______________________ 7

Date: ______________________ 8

Result: ______________________ 9

10

Test for residual host-cell protein (if relevant) 11

Method: ______________________ 12

Specification: ______________________ 13

Date: ______________________ 14

Result: ______________________ 15

16

Test for residual host-cell DNA (if applicable) 17

Method: ______________________ 18

Specification: ______________________ 19

Date: ______________________ 20

Result: ______________________ 21

22

Test for viral clearance (if relevant) 23

Method: ______________________ 24

Specification: ______________________ 25

Date: ______________________ 26

Result: ______________________ 27

28

Control of adsorbed antigen bulk (if applicable) 29

Lot number of adsorbed antigen bulk: ______________________ 30

Date of adsorption: ______________________ 31


and approved storage period: ______________________ 33

Sterility test for bacteria and fungi 34

Method: ______________________ 35

Media: ______________________ 36



WHO/BS/2018.2348 of 60


Result: ______________________ 2

3

Bacterial endotoxin 4

Method: ______________________ 5

Specification: ______________________ 6

Date: ______________________ 7

Result: ______________________ 8

9

Identity test 10

Method: ______________________ 11

Specification: ______________________ 12

Date: ______________________ 13

Result: ______________________ 14

15

Adjuvant content 16

Method: ______________________ 17

Specification: ______________________ 18

Date: ______________________ 19

Result: ______________________ 20

21

Degree of adsorption 22

Method: ______________________ 23

Specification: ______________________ 24

Date: ______________________ 25

Result: ______________________ 26

27

pH 28

Method: ______________________ 29

Specification: ______________________ 30

Date: ______________________ 31

Result: ______________________ 32

33

Antigen content 34

Method: ______________________ 35

Specification: ______________________ 36

Date: ______________________ 37

Result: ______________________ 38

WHO/BS/2018.2348 of 60

1

Control of final bulk 2

Identification (Lot number): ______________________ 3

Date of manufacture/blending: ______________________ 4


and approved storage period ______________________ 6

7

Blending: Prescription (SHD) Added 8

HEV antigen (mg): ____________ ____________ 9

Adjuvant: ____________ ____________ 10

Preservative (specify): ____________ ____________ 11

Others (salt): ____________ ____________ 12

Final volume (ml): ____________ ____________ 13

14

Sterility tests for bacteria and fungi 15

Method: ______________________ 16

Media: ______________________ 17




Result: ______________________ 21

22

Adjuvant content 23

Method: ______________________ 24

Specification: ______________________ 25

Date: ______________________ 26

Result: ______________________ 27

28


Method: ______________________ 30

Specification: ______________________ 31

Date: ______________________ 32

Result: ______________________ 33

34

Preservative content 35

Method: ______________________ 36

Specification: ______________________ 37

Date: ______________________ 38

Result: ______________________ 39

40

Potency test 41

WHO/BS/2018.2348 of 60

in vivo assay (may be performed at final bulk stage) 1

Species, strain, sex and weight specifications: ______________________ 2

Number of mice tested: ______________________ 3

Dates of vaccination, bleeding: ______________________ 4

Date of assay: ______________________ 5

Lot number of reference vaccine and 6

assigned potency: ______________________ 7

Vaccine doses (dilutions) and number of 8

animals responding at each dose: ______________________ 9

ED50 of reference and test vaccine: ______________________ 10

Potency of test vaccine (with 95% fiducial limits): ______________________ 11

12

If an in vitro assay is used 13

Method: ______________________ 14

Specification: ______________________ 15

Date: ______________________ 16

Result: ______________________ 17

18

Osmolarity test 19

Method: ______________________ 20

Specification: ______________________ 21

Date: ______________________ 22

Result: ______________________ 23

24

Control of final lot 25

Lot number: ______________________ 26

Date of filling: ______________________ 27

Type of container: ______________________ 28

Filling volume: ______________________ 29

Number of containers after inspection: ______________________ 30

Number and percentage of containers rejected: ______________________ 31

32

Appearance 33

Method: ______________________ 34

Specification: ______________________ 35

Date: ______________________ 36

Result: ______________________ 37

38

Identity test 39

Method: ______________________ 40

Specification: ______________________ 41

Date: ______________________ 42

WHO/BS/2018.2348 of 60

Result: ______________________ 1

2

Sterility tests for bacteria and fungi 3

Method: ______________________ 4

Media: ______________________ 5




Result: ______________________ 9

10

Osmolarity test 11

Method: ______________________ 12

Specification: ______________________ 13

Date: ______________________ 14

Result: ______________________ 15

16

pH 17

Method: ______________________ 18

Specification: ______________________ 19

Date: ______________________ 20

Result: ______________________ 21

22

Preservative content 23

Method: ______________________ 24

Specification: ______________________ 25

Date: ______________________ 26

Result: ______________________ 27

28

Test for pyrogenic substances 29

Method: ______________________ 30

Specification: ______________________ 31

Date: ______________________ 32

Result: ______________________ 33

34

Adjuvant content 35

Method: ______________________ 36

Specification: ______________________ 37

Date: ______________________ 38

Result: ______________________ 39

40

Extractable volume 41

WHO/BS/2018.2348 of 60

Method: ______________________ 1

Specification: ______________________ 2

Date: ______________________ 3

Result: ______________________ 4

5


Method: ______________________ 7

Specification: ______________________ 8

Date: ______________________ 9

Result: ______________________ 10

11

Potency test 12

in vivo assay (may be performed at final bulk stage) 13

Species, strain, sex and weight specifications: ______________________ 14

Number of mice tested: ______________________ 15

Dates of vaccination, bleeding: ______________________ 16

Date of assay: ______________________ 17

Lot number of reference vaccine and 18

assigned potency: ______________________ 19

Vaccine doses (dilutions) and number of 20

animals responding at each dose: ______________________ 21

ED50 of reference and test vaccine: ______________________ 22

Potency of test vaccine (with 95% fiducial limits): ______________________ 23

24

If an in vitro assay is used 25

Method: ______________________ 26

Lot number of reference and assigned potency: ______________________ 27

Specification: ______________________ 28

Date: ______________________ 29

Result: ______________________ 30

31

Innocuity (general safety) test (unless omission authorized) 32

Method: ______________________ 33

Specification: ______________________ 34

Date: ______________________ 35

Result: ______________________ 36

37

Certification by the manufacturer 38

Name of Head of Quality Control (typed) ______________________ 39

Certification by the person from the control laboratory of the manufacturing company taking 40

overall responsibility for the production and quality control of the vaccine. 41

WHO/BS/2018.2348 of 60

I certify that lot no. ______________________ of recombinant hepatitis E vaccine, whose 1

number appears on the label of the final containers, meets all national requirements and/or 2

satisfies Part A (1) of the WHO Recommendations to assure the quality, safety and efficacy of 3

recombinant hepatitis E vaccines (2). 4

Signature ______________________ 5

Name (typed) ______________________ 6

Date ______________________ 7

Certification by the NRA 8

If the vaccine is to be exported, attach the NRA Lot Release Certificate (as shown in 9

Appendix 2), a label from a final container and an instruction leaflet for users. 10

11

12

Notes: 13

1. With the exception of provisions on distribution and shipping, which the NRA may not be 14

in a position to assess. 15

2. WHO Technical Report Series, No. xxxx, Annex x. 16

WHO/BS/2018.2348 of 60

Appendix 2. Model NRA lot release certificate for recombinant 1

hepatitis E vaccines 2

3

Certificate No.________________ 4

5

The following lot(s) of recombinant hepatitis E vaccine produced by _____________ 6

________________________1 in _______________________________________,

2 whose 7

numbers appear on the labels of the final containers, meet all national requirements3 and Part 8

A4 of the WHO Recommendations to assure the quality, safety and efficacy of recombinant 9

hepatitis E vaccines5 and comply with WHO good manufacturing practices for 10

pharmaceutical products: main principles;6WHO good manufacturing practices for biological 11

products;7 and Guidelines for independent lot release of vaccines by regulatory authorities.

8 12

13

The release decision is based on ___________________________________9 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 and packaging lot numbers if necessary); 21

type of container used; 22

number of doses per container; 23

number of containers or lot size; 24

date of start of period of validity (for example, manufacturing date) and expiry date; 25

storage conditions; 26

signature and function of the person authorized to issue the certificate; 27

date of issue of certificate; 28

certificate number. 29

The Director of the NRA (or other appropriate authority) 30

Name (typed) ________________________________________ 31

Signature ________________________________________ 32

Date ________________________________________ 33

1 Name of manufacturer. 34 2 Country of origin. 35 3 If any national requirements have not been met, specify which one(s) and indicate why the release of the lot(s) has 36 nevertheless been authorized by the NRA. 37 4 With the exception of provisions on distribution and shipping, which the NRA may not be in a position to assess. 38 5 WHO Technical Report Series, No. 000, Annex 0. 39 6 WHO Technical Report Series, No. 986, Annex 2. 40 7 WHO Technical Report Series, No. 999, Annex 2. 41 8 WHO Technical Report Series, No. 978, Annex 2. 42 9 Evaluation of the summary protocol, independent laboratory testing and/or procedures specified in a defined document etc., 43 as appropriate.1 44 1

recommendations to assure the quality, safety and efficacy ......14 paper on hepatitis e in 2015...

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