Guidelines on the quality, safety and efficacy of typhoid ... · 1 2 WHO/BS/2013.2215 3 ENGLISH...

1 WHO/BS/2013.2215 2

ENGLISH ONLY 3

4

Guidelines on the quality, safety and efficacy of typhoid conjugate 5

vaccines 6

7

Proposed Guidelines 8

9 NOTE: 10

11

This document has been prepared for the purpose of inviting comments and suggestions on the 12

proposals contained therein, which will then be considered by the Expert Committee on 13

Biological Standardization (ECBS). Publication of this draft is to provide information about the 14

proposed WHO Guidelines on the quality, safety and efficacy of typhoid conjugate vaccine to a 15

broad audience and to improve transparency of the consultation process. 16

17

The text in its present form does not necessarily represent an agreed formulation of the Expert 18

Committee. Written comments proposing modifications to this text MUST be received by 30 19 September 2013 in the Comment Form available separately and should be addressed to the 20

World Health Organization, 1211 Geneva 27, Switzerland, attention: Department of Essential 21

Medicines and Health Products (EMP). Comments may also be submitted electronically to the 22

Responsible Officer: Dr Jinho Shin at email: [email protected]. 23

24

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

Technical Report Series. The final agreed formulation of the document will be edited to be in 26

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

28

© World Health Organization 2013 29

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health 30 Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: 31 [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for 32 non-commercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-33 mail: [email protected]). 34

The designations employed and the presentation of the material in this publication do not imply the expression of any 35 opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, 36 city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps 37 represent approximate border lines for which there may not yet be full agreement. 38 39 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 40 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. 41 Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 42 43 All reasonable precautions have been taken by the World Health Organization to verify the information contained in 44 this publication. However, the published material is being distributed without warranty of any kind, either expressed or 45 implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the 46 World Health Organization be liable for damages arising from its use. 47

48 The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. 49 50

mailto:[email protected]

WHO/BS/2013. 2215

1

2

3

Recommendations and guidelines published by WHO are intended to be scientific and advisory

in nature. Each of the following sections constitutes guidance for national regulatory authorities

(NRAs) and for manufacturers of biological products. If a NRA so desires, these Guidelines may

be adopted as definitive national requirements, or modifications may be justified and made by

the NRA. It is recommended that modifications to these Guidelines made only on condition that

modifications ensure that the product is at least as safe and efficacious as that prepared in

accordance with the guidelines set out below.

WHO/BS/2013. 2215

Contents 1

2

Abbreviations 4 3

Introduction 5 4

General considerations 5 5

Part A. Guidelines on manufacture and control 13 6

A.1 Definitions 13 7

A.2 Guidelines on general manufacturing 15 8

A.3 Control of starting material 15 9

A.4 Control of vaccine production 16 10

A.5 Filling and containers 25 11

A.6 Control of final product 25 12

A.7 Records 28 13

A.8 Samples 28 14

A.9 Labelling 28 15

A.10 Distribution and shipping 29 16

A.11 Stability, storage and expiry date 29 17

Part B. Nonclinical evaluation of new typhoid conjugate vaccines 31 18

B.1 General Principles 31 19

B.2 Product characterization and process development 31 20

B.3 Nonclinical immunogenicity and animal challenge studies 31 21

B.4 Nonclinical toxicity and safety 32 22

Part C. Clinical evaluation of new typhoid conjugate vaccines 33 23

C.1 General principles 33 24

C.2 Assessment of the immune response 35 25

C.3 Clinical study designs 38 26

C.4 Pre-licensure assessment of safety 42 27

C.5 Post-marketing studies and surveillance 42 28

Part D. Guidelines for national regulatory authorities 44 29

D.1 General guidelines 44 30

D.2 Official release and certification 44 31

Authors & acknowledgments 44 32

References 48 33

Appendix 1: Model summary protocol for manufacture and control of typhoid 34

conjugate vaccines 55 35

Appendix 2: Model certificate for the release of typhoid conjugate vaccines 69 36

37 38

WHO/BS/2013. 2215

Abbreviations 1

2

ADH adipic acid dihydrazide 3

AH adipic acid hydrazide 4

C. freundii s.l. Citrobacter freundii sensu lato 5

CI confidence interval 6

CRM197 cross-reactive material, a non-toxic mutant of diphtheria toxin 7

CTAB hexadecyltrimethylammonium bromide 8

DT diphtheria toxoid 9

EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (also abbreviated EDAC) 10

ELISA enzyme-linked immunosorbent assay 11

ELISPOT enzyme-linked immunosorbent spot assay 12

EPI Expanded Programme on Immunization 13

EU ELISA unit 14

GALT gut-associated lymphoid tissue 15

GMC geometic mean concentration 16

HPAEC–PAD high performance anion exchange chromatography with pulsed amperometric 17

detection 18

HPLC high-performance liquid chromatography 19

HPSEC high-performance size-exclusion chromatography 20

IU international unit 21

Lf limit of flocculation 22

LPS Lipopolysaccharide 23

MALLS multiple angle laser light scattering 24

MW molecular weight 25

NMR nuclear magnetic resonance 26

NRA national regulatory authority 27

OPA opsonophagocytic antibody 28

rEPA recombinant Pseudomonas aeruginosa exoprotein A 29

S. Typhi Salmonella enterica subspecies enterica serovar Typhi 30

SBA serum bactericidal antibody 31

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis 32

SPDP N-Succinimidyl 3-(2-pyridyldithio)-propionate 33

TLR toll like receptor 34

TT tetanus toxoid 35

VVM vaccine vial monitor36

WHO/BS/2013. 2215

Introduction 1

2

These guidelines are intended to assist national regulatory authorities (NRAs) in their evaluation 3

of the scientific issues connected with the quality, safety and efficacy of typhoid conjugate 4

vaccines based on Vi polysaccharide covalently linked to a carrier protein. The currently 5

available guidelines for Vi polysaccharide typhoid vaccine (1) and for live-attenuated Ty21a 6

vaccines (2) are not applicable to typhoid conjugate vaccines consisting of Vi polysaccharide 7

(derived from Salmonella Typhi, Citrobacter freundii sensu lato or other bacterial sources) 8

conjugated with a carrier protein, such as diphtheria toxoid, tetanus toxoid, recombinant 9

Pseudomonas aeruginosa exoprotein A (rEPA), non-toxic mutated or recombinant form of 10

diphtheria toxin (e.g. CRM197), or any suitable protein. 11

12

These guidelines are based on experience gained from the development of experimental typhoid 13

conjugate vaccines as well as potentially relevant information obtained from the evidence of 14

other types of bacterial polysaccharide-protein conjugate vaccines such as Hib, meningococcal 15

and pneumococcal conjugate vaccines. The evdience gathered thus far indicates that typhoid 16

conjugate vaccines will overcome several of the limitations of unconjugated, plain Vi 17

polysaccharide vaccines and are anticipated to demonstrate (i) greater efficacy and effectiveness, 18

(ii) longer persistence of immunity, (iii) immunogenicity across all age groups including infants 19

and toddlers < 2 years old, (iv) perhaps some degree of herd immunity, (v) lack of hypo-20

responsiveness, which has been observed with repeated administrations of Vi polysaccharide 21

vaccineantigens and vi) induction of immune memory with initial dosing, leading to anamnestic 22

responses to subsequent dose(s). 23

24

Part A sets out guidance on manufacturing and quality control while Parts B and C address the 25

nonclinical and clinical evaluation, respectively. Part D provides guidance for the NRA. 26

27

General considerations 28

29

This section provides a brief overview of scientific knowledge that underpins the guidance given 30

in Parts A, B and C. A comprehensive review of immunological basis for typhoid vaccines is 31

also available from WHO (3). 32

33

Typhoid fever is an acute generalized infection of the reticuloendothelial system, intestinal 34

lymphoid tissue, and gall bladder caused by Salmonella enterica subspecies enterica serovar 35

Typhi (typically shortened to “S. Typhi”). Paratyphoid fever is a clinically indistinguishable 36

illness caused by S. Paratyphi A or B (more rarely C) (4, 5). Typhoid and paratyphoid fevers are 37

often referred to collectively as enteric fever. In most endemic areas, typhoid comprises 38

approximately 75–80% of enteric fever. However, in some regions, particularly in Asia, 39

Paratyphi A contributes a relatively larger proportion of all enteric fevers (6, 7). 40

41

Pathogen 42

43

S. Typhi is a member of the family Enterobacteriaceae. It is a Gram-negative, non-lactose 44

fermenting bacillus that produces trace amounts of hydrogen sulphide. Its antigenic 45

characteristics include an immunodominant lipopolysaccharide (LPS) antigen O9, flagellar 46

antigen Hd and capsular polysaccharide antigen Vi. 47

48

WHO/BS/2013. 2215

Vi acts as a virulence factor by preventing anti-O antibody from binding to the O antigen and 1

also inhibits the C3 component of complement from fixing to the surface of S. Typhi. The Vi 2

antigen is not unique to S. Typhi, but is also expressed by S. Paratyphi C, C. freundii s.l. and S. 3

Dublin. The genes responsible for the biosynthesis of Vi polysaccharide are located in a locus 4

(viaB) within Salmonella Pathogenicity Island-7 (SPI-7) in the S. Typhi chromosome. Several 5

other loci participate in the complex regulation of Vi expression. Almost all S. Typhi isolates 6

from blood cultures can be shown to expressing Vi. Nevertheless, occasional Vi-negative strains 7

have been identified both in sporadic cases as well as outbreaks (8). Some of the strains are 8

regulatory mutants that can revert to a Vi-positive state (9). However, some Vi-negative isolates 9

from blood have been shown to harbor deletion mutations in critical genes (e.g. tviB) within the 10

viaB locus that render the strains unable to synthesize Vi. This raises the theoretical concern that 11

large-scale usage of Vi-containing (polysaccharide or conjugate) vaccines could create selective 12

pressure creating a biological advantage for the emergence of Vi-negative strains (10). 13

14

Pathogenesis 15

16 Typhoid infection begins with ingestion of S. Typhi in contaminated food or water. In the small 17

intestine, the bacteria penetrate the mucosal layer and ultimately reach the lamina propria. 18

Translocation from the intestinal lumen mainly occurs by S. Typhi targeting M cells overlying 19

gut-associated lymphoid tissue (GALT). Within the GALT and in the lamina propria S. Typhi 20

invokes an influx of macrophages and dendritic cells that ingest the bacteria but fail to destroy 21

them. Thus some bacteria remain within macrophages of the small intestinal lymphoid tissue and 22

flow into mesenteric lymph nodes where there is an inflammatory response mediated by the 23

release of various cytokines. Bacteria enter the bloodstream via lymphatic drainage thereby 24

seeding organs of the reticuloendothelial system (e.g. spleen, liver, bone marrow) and gall 25

bladder by means of a silent primary bacteremia. After an incubation period of typically 8-14 26

days the clinical illness begins usually with the onset of fever, abdominal discomfort and 27

headache. An accompanying low level secondary bacteremia occurs. 28

Before the availability of fluoroquinolone antibiotics, clinical relapses were observed in 5–30% 29

of patients treated with antibacterial agents such as chloramphenicol and 30

trimethoprim/sulfamethoxazole. These post-treatment relapses occurred when typhoid bacilli re-31

emerged from their protected intracellular niches within macrophages of the reticuloendothelial 32

system into which these antibacterial agents could not penetrate. 33

In a small proportion of S. Typhi-infected patients who have pre-morbid abnormalities of the gall 34

bladder mucosa, such as consequent to gallstones, gallbladder infection becomes chronic 35

(excretion > 12 months) (11). Such chronic carriers, who are not clinically affected by the 36

presence of typhoid bacilli in their system, may excrete the pathogen in their feces for decades 37

(12). They serve as a long-term epidemiologic reservoir in the community who can lead to 38

transmission of typhoid wherever there is inadequate sanitation, untreated water supplies or 39

improper food handling. 40

41

Epidemiology 42

43

Typhoid is restricted to human hosts, and chronic carriers constitute the reservoir of infection. In 44

the late nineteenth and early twentieth century, typhoid was endemic in virtually all countries 45

within Europe and the Americas. Subsequently the widespread use of chlorination, sand 46

WHO/BS/2013. 2215

filtration, and other means of water treatment reduced the incidence of typhoid fever drastically, 1

despite the high prevalence of chronic carriers in the community (11). At present, typhoid is still 2

endemic in most developing countries mainly because large segments of the population lack 3

access to safe water supply and basic sanitation services. In addition, there are limited 4

programmes for detecting carriers and restricting them from food-handling. 5

6

Disease burden 7

8

Variable estimates of typhoid fever have been published in scientific literature. The true 9

incidence of typhoid fever in most regions of developing countries is not known. A study 10

published in 2004 estimated that 22-million cases occur each year causing 216,000 deaths, 11

predominantly in school-age children and young adults with the annual incidence 10–100 per 12

100,000 population (13). A systematic review of population-based studies from 1984 through 13

2005 reported an annual incidence of 13–976 per 100,000 per year based on diagnosis by blood 14

culture (14). 15

16

Several factors affect calculation of typhoid disease burden. In the absence of a rapid, affordable, 17

and accurate diagnostic test, blood culture is recognized as the current gold standard. However, 18

blood culture alone detects only 60–70% of cases that are detectable using bone marrow culture 19

or bile fluid culture (14). Prior treatment with antibacterial agents also affects culture results. On 20

the other hand, relying on clinical diagnosis alone can over-estimate the burden because several 21

febrile syndromes caused by other micro-organisms, such as malaria, dengue, and leptospirosis 22

can be confused with typhoid, particularly in children. 23

24

The incidence of typhoid, its age-specific distribution and the severity of clinical disease gleaned 25

from passive health facility-based surveillance often appears quite different from data acquired 26

through active surveillance where households are visited systematically once or twice weekly to 27

detect fever among household members. A recent study reported the incidence of typhoid 28

detected through passive surveillance (and “modified passive” surveillance in two countries 29

where additional health clinics were introduced in the community) in five Asian countries. The 30

incidence of typhoid fever ranged from 15.3 per 100,000 person-years in 5–60 year old persons 31

in China to 451.7 per 100,000 person-years in 2–15 year old children in Pakistan (15). Incidence 32

data from the placebo control groups of vaccine trials have also provided data on the incidence 33

of typhoid fever in multiple geographic areas and venues. However, as vaccine efficacy trials are 34

typically carried out in areas with high endemicity, caution must be taken in extrapolating such 35

incidence rates to other populations. 36

37

In general, there is less information on the burden of disease in children less than 2 years of age 38

than in older age groups. In the 5-Asian site surveillance study of Ochiai et al, two sites 39

(Kolkata, India and North Jakarta, Indonesia) included surveillance among children < 2 years of 40

age. In Kolkata the recorded annual incidence in children < 2 years of age was 89 cases per 41

100,000 child-years (15) and in North Jakarta the annual incidence was 0 cases per 100,000 child 42

years. In Kolkata only 1 of 145 blood cultures from febrile children < 2 years of age was positive 43

for S. Typhi (0.69%) (16) and in Jakarta 0 of 404 blood cultures were positive in febrile children 44

of this age. By comparison, the incidence of culture-confirmed typhoid fever in Kolkata was 45

340.1 cases per 100,000 child-years in children 2–4 years of age and was 493.5 cases per 46

100,000 child-years in children 5–15 years of age. In North Jakarta the annual incidence of 47

typhoid was 148.7 cases per 100,000 child-years among 2–4 year olds and 180.3 cases per 48

100,000 child-years among children 5–15 years of age. 49

50

WHO/BS/2013. 2215

Prior to the availability of antibacterial agents, typhoid resulted in a case-fatality rate of 1

approximately 10–20% (17). The 2008 WHO position paper on typhoid fever estimated 2

216,000–600,000 annual deaths in recent years (18). Most of the mortality occurs in developing 3

countries, and 80% of deaths occur in Asia. A review by Crump et al reported community-based 4

mortality ranging from 0–1.8% across 5 studies in developing countries and hospital-based 5

mortality ranging from 0–13.9% (across all ages in 12 studies) and 0–14.8% in children less than 6

15 years (across 13 studies) (14). 7

8

Few studies have estimated the prevalence of chronic typhoid and paratyphoid carriers in 9

developing countries. A survey in Santiago, Chile when typhoid fever was highly endemic there 10

in the 1970s estimated a crude prevalence of 694 typhoid carriers/100,000 population (19). In 11

Kathmandu, Nepal, among 404 patients (316 female and 88 male) with gallbladder disease 12

undergoing cholecystectomy, S. Typhi was isolated from 3.0% and S. Paratyphi A from 2.2% of 13

bile cultures (20). Since the overall prevalence of cholelithiasis in the Kathmandu population 14

was not known, an overall prevalence of chronic carriage in that population could not be 15

calculated. 16

17

Clinical features 18

19

S. Typhi infection results in a broad spectrum of clinical features most often characterized by 20

persisting high-grade fever, abdominal discomfort, malaise and headache. Important clinical 21

signs in hospitalized patients include hepatomegaly (41%), toxicity (33%), splenomegaly (20%), 22

obtundation (2%) and ileus (1%) (21). Gross bleeding from the GI tract and perforations 23

occurred in 1–3% of untreated patients before antibacterial agents became available but this case 24

is now rarely observed except in settings with poor access to health-care. 25

26

Typhoid fever has the potential for serious complications. Hospital based reports suggest that 27

over 50% patients can have complications. Huang et al (2005) summarized various 28

complications as follows: central nervous system (3–55%), hepato-biliary system (1–26%), 29

cardiovascular system (1–5%), pulmonary system (1–6%), bone and joint (< 1%), and 30

hematologic system (rare) (22). Intestinal perforations leading to peritonitis and even death 31

continue to be reported is some settings even today. 32

33

Immune responses to natural infection 34

35

Natural typhoid infection is usually associated with the detection of serum antibodies and 36

mucosal secretory IgA intestinal antibody against various S. Typhi antigens and cell-mediated 37

immune responses are also measureable (23-27). In typhoid endemic areas, there is an age-38

related increase in the prevalence and geometric mean titer of anti-Vi antibodies (28). Anti-39

flagella (H-antigen) serum IgG antibodies following natural infection are long-lived and have 40

been studied for seroepidemiologic surveys (29). 41

42

While serological responses to LPS and flagella antigens tend to be fairly strong and are 43

commonly found in patients with culture-confirmed acute typhoid fever, curiously, only about 44

20% of such patients exhibit significant levels of anti-Vi antibody (30, 31). In contrast, very high 45

concentrations of anti-Vi serum IgG antibody are detected in 80–90% of chronic carriers (30, 46

31). 47

48

WHO/BS/2013. 2215

Cell-mediated immunity also appears to play a role in protection because it has been observed 1

that peripheral blood mononuclear leukocytes of otherwise healthy adults residing in typhoid-2

endemic areas with no history of typhoid proliferate on exposure to S. Typhi antigens (32). 3

4

Disease control 5

6

Similar to other enteric and diarrheal diseases, typhoid fever exists predominantly in populations 7

with inadequate access to safe water and basic sanitation. Effective typhoid control requires a 8

comprehensive approach that combines immediate measures, such as accurate and rapid 9

diagnostic confirmation and timely administration of appropriate antibiotic treatment as well as 10

sustainable long-term solutions like access to safe water and basic sanitation. 11

12

Other interventions, such as treatment of household water, proper food handling, hand washing 13

with soap and discouraging open defecation can also be effective control measures (33-35). The 14

most effective strategy to improve overall public health in typhoid-affected populations is the 15

implementation and maintenance of municipal water and sanitation systems. 16

17

Vaccination against typhoid has been proven an effective preventive intervention, especially 18

when coupled with hand washing, household water treatment, adequate sanitation and other 19

preventive measures. A detailed review of the immunological basis for typhoid vaccination has 20

been published recently (36). 21

22

Typhoid vaccines 23

24

Inactivated whole cell vaccine 25

26

Inactivated S. Typhi (heat-inactivated and phenol-preserved) bacteria were first utilized for the 27

preparation of parenteral vaccines over 100 years ago. In the 1960s WHO-sponsored field trials 28

evaluated the efficacy of inactivated parenteral whole cell vaccines in several countries (37, 38) 29

and documented a moderate level of efficacy lasting up to seven years (39). Human immune 30

response data and immunogenicity studies in rabbits suggested that anti-H antibodies might 31

represent a correlate (40), while years later extrapolation from results of mouse protection 32

studies suggested that responses to Vi antigen may have correlated with protection (41). These 33

vaccines were associated with considerable rates of systemic adverse reactions (42) and are no 34

longer in use. 35

36

Live-attenuated Ty21a oral vaccine 37

38 In the early 1970s, an attenuated strain of S. Typhi was developed through chemical induced 39

mutagenesis of pathogenic S. Typhi strain Ty2 (43). The resultant mutant strain lost activity of 40

the epimerase enzyme encoded by the galE gene and was also no longer capable of expressing 41

Vi polysaccharide. The vaccine was found to be stable, safe and efficacious in adults as well as 42

children (44-48) but the level of protective immunity achieved varied with the vaccine 43

formulation, the number of doses administered and the interval between doses. 44

45

For example, three doses of a provisional formulation of vaccine or placebo administered to 46

about 32,000 children (6–7 year olds) in Alexandria, Egypt, gave a point estimate of efficacy of 47

WHO/BS/2013. 2215

95.6% over a three-year follow-up (49). Three doses of the enteric-coated capsule preparation 1

administered to 6–19 year old Chilean school children using two dosage intervals (either 2

alternate days or 21 days between doses) demonstrated 67% point estimate of efficacy over a 3-3

year follow-up and 62% protection over seven years (44, 50) in the group receiving doses on 4

alternate days. The estimate of protection was 49% with the 21-day interval between doses. 5

Another trial using four doses administered within seven days to Chilean school children 6

demonstrated even greater protection (51). Only 5% of 6–7 year old children had difficulty in 7

swallowing the capsules (51). Currently, almost all countries where Ty21a is licensed utilize a 8

three-dose course of enteric-coated capsules taken on alternate days except the United States and 9

Canada that recommend a 4-dose course. 10

11

Two other field trials in Chile (48) and Indonesia (47) compared the enteric coated capsules with 12

three doses of the “liquid” formulation. The liquid formulation conferred greater efficacy than 13

the capsules in both trials. In Chile, where doses were given on alternate days, results with the 14

liquid formulation were superior to Indonesia where the doses were administered one week apart 15

(respective point estimates of efficacy 77% and 53%). In Chile, 78% protection was documented 16

up to five years after vaccination with the liquid formulation (50). There is also indirect evidence 17

that large-scale vaccination with Ty21a can provide some degree of protection against typhoid to 18

non-vaccinated subjects through herd immunity. 19

20

Vi polysaccharide vaccine 21

22

Technological advances made it feasible to purify Vi polysaccharide and to prepare vaccines 23

almost totally free of contaminating LPS (52) that are associated with low rates (1–2%) of febrile 24

reactions. 25

26

The immunologic basis of purified Vi polysaccharide parenteral vaccines is the generation of 27

serum anti-Vi IgG antibodies in 85–90% vaccine recipients over 2 years old. Some experts have 28

tried to define threshold levels of anti-Vi IgG that correlate with protection (such as 1.0 µg/ml 29

and 1.5 µg/ml). 30

31

Clinical trials with the vaccine showed a rise in anti-Vi antibody titre in adults and children (53-32

55). However, subsequent inoculations with Vi did not ‘boost’ the antibody response. Although a 33

single dose was associated with persistence of antibodies for up to 3 years in some vaccine 34

recipients, many adult recipients in non-endemic areas showed a marked drop in antibody levels 35

after 2 years (56, 57). A typhoid fever epidemic among French soldiers deployed in the Ivory 36

Coast showed that the risk of typhoid fever was significantly higher in persons vaccinated more 37

than 3 years previously (58). 38

39

Field trials in children and adults in Nepal (53) and school children in South Africa with a single 40

(25 μg) dose showed 72% vaccine efficacy over 17 months follow-up and 60% protection over 41

21 months follow-up, respectively (54). In South Africa, protection declined to 55% at three 42

years (59). Another field trial in Chinese subjects from 3–50 years of age with a single 30 μg 43

dose showed 69% efficacy over 19 months of follow-up (60). Thus the main advantage of the Vi 44

vaccine is that it could provide moderate protection with a single dose. The disadvantage is that 45

there are no data suggesting protective efficacy beyond three years, necessitating re-46

vaccination(s) within three years. 47

48

Most data suggest that young children under 5 years old respond poorly to Vi polysaccharide 49

vaccines (61). However, one cluster-randomized trial in Kolkata India (62) showed that 50

protective efficacy among young children (2–4 years) was higher than that observed in 5–14 year 51

WHO/BS/2013. 2215

old children and older persons (80%, 56% and 46% respectively). In contrast, a cluster-1

randomized trial field trial of very similar design and using the same Vi vaccine in Karachi, 2

Pakistan reported an adjusted total vaccine protective effectiveness of -38% (95% CI: -192%, 3

35%) for children aged 2–5 years compared to 57% (95% CI: 6%, 81%) for children 5–16 years 4

old (61). 5

6

In summary Vi vaccines can provide moderate protection with a single dose for a limited 7

duration but have the usual limitations associated with polysaccharide vaccines, including poor 8

immunogenicity in infants and young children, short-lived immunity and lack of anamnestic 9

immune responses to subsequent doses (56, 63, 64). 10

11

Vi polysaccharide–protein conjugate vaccine 12

13 Experience with several polysaccharide-protein conjugate vaccines (such as Hib, meningococcal, 14

pneumococcal vaccines) has shown that conjugation overcomes many of the limitations 15

associated with unconjugated bacterial polysaccharides. On the basis of this experience and to 16

try to address the limitations of the various typhoid vaccines described above, several Vi 17

polysaccharide—protein conjugate vaccines have been developed. 18

19

A preparation of Vi polysaccharide conjugated to rEPA (Vi-rEPA) was evaluated in a series of 20

studies in endemic as well as other areas. Children of school and pre-school age from highly 21

endemic areas who received the Vi conjugate vaccine achieved and maintained higher levels of 22

anti-Vi IgG serum antibodies compared to those who received Vi polysaccharide vaccine (65-23

67). The immunogenicity of this Vi conjugate vaccine was observed to be dose-dependent (67). 24

Following the administration of a single dose, detectable antibody levels were maintained for as 25

long as 10 years in adults and 8 years in children. 26

27

A placebo-controlled, randomized, double-blind study in Vietnamese children aged 2–5 years in 28

the high endemic area of Megon Delta gave an estimated vaccine efficacy of 89% after nearly 4 29

years (65, 67). 30

31

Vietnamese infants who received Vi-rEPA at 2, 4 and 6 months of age showed a rise in anti-Vi 32

level from a GMC at 0.66 ELISA units (EU) in cord blood to 17.4 EU at seven months (i.e. one 33

month after the third dose) (68). By 12 months of age, the GMC had declined to 4.76 EU. An 34

additional dose at this age resulted in a boosting effect with a GMC at 50.1 EU one month later. 35

At this time, over 95% of infants had levels of greater than 3.5 EU, which was a putative 36

antibody concentration associated with protection using the assay described in the study. 37

Antibody responses to the routine EPI vaccines (administered simultaneously at 2, 4, 6 months) 38

were comparable in all groups. 39

40

A typhoid conjugate vaccine that uses Vi prepared from C. freundii s.l. and CRM197 as carrier 41

protein has been demonstrated to elicit a significantly higher level of anti-Vi IgG in European 42

adults who had never been exposed to typhoid fever (69). Vi preparations from C. freundii s.l. 43

have been shown to be immunologically indistinguishable and structurally similar (70, 71), 44

although size differences have been observed for Vi polysaccharide of S. Typhi and C. freundii s. 45

l. by size exclusion high-performance liquid chromatography (SEC-HPLC). 46

47

Animal challenge studies 48

49

WHO/BS/2013. 2215

In the 1950s and 1960s, WHO has encouraged research on the evaluation of inactivated typhoid 1

vaccines in various passive and active mouse protection models to assess whether a model could 2

be identified that predicted and correlated with the results of the large-scale field trials of the 3

vaccines (see above) in humans (72), Spaun and Uemura (73) and Cvjetanovic and Uemura (74). 4

5

More recent evaluation of Vi conjugate vaccines in bacterial challenge models have been 6

reported (71). Hale and colleagues used a tranformed Vi producing S. Typhimurium strain 7

(C5.507) in a challenge model with BALB/c mice. Vaccination with Vi polysaccharide 8

conjugated to the Klebsiella pneumoniae outer membrane 40 kDa protein (rP40) provided partial 9

protection from infection against C5.507. Opsonisation assays demonstrated post-vaccination 10

enhancement of Vi-positive bacterial uptake by cultured murine bone marrow derived 11

macrophages. Rondini and colleagues also showed protection in BALB/c mice against challenge 12

with Vi-positive C5.507 subsequent to vaccination with C. freundii s.l. derived Vi conjugated 13

with CRM197 (75). 14

15

Historically animal models could not closely mimic the disease processes of human typhoid. 16

Recently Libby and colleagues (76) engrafted human hematopetic stem cells into (NOD)-SCID-17

IL12rnull

diabetic mice. A ten fold increase in liver bacterial burden was reported subsequent to 18

IP infection with S. Typhi. In another studies with engrafted immunocompromised Rag2-/-c-/- 19

mice with human fetal liver hematopoietic stem and progenitor cells or with human umbilical 20

cord blood cells, a more human-like disease was observed that included dissemination and 21

replication of bacteria in liver and spleen (77-79). Other murine typhoid animal models are in 22

development such as those in TLR4 (80) and TLR11 (81, 82) deficient mice. The TLR mouse 23

models may provide an advantage over human immune system mice as variability due to 24

engraftment is not present. 25 26

27

WHO/BS/2013. 2215

Part A. Guidelines on manufacture and control 1

A.1 Definitions 2

A.1.1 International name and proper name 3

4

The international name of the vaccine should be “typhoid conjugate vaccine”[1]

. The proper 5

name should be the equivalent of the international name in the language of the country of origin. 6

The use of the international name should be limited to the vaccines that satisfy the specifications 7

formulated below. 8

9

A.1.2 Descriptive definition 10

11

A typhoid conjugate vaccine is a preparation of S.Typhi or C. freundii s.l. Vi polysaccharide 12

covalently linked to a carrier protein. It may be formulated with a suitable adjuvant. It may be 13

presented as a sterile, aqueous suspension or as freeze-dried material. The preparation should 14

satisfy all the specifications given below. 15

16

A.1.3 International reference materials 17

18

There are no international reference materials commonly applicable for measuring 19

polysaccharide content, molecular mass/size distribution, and/or animal immunogenicity of Vi-20

polysaccharide-based typhoid conjugate vaccines under clinical development. Working standards 21

for Vi polysaccharide either from S. Typhi or C. freundii s.l. are under development. 22

23

An international reference material to standardize antibody responses to Vi polysaccharide 24

conjugate vaccines against typhoid is under development and expected to be available in the near 25

future. A national reference preparation of purified human anti-Vi polysaccharide IgG is 26

currently available for the standardization of ELISA to evaluate immune response of 27

experimental vaccines in clinical studies (83) (see section C.2.1). 28

29

A.1.4 Terminology 30

31

The definitions given below apply to the terms used in these guidelines. They may have different 32

meanings in other contexts. 33

34

Carrier protein: 35

36

The protein to which the Vi polysaccharide is covalently linked for the purpose of eliciting a T-37

cell-dependent immune response to the Vi polysaccharide. 38

39

1 Point of Discussion: proposal for an alternative international name, “Salmonella enterica serovar Typhi

conjugate vaccine”

WHO/BS/2013. 2215

Final bulk: 1

The homogeneous preparation present in a single container from which the final containers are 2

filled, either directly or through one or more intermediate containers derived from the initial 3

single container. 4

5

Final lot: 6

7

A number of sealed, final containers that are equivalent with respect to the risk of contamination 8

during filling and freeze-drying (if performed). A final lot should therefore have been filled from 9

a single container and freeze-dried in one continuous working session. 10

11

Master seed lot: 12

13

Bacterial suspensions for the production of Vi polysaccharide or carrier protein should be 14

derived from a strain that has been processed as a single lot and is of uniform composition. It is 15

used for the preparation of the working seed lots. Master seed lots should be maintained in the 16

freeze-dried form or be frozen below -45 C. 17

18

Modified carrier protein: 19

20 Chemically or physically modified carrier protein prepared for conjugation to the 21

polysaccharide. 22

23

Modified polysaccharide: 24

25

Purified polysaccharide that has been modified by chemical reaction or a physical process in 26

preparation for conjugation to the carrier protein. 27

28

Purified bulk conjugate: 29

30

A bulk conjugate prepared from a single lot or pool of lots of modified polysaccharide and a 31

single lot or a pool of lots of carrier protein. This is the parent material from which the final bulk 32

is prepared. 33

34

Purified polysaccharide: 35

36

The material obtained after final purification. The lot of purified polysaccharide may be derived 37

from a single harvest or a pool of single harvests processed together. 38

39

WHO/BS/2013. 2215

Single harvest: 1

2

The material obtained from one batch of culture that has been inoculated with the working seed 3

lot (or with the inoculum derived from it), harvested and processed together in one production 4

run. 5

6

Working seed lot: 7

8

A quantity of live strains for the production of Vi polysaccharide or carrier protein of uniform 9

composition derived from the master seed lot by growing the organisms and maintaining them in 10

aliquots in the freeze-dried form or the frozen state at or below -45 C. The working seed lot is 11

used for the inoculation of production medium. 12

13

A.2 Guidelines on general manufacturing 14

15

The general manufacturing recommendations contained in good manufacturing practices for 16

pharmaceutical (84) and biological products (85) should be applied to the establishments 17

manufacturing Vi polysaccharide conjugate vaccines. 18

19

Details of standard operating procedures for the preparation and testing of Vi polysaccharide 20

conjugate vaccines adopted by the manufacturer, together with evidence of appropriate 21

validation of each production step, should be submitted for the approval of the NRA. All assay 22

procedures used for quality control of the conjugate vaccine and vaccine intermediates should be 23

validated. When they are required, proposals for the modification of the manufacturing and 24

control methods should also be submitted for approval to the NRA before they are implemented. 25

26

Production strains for Vi polysaccharide and the carrier proteins should be handled according 27

their biosafety level specifications and depending on the requirements of the NRA (86). Standard 28

operating procedures should be developed to deal with emergencies arising from the accidental 29

spillage, leakage or other dissemination. Personnel employed in the production and control 30

facilities should be adequately trained. Appropriate protective measures including vaccination 31

should be implemented if available. 32

33

34

A.3 Control of starting material 35

A.3.1 Certification of bacterial strain 36

A.3.1.1 Bacterial strain for preparing Vi polysaccharide 37

38

The bacterial strain used for preparing Vi polysaccharide should be from single well 39

characterized stock identified by a record of its history, including the source from which it was 40

obtained and the tests used to determine the characteristics of the strain. 41

42

The strain should be capable of stably producing Vi polysaccharide. 43

WHO/BS/2013. 2215

S. Typhi and C. freundii s.l. have been shown to be suitable source for Vi polysaccharide. 1H 1

nuclear magnetic resonance (NMR) spectroscopy and immunochemical tests are suitable 2

methods for the confirmation of the identity of the polysaccharide. 3 4

A.3.1.2 Bacterial strain for preparing carrier protein 5

6

The bacterial strains used for preparing carrier protein should be identified by a record of its 7

history, including the source from which it was obtained and the tests used to determine the 8

characteristics of the strain. 9 10

A.3.2 Bacterial seed lot system 11

12

The production of both Vi polysaccharide and carrier protein should be based on seed lot system 13

involving a master and working seed. Cultures derived from the working seed should have the 14

same characteristics as the cultures of the strain from which the master seed lot was derived 15

(A.3.1.1 and A.3.1.2). 16

17

Each new seed lot prepared should be characterized for Vi production by appropriate methods. 18

19

If materials of animal origin are used in the medium for instance, for seed lot preparation for the 20

preservation of strain viability, for freeze-drying, or for frozen storage, they should comply with 21

the WHO Guidelines on transmissible spongiform encephalopathies in relation to biological and 22

pharmaceutical products (87) and should be approved by the NRAs. 23

24

Manufacturers are encouraged to avoid the use of materials of animal origin wherever possible. 25

26

A.3.3 Bacterial culture media 27

28

Basal medium must be sterilised and manufacturers are encouraged to use semi synthetic or 29

chemically defined media without addition of ingredients of animal origin. 30

The liquid culture medium used for preparation of bacterial seed lots and for production of 31

polysaccharide antigen should be free from ingredients that will form a precipitate upon addition 32

of hexadecyltrimethylammonium bromide (CTAB) to a concentration subsequently used in the 33

manufacturing process. 34

35

Culture media should be free from substances likely to cause toxic or allergic reactions in 36

humans. If materials of animal origin are used they should comply with the WHO Guidelines on 37

transmissible spongiform encephalopathies in relation to biological and pharmaceutical 38

products (87) and should be approved by the NRA. 39

40

A.4 Control of vaccine production 41

42

A.4.1 Control of polysaccharide antigen production 43

44

WHO/BS/2013. 2215

Vi polysaccharide, used in licensed vaccines, are defined chemical substances if prepared to 1

similar specifications. As a result, it is expected that they would have comparable potencies 2

independent of the manufacturing process. 3

4

A.4.1.1 Single harvests for preparing Vi polysaccharide antigen 5

6

Consistency of the production process should be demonstrated by monitoring growth of the 7

organisms and yield of the Vi polysaccharide. 8

9

A.4.1.1.1 Consistency of microbial growth for antigen production 10

11

Consistency of growth of production strains should be demonstrated by monitoring, but not 12

limited to, growth rate, pH and the final yield of Vi polysaccharide 13

14

A.4.1.1.2 Bacterial purity 15

16

Samples of the culture should be taken before inactivation if required andexamined for microbial 17

contamination. The purity of the culture should be verified by suitable methods that should 18

include inoculation on appropriate culture media. If any contamination is found, the culture and 19

any product derived from it should be discarded. 20

21

A.4.1.2 Bacterial inactivation and antigen purification 22

23

Generally, S. Typhi is inactivated by formaldehyde or by use of a suitable inactivating agent or 24

alternative methods (for instance heating). The inactivation process should be adequately 25

validated. 26

27

The biomass is removed by centrifugation or tangential flow filtration. The Vi polysaccharide is 28

purified from the supernatant by precipitation with CTAB. All reagents should be 29

pharmaceutical grade and sterile. Controls should be in place to monitor the bioburden during 30

purification. Methods used for further purification of this intermediate should be agreed upon 31

with the NRA. To ensure stability, purified Vi polysaccharide in powder form should be stored at 32

2–8 °C and purified Vi polysaccharide in solution should be stored preferably below -20 °C. 33

Polysaccharide stability during the hold time should be validated. 34

35

A.4.1.3 Control of purified Vi polysaccharide antigen 36

37

Each lot of purified Vi polysaccharide should be tested for identity and purity. These test should 38

be validated. In case tests to determine polysaccharide identity and purity give complementary 39

but incomplete information, the combination of methods employed should be agreed by the 40

NRA. 41

42

A.4.1.3.1 Purity 43

WHO/BS/2013. 2215

1

Vi polysaccharide is a linear homopolymer composed of (l→4)-2-acetamido-2-deoxy- α-D-2

galacturonic acidwhich is O-acetylated at carbon-3 (88). 3

4

Each lot of purified polysaccharide should be tested for purity. The limits given below are 5

expressed with reference to the polysaccharide in its fully O-acetylated acid form corrected for 6

moisture. Each manufacturer should define the limits for its own product and they should be 7

agreed by the NRA. 8 9

A.4.1.3.2 Identity 10

11

A test should be performed on the purified polysaccharide to verify its identity. NMR 12

spectroscopy (89) or a suitable immunoassay provide convenient methods. 13 14 15

A.4.1.3.3 Molecular size/mass distribution 16

17

The molecular size/mass distribution of each lot of purified polysaccharide should be estimated 18

to assess the consistency of each batch. The distribution constant (KD) should be determined by 19

measuring the molecular size distribution of the polysaccharide at the main peak of the elution 20

curve obtained by a suitable chromatographic method. The KD value and/or the mass distribution 21

limits should be established and shown to be consistent from lot to lot for a given product. For 22

gel filtration, typically at least 50% of the Vi polysaccharide should be eluted before an 23

appropriate KD value dependent on the chromatographic method used. 24

25

An acceptable level of consistency should be agreed with the NRA. Alternatively calculation of 26

peak width at the 50% level can be used to analyse MW distribution. 27

Suitable methods for this purpose are: gel filtration using i) a refractive index detector (90), ii) a 28

colorimetric assay, or iii) a light scattering detector (91). Manufacturers are encouraged to 29

produce Vi polysaccharide with a consistent molecular size distribution . 30

31

A.4.1.3.4 Polysaccharide content 32

33 The concentration of the Vi polysaccharide in its fully O-acetylated / acid form in eluted 34

fractions can be measured by the method of Hestrin (92) or a suitable method such as NMR (89). 35

The acridine orange method (88, 93) and high performance anion exchange chromatography 36

with pulsed amperometric detection (HPAEC-PAD) method (93) have been reported to produce 37

comparable results for Vi polysaccharide in a range of 15–200 µg/mL. A suitable immunoassay 38

may also be considered. A suitable reference preparation of Vi polysaccharide should be used. 39

These methods should be agreed with the NRA and should be validated. 40

41

A.4.1.3.5 O-acetyl content 42

43

The O-acetyl content of the purified Vi polysaccharide is important for the immunogenicity of 44

Vi and should not be less than 2.0 mmol/g polysaccharide (52% O-acetylation) (88, 90, 94). 45

46

WHO/BS/2013. 2215

The Hestrin method (92) or NMR (89, 95) may also be used to quantitatively determine the O-1

acetylation. The methods and acceptance criteria should be agreed with the NRA. 2

3

A.4.1.3.6 Moisture content 4

5

If the purified polysaccharide is to be stored as a powder, the moisture content should be 6

determined by suitable validated methods agreed by the NRA and shown to be within agreed 7

limits. 8 9

A.4.1.3.7 Protein impurity 10

11

Each lot of purified polysaccharide should contain not more than 1% (weight/weight) of protein 12

as determined by a suitable validated assay using bovine serum albumin as a reference (96). 13

14

Sufficient polysaccharide should be assayed to detect 1% protein contamination accurately. 15

16

A.4.1.3.8 Nucleic acid impurity 17

18

Each lot of purified polysaccharide should contain not more than 2% of nucleic acid by weight 19

as determined by ultraviolet spectroscopy, on the assumption that the absorbance of a 10 g/L 20

nucleic acid solution contained in a cell of 1 cm path length at 260 nm is 200 (90) or by another 21

validated method (for instance, PicoGreen, Threshold method). 22

23

Sufficient polysaccharide should be assayed to detect 2% nucleic acid contamination accurately. 24

25

A.4.1.3.9 Phenol content 26

27

If phenol has been used in the preparation, each lot should be tested for phenol content, using a 28

validated method approved by the NRA. The phenol content should be expressed in microgram 29

per mg purifed Vi antigen and shown to be consistent and within the limits approved by the 30

NRA. 31 32

A.4.1.3.10 Endotoxin/pyrogen content 33

34

To ensure an acceptable level of pyrogenic activity of the final product, the endotoxin content of 35

each lot of purified Vi polysaccharide should be determined, and shown to be within limits 36

agreed as being acceptable by the NRA. 37

38

An endotoxin content not more than 150 International Units (IU) per microgram of Vi 39

polysaccharide (not more than 3,750 IU per SHD) measured by the Limulus amoebocyte lysate 40

test should be achievable. Alternatively a rabbit pyrogen test can be performed. 41 42

WHO/BS/2013. 2215

A.4.1.3.11 Residues of process related contaminants 1

2

The residues of process-related contaminants (for instance, CTAB, formaldehyde and antifoam) 3

in the purified polysaccharide should be determined, and shown to be within limits agreed as 4

being acceptable by the NRA. Routine release testing for residual process related contaminants 5

on each lot can be omitted once consistency has been demonstrated on a sufficient number of 6

lots agreed with NRA or evaluate via process validation. 7 8

A.4.1.3.12 Modified polysaccharide preparations 9

10

Several registered and candidate polysaccharide-conjugate vaccines use modified 11

polysaccharides chains. Subsequent modification or truncation of Vi may be considered for use 12

if adequately characterized. 13

14

A.4.1.3.12.1 Chemical modification 15

16

Several methods for the chemical modification of polysaccharides prior to conjugation have been 17

found to be satisfactory. The chosen method should be approved by the NRA. As part of the in-18

process controls, the processed polysaccharide to be used in the conjugation reaction may be 19

assessed for the number of functional groups introduced. 20

21

A.4.1.3.12.2 Molecular size/mass distribution 22

23

The degree of size reduction of the polysaccharide will depend upon the manufacturing process. 24

The average size/mass distribution (degree of polymerization) of the processed polysaccharide 25

should be measured by a suitable method. The size/mass distribution should be specified for each 26

type of conjugate vaccine with appropriate limits for consistency, as the size may affect the 27

reproducibility of the conjugation process. 28

29

A.4.2 Control of carrier protein production 30

31

A.4.2.1 Consistency of microbial growth for carrier protein 32

33

Consistency of growth of the microorganisms used should be demonstrated using methods such 34

as, but not limited to, pH and final yield of appropriate protein(s). 35 36

A.4.2.2 Characterization and purity of carrier protein 37

38 Proteins that have been used as carriers in conjugate vaccines include licensed carrier proteins 39

such as tetanus toxoid (TT), diphtheria toxoid (DT) and CRM197 but could also include other 40

approved proteins. Manufacturers have a choice of carrier proteins to use for conjugation 41

provided the vaccine is safe and immunogenic. 42

43

WHO/BS/2013. 2215

The test methods used to characterize such proteins, to ensure that they are non-toxic, and to 1

determine their purity and concentration should be agreed by the NRA. 2

3

TT and DT should be of high purity and satisfy the relevant recommendations published by 4

WHO (97, 98). The purity should be at least 1,500 Lf/mg protein nitrogen (Lf = limit of 5

flocculation) (97, 98). 6

7

Either classical CRM197 or recombinant CRM197 produced by genetically modified micro-8

organisms may be used. CRM197 with a purity not less than 90% as determined by high-9

performance liquid chromatography (HPLC) should be prepared by column chromatographic 10

methods. A higher level of purity may be required if this is specified for carrier proteins already 11

in use. The content of residual host DNA should be performed and should be within the limits 12

approved for the particular product by the NRA. When produced in the same facility as DT, 13

methods should be in place to distinguish the CRM197 protein from the active toxin. 14

15

The identity of the carrier protein should be determined serologically and characterized by a 16

combination of the following physicochemical methods such as sodium dodecyl sulfate-17

polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, HPLC, amino acid 18

analysis, amino acid sequencing, circular dichroism, fluorescence spectroscopy, peptide 19

mapping, and mass spectrometry as appropriate (99). The outcome should be consistent with the 20

reference material. 21 22

A.4.2.3 Degree of activation of modified carrier protein 23

24

Adipic acid dihydrazide (ADH) or other appropriate linkers such as N-Succinimidyl 3-(2-25

pyridyldithio)-propionate (SPDP) can be used to modify the carrier protein. The level of protein 26

modification should be monitored, quantified and consistent. An in-process control may be 27

required. Reproducibility of the method for modification should be validated. 28

29

The level of modification of the carrier protein by ADH can be assessed by determining the 30

amount of hydrazide (AH) by a colorimetric reactions with 2,4,6-trinitrobenzenesulfonic acid 31

with ADH as a standard (100-102). Fluorescent tagging followed by HPLC or quadrupole time 32

of flight mass spectrometry may also be suitable methods. 33

34

A.4.3 Conjugation and purification of conjugate 35

36

A number of methods of conjugation are currently in use; all involve multi-step processes (93, 37

100-102). Both the method and the control procedures used to ensure the reproducibility, 38

stability and safety of the conjugate should be established prior to demonstrating the 39

immunogenicity of the Vi polysaccharide conjugate vaccine in clinical trials. The derivatization 40

and conjugation process should be monitored by analysis for unique reaction products or by 41

other suitable means. Residual unreacted functional groups or their derivatives are potentially 42

capable of reacting in vivo and may be present following the conjugation process. The 43

manufacturing process should be validated and the limits for unreacted activated functional 44

groups (known to be clinically relevant) at the conclusion of the conjugation process should be 45

agreed by the NRA. 46

47

After the conjugate has been purified, the tests described below should be performed in order to 48

assess consistency of manufacture. The tests are critical for assuring lot-to-lot consistency. 49

50

WHO/BS/2013. 2215

A.4.4 Control of purified bulk conjugate 1

2

Tests for releasing purified bulk conjugate should be validated. 3

4

A.4.4.1 Identity 5

6

A suitable immunoassay should be performed on the purified bulk conjugate to verify its 7

identity. 8

9

Depending on the buffer used, NMR spectroscopy may be used to confirm the identity and 10

integrity of the polysaccharide in the purified bulk conjugate (95, 103-105). 11 12

A.4.4.2 Endotoxin/pyrogen content 13

14

The endotoxin/pyrogen content of the purified bulk conjugate should be determined, and shown 15

to be within limits agreed as being acceptable by the NRA. 16 17

A.4.4.3 O-acetyl content 18

19

The O-acetyl content of the purified bulk conjugate should be determined by NMR or by other 20

appropriate methods. The O-acetyl content of the purified bulk conjugate should be agreed by 21

the NRA. 22 23

A.4.4.4 Residual reagents 24

25

The conjugate purification procedures should remove residual reagents used for conjugation and 26

capping. The removal of reagents, their derivatives and reaction by-products such as ADH, 27

phenol and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC also known as EDAC or 28

EDCI) should be confirmed by suitable tests or by validation of the purification process. 29 30 Process specifications and quantifiable methods should be agreed upon in consultation with the 31

NRA. 32

33

The process should also demonstrate no significant covalent modification of the Vi itself (e.g. < 34

5% of Vi monosaccharides modified). For example, commonly used conjugation procedures use 35

EDC and a frequent side reaction can result in Vi carboxylates being covalently modified to form 36

an N-acyl urea. Such modification may alter the structure of the Vi and importantly is known to 37

be immunogenic leading to antibodies that cross react with other EDC modified polysaccharides 38

such as those in Hib, pneumococcal and meningococcal conjugate vaccines, and thus may 39

interfere with these vaccines. N-acyl urea content can be readily measured using NMR. 40

41

A.4.4.5 Polysaccharide content 42

43

The content of Vi polysaccharide should be determined by means of an appropriate validated 44

assay. 45

46

WHO/BS/2013. 2215

As an example, methods that have been used for the determination of the Vi polysaccharide 1

content include the colorimetric assay with acridine orange or HPAEC-PAD (93), with the latter 2

method having superior reproducibility. 3

4

A.4.4.6 Conjugated and unbound (free) polysaccharide 5

6

A limit for the presence of unbound (free) Vi polysaccharide relative to total Vi polsaccharide 7

should be set for each purified bulk conjugate as agreed by the NRA. The upper limit is specific 8

for the polysaccharide conjugate formulation and the limit should not be exceeded during the 9

shelf-life of the batch. Methods that have been used to assay unbound polysaccharide include gel 10

filtration; ultrafiltration and hydrophobic chromatography; ultracentrifugation followed by 11

HPAEC-PAD, or colorimetric detection (90, 101); other suitable methods can be developed and 12

validated. 13 14

A.4.4.7 Protein content 15

16

The protein content of the purified bulk conjugate should be determined with an appropriate 17

validated assay. Each batch should be tested for conjugated and unbound protein. The 18

unconjugated protein content of the purified bulk conjugate should comply with the limit for the 19

particular product as approved by the NRA. 20

21

Appropriate methods for the determination of unbound protein include HPLC or capillary 22

electrophoresis. 23 24

A.4.4.8 Conjugation markers 25

26

The success of the conjugation process can be assessed by characterisation of the conjugate by 27

suitable methods. For example an increase in MW of the protein component of the conjugate 28

compared to the carrier protein should be determined by coomassie blue stain of an SDS-PAGE 29

gel and an increase in MW of the conjugate compared to both the Vi polysaccharide and protein 30

components should be evidenced by the gel filtration profile. The conjugate should retain the 31

antigenicity for both Vi and the carrier protein as demonstrated by dot blot or Western blot. 32

33

Where the chemistry of the conjugation reaction results in the creation of a unique linkage 34

marker such as a unique amino acid, the validation batch should be assessed to quantify the 35

extent of covalent reaction of the Vi polysaccharide with the carrier protein, so that the 36

frequency of the covalent bond is given as a function of the number of polysaccharide repeating 37

units or overall polysaccharide content. 38

39

A unique linkage marker could be assessed for the validation batch or, alternatively, the 40

manufacturing process should be validated to demonstrate that it yields conjugate with a level of 41

substitution that is consistent from batch to batch. 42

43

A.4.4.9 Absence of reactive functional groups 44

45

The validation batch should be shown to be free of reactive functional groups or their derivatives 46

suspected to be clinically relevant on either the polysaccharide or on the carrier protein suspected 47

to be clinically relevant. 48

WHO/BS/2013. 2215

1

Where possible, reactive functional groups, for example, derived by ADH treatment should be 2

assessed for each batch. Alternatively, the product of the capping reaction can be monitored or 3

the capping reaction can be validated to show removal of reactive functional groups. 4

5

A.4.4.10 Polysaccharide to carrier protein ratio 6

7

The polysaccharide to carrier protein ratio of the purified bulk conjugate should be calculated. 8

The content of each of the conjugate components prior to their use should be known for this ratio 9

to be a suitable marker for conjugation. For each purified bulk conjugate, the ratio should be 10

within the range approved for that particular conjugate by the NRA and should be consistent 11

with the ratio in vaccine that have been shown to be effective in clinical trials. 12

13

A.4.4.11 Molecular size/mass distribution 14

15

The molecular size/mass of the polysaccharide–protein conjugate is an important parameter to 16

establish consistency of production, for product homogeneity, and stability during storage. 17

18

The relative molecular size of the polysaccharide–protein conjugate should be determined for 19

each purified bulk conjugate, using a gel matrix appropriate to the size of the conjugate (106). 20

The method should be validated with an emphasis on its specificity to distinguish the 21

polysaccharide–protein conjugate from other components that may be present (for instance, 22

unbound protein or polysaccharide). The molecular size/mass distribution specification should be 23

vaccine-specific and consistent with that of lots shown to be immunogenic in clinical trials. 24

25

Typically the size of the polysaccharide–protein conjugate may be examined by methods such as 26

gel filtration for example HPSEC on an appropriate column. Since the polysaccharide to protein 27

ratio is an average value, characterization of this ratio over the molecular size/mass distribution 28

(for instance, by dual monitoring of the column eluent) can be used to provide further proof of 29

manufacturing consistency (99, 107). 30

31

A.4.4.12 Bacterial and mycotic bioburden 32

33

The purified bulk conjugate should be tested for bacterial and mycotic bioburden in accordance 34

with the requirements of Part A, sections 5.1 and 5.2, of the revised Requirements for Biological 35

Substances (108, 109), or by a method approved by the NRA. If a preservative has been added to 36

the product, appropriate measures should be taken to prevent it from interfering with the test. 37 38

A.4.4.13 Specific toxicity of carrier protein 39

40

The bulk conjugate should be tested for the absence of specific toxicity of the carrier protein 41

where appropriate (for instance, when TT or DT have been used). 42

43

A.4.4.14 pH 44

45

WHO/BS/2013. 2215

If the purified bulk conjugate is a liquid preparation, the pH of each batch should be tested and 1

shown to be within the range of values shown to be safe in the clinical trials and in stability 2

studies. For a lyophilized preparation, the pH should be measured after reconstitution with the 3

appropriate diluent. 4

5

A.4.4.15 Appearance 6

7

The bulk purified conjugate should be examined for appearance. It should be clear to moderately 8

turbid and colorless to pale yellow. 9

10 11

A.4.5 Preparation and control of final bulk 12

13

A.4.5.1 Preparation 14

15

The final bulk is prepared by mixing a preservative and/or stabilizer (if used) with a suitable 16

quantity of the bulk conjugate so as to meet the specifications of vaccine lots that have been 17

shown to be safe and efficacious in clinical trials. If an adjuvant is used then it should be mixed 18

with the final bulk at this stage. 19

20

A.4.5.2 Test for bacterial and mycotic sterility 21

22

Each final bulk should be tested for bacterial and mycotic sterility as indicated in section 23

A.4.4.12. If a preservative has been added to the final bulk, appropriate measures should be 24

taken to prevent it from interfering with the test. 25

26

A.4.5.3 Sterile filtration 27

28

It is preferred that the bulk conjugate be sterile filtered before final bottling. Concentration of 29

both Vi and carrier protein should be analyzed after filtration and the integrity of the covalent 30

binding should be verified in the final filtrate. 31 32

A.5 Filling and containers 33

34

The recommendations concerning filling and containers given in annex 1, section 4 of Good 35

manufacturing practices for biological products (85) should be applied. 36 37

A.6 Control of final product 38

39

A.6.1 Inspection of final containers 40

41

Each container of a final lot should be inspected visually (manually or with automatic inspection 42

systems), and those showing abnormalities such as improper sealing, lack of integrity, clumping 43

or the presence of particles should be discarded. 44 45

WHO/BS/2013. 2215

A.6.2 Control tests on final lot 1

2 Tests for releasing final lot should be validated. 3 4

A.6.2.1 Identity 5

6

Identity test on Vi polysaccharide and carrier protein should be performed on each final lot. An 7

immunological test or a physicochemical assay for the Vi polysaccharide and for the carrier 8

protein may be used. 9 10

A.6.2.2 Sterility 11

12

The contents of final containers should be tested for bacterial and mycotic sterility in accordance 13

with the requirements of Part A, sections 5.1 and 5.2, of the revised Requirements for Biological 14

Substances (108, 109), or by a method approved by the NRA. If a preservative has been added, 15

appropriate measures should be taken to prevent it from interfering with the sterility test. 16 17

A.6.2.3 Polysaccharide content 18

19

The amount of Vi polysaccharide conjugate in the final containers should be determined and 20

shown to be within the limits agreed by the NRA. 21

22

The conjugate vaccines produced by different manufacturers may differ in formulation. A 23

quantitative assay for the Vi polysaccharide should be carried out. The specification is likely to 24

be product-specific. Colorimetric methods, chromatographic methods (including HPLC or 25

HPAEC-PAD), or immunological methods (including rate nephelometry) may be used. 26

27

If the conjugate is dried then the level of residual moisture should be established and the limit 28

should be agreed by the NRA. 29

30

A.6.2.4 Unbound (free) polysaccharide 31

32

A limit for the presence of free Vi polysaccharide should be set for each type of conjugate 33

vaccine. Assessment of the level of unconjugated polysaccharide in the final lot may be 34

technically demanding, and alternatively the molecular size of the conjugate could be determined 35

on the final lot to confirm integrity of the conjugate. An acceptable value should be consistent 36

with the value seen in clinical trial batches which showed adequate immunogenicity and be 37

approved by the NRA. 38

39

A.6.2.5 O-acetyl content 40

41

The O-acetyl content of Vi polysaccharide conjugate in the final container should be determined 42

for each final lot by NMR (89) or other appropriate methods, such as Hestrin method (92). If the 43

O-acetyl is measured at level of conjugate bulk and provided process validation data during 44

product development confirm that formulation and filling do not alter the integrity of the 45

functional groups, routine release testing of each lot for O-acetyl content in the final product can 46

be omitted following agreementby the NRA. A limit should be approved by the NRA (94). 47

WHO/BS/2013. 2215

1

A.6.2.6 Molecular size/mass distribution 2

3 The molecular size/mass molecular size of the polysaccharide conjugate should be determined 4

for each final lot, using a gel matrix appropriate to the size of the conjugate, for example 5

HPSEC-MALLS (106). The molecular size/mass distribution analysis for each final lot may be 6

omitted provided the test has been performed on the conjugate bulk level and this approach has 7

been agreed by the NRA (see section A.4.4.11). 8 9

A.6.2.7 Endotoxin/pyrogen content 10

11

The pyrogenic activity of the vaccine in final container should be tested in rabbits. The 12

endotoxin should be tested by a validated Limulus amoebocyte lysate test or a suitable in vitro 13

assay. The pyrogen content and/or the endotoxin content should be within limits agreed upon as 14

being acceptable by the NRA. 15 16

A.6.2.8 Adjuvant content 17

18

If an adjuvant has been added to the vaccine, its content should be determined by a method 19

approved by the NRA. The amount and nature of the adjuvant should also be agreed with the 20

NRA. If aluminium compounds are used as adjuvants, the amount of aluminium should not 21

exceed 1.25 mg per single human dose. 22

23

The consistency of adsorption of the antigen to the adjuvant is important and the degree of 24

adsorption should be demonstrated in production lots and should be within the range of values 25

measured in vaccine lots shown to be clinically effective. 26 27

A.6.2.9 Preservative content 28

29

If a preservative has been added to the vaccine, its content should be determined by a method 30

approved by the NRA. 31

32

The amount of preservative in the vaccine dose should be shown not to have any deleterious 33

effect on the antigen or to impair the safety of the product in humans. Preservative efficacy 34

should be demonstrated.The preservative and its concentration should be approved by the NRA. 35 36

A.6.2.10 General safety (innocuity) 37

38

The need to test final lots of Vi polysaccharide conjugate vaccine for unexpected toxicity 39

(abnormal toxicity) should be agreed with the NRA. The general safety test may not be required 40

if another animal test is performed (for instance, for immunogenicity). It can be omitted for 41

routine lot release once consistency of production has been established to the satisfaction of the 42

NRA and when reliable good manufacturing practice is in place. 43 44

A.6.2.11 pH 45

46

WHO/BS/2013. 2215

If the vaccine is a liquid preparation, the pH (preferably near 7.2) of each final lot should be 1

tested and shown to be within the range of values shown to be safe and effective for vaccine lots 2

in the clinical trials and in stability studies. For a lyophilized preparation, the pH should be 3

measured after reconstitution with the appropriate diluent. 4 5

A.6.2.12 Osmolality 6

7

The osmolality of final lots should be determined and shown to be within limits agreed upon as 8

being acceptable by the NRA. 9 10

A.6.3 Control of diluents 11

12

The recommendations given in the Good manufacturing practices for pharmaceutical products 13

(85) should apply for the manufacturing and control of diluents used to reconstitute conjugate 14

typhoid vaccines. An expiry date should be established for the diluent based upon stability data. 15

For lot release of the diluent, tests for appearance, identity, volume, sterility, and the content of 16

key components should be done. 17

18

A.7 Records 19

20 The recommendations of the Good manufacturing practices for biological products (85) should 21

apply, as appropriate for the level of development of the candidate vaccine. 22

23

A.8 Samples 24

25

A sufficient number of product lot samples should be retained for future studies and needs. 26

Vaccine lots that are to be used for clinical trials may serve as reference materials in the future, 27

and a sufficient number of vials should be reserved and appropriately stored for that purpose. 28

29

A.9 Labelling 30

31

The recommendations in the Good manufacturing practices for biological products (85) 32

appropriate for a candidate vaccine should be applied with the addition of the following. 33

34

The label on the carton enclosing one or more final containers, or the leaflet accompanying the 35

container, should indicate: 36

37

a statement that the candidate vaccine fulfills Part A of these guidelines; 38

that if the vaccine is a dried powder, it should be used immediately after its reconstitution 39

unless data have been provided to the licensing authority to indicate that it may be stored 40

for a limited time; and 41

the volume and nature of the diluent to be added in order to reconstitute a dried powder 42

vaccine, specifying that the diluent should be supplied by the manufacturer and approved 43

by the NRA. 44

45

WHO/BS/2013. 2215

A.10 Distribution and shipping 1

2

The recommendations given in the Good manufacturing practices for biological products (85) 3

appropriate for a candidate vaccine should apply. 4

5

Shipments should be maintained within specified temperature ranges and packages should 6

contain cold-chain monitors (110). 7

8

A.11 Stability, storage and expiry date 9

10

The recommendations given in the Good manufacturing practices for biological products (85) 11

and the Guidelines on stability evaluation of vaccines (111) appropriate for a candidate vaccine 12

should apply. The statements concerning storage temperature and expiry date that appear on the 13

primary or secondary packaging should be based on experimental evidence and should be 14

submitted for approval to the NRA. 15

16

A.11.1 Stability testing 17

18

Stability testing should be performed at different stages of production, namely on stored 19

intermediates (such as purified polysaccharide, carrier protein and purified bulk conjugate) and 20

final product. Stability-indicating parameters should be defined or selected appropriately 21

according to the stage of production. A stability protocol should be established for intermediates 22

and final product and include release assays as agreed upon with the NRA. It is advisable to 23

assign a shelf-life to all in-process materials during vaccine production, in particular stored 24

intermediates. 25

26

The stability of the vaccine in its final container and at the recommended storage temperatures 27

should be demonstrated to the satisfaction of the NRAs on at least three lots of final product 28

manufactured from different independent bulk conjugates. 29

30

In addition, a real-time real-condition stability study should be conducted on at least one final 31

container lot produced per year. 32

33

The formulation of vaccine and adjuvant (if used) should be stable throughout its shelf-life. 34

Acceptable limits for stability should be agreed with the NRA. 35

36

The polysaccharide component of conjugate vaccines may be subject to gradual hydrolysis at a 37

rate which may vary depending upon the type of conjugate, the type of formulation or adjuvant, 38

the types of excipient and conditions of storage. The hydrolysis may result in reduced molecular 39

size of the Vi polysaccharide component, a reduction in O-acetyl content, a reduction in the 40

amount of the polysaccharide bound to the carrier protein and/or in a reduced molecular size of 41

the conjugate. 42

43

The O-acetyl content should be a monitored quantity for stability and release testing 44

The quantity of free protein should be monitored for stability and release testing. The molecular 45

size/mass distribution should be monitored for stability and release testing. 46

If applicable, the residual moisture should be monitored for stability and release testing. 47

Tests should be conducted before licensing to determine the extent to which the stability of the 48

product has been maintained throughout the proposed validity period. The free saccharide 49

WHO/BS/2013. 2215

content as a percentage of the total saccharide should be determined and should meet 1

recommendations for final product as established by manufacture as defined in section A.6.2 2

until the expiry date. 3

4

The level of adsorption of conjugate to adjuvant, where applicable, should be shown to be within 5

limits agreed by the NRA, unless data are available to show that the immunogenicity of the final 6

product is not dependent upon adsorption of the antigen to the adjuvant. 7

8

Accelerated stability studies may provide additional supporting evidence of the stability of the 9

product and/or consistency of manufacture but are not recommended for establishing a shelf-life 10

of the vaccine in a defined storage condition. 11

12

When any changes are made in the production process that may affect the stability of the 13

product, the vaccine produced by the new method should be shown to be stable. 14

15

If the manufacturers consider incorporating a vaccine vial monitor (VVM) into the label, they 16

should provide appropriate study data to justify correlation between the stability kinetics of the 17

vaccine and the selected VVM (112). 18

19

A.11.2 Storage conditions 20

21

Before being distributed by the manufacturing establishment or before being issued from a 22

storage site, the vaccine should be stored at a temperature shown by the manufacturer to be 23

compatible with a minimal loss of titer. The maximum duration of storage and storage conditions 24

should be based on stability studies and should be fixed with the approval of the NRA and 25

should be such as to ensure that all quality specifications for final product including the 26

minimum titre specified on the label of the container (or package) will still be maintained until 27

the end of the shelf-life. 28

29

A.11.3 Expiry date 30

31

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

with the approval of the NRA. The expiry dates for the vaccine and the diluent may be different. 33

34

A.11.4 Expiry of reconstituted vaccine (if applicable) 35

36

For single dose containers, the reconstituted vaccine should be used immediately. For multi-dose 37

containers, the container should be kept in the dark at 2–8 ºC unless absence of such requirement 38

is evidenced by photostability study and the expiry time for use of an opened container should be 39

defined by stability studies, approved by the NRA, but not more than 6 hours. 40

WHO/BS/2013. 2215

Part B. Nonclinical evaluation of new typhoid conjugate vaccines 1

2

B.1 General Principles 3

4

Detailed WHO guidelines on the design, conduct, analysis and evaluation of nonclinical studies 5

of vaccines are available separately (113) and they should be read in connection with the 6

following Part B of these guidelines. Specific issues to be considered in relation to candidate Vi 7

conjugate vaccines are considered in section B3. The plan for nonclinical studies conducted 8

during development of the vaccine should be discussed with the NRAs early in the review 9

process. 10

11

B.2 Product characterization and process development 12

13

It is critical that the vaccine production processes are appropriately standardized and controlled 14

to ensure consistency of manufacture in support of nonclinical data suggesting potential safety 15

and efficacy in humans. 16

17

Candidate Vi conjugate vaccine formulation(s) should be characterized to define the critical 18

structural and chemical attributes that indicate the polysaccharide, conjugating protein, and the 19

conjugate product are sufficiently pure and stable and their properties consistent. The extent of 20

product characterization may vary depending on the stage of development. Vaccine lots used in 21

nonclinical studies should be adequately representative of those intended for clinical 22

investigation and ideally should be the same lots as those used in the clinical studies. 23 24

B.3 Nonclinical immunogenicity and animal challenge studies 25

26

Immunization studies in animal models should be conducted because they provide valuable 27

“proof of concept” information to support a clinical development plan. In addition, 28

immunogenicity data derived from appropriate animal models are useful in establishing the 29

immunological characteristics of the Vi polysaccharide conjugate product and may guide 30

selection of the doses, schedules and routes of administration to be evaluated in clinical trials. To 31

ensure the immunogenicity in nonclinical testing — weaning mice (< 6 weeks old) should be 32

used to receive 2 injections (2 weeks apart) intramuscularly, using Vi as the control group. The 33

anti-Vi IgG should then be measured. The conjugate should show at least 4 times higher 34

response than Vi and a booster response after second dose (100). Immunogenicity studies of Vi 35

polysaccharide conjugates have been conducted in mice (71, 93, 114-116) and correlation has 36

been made in humans between the level of anti-Vi IgG and protection against clinical disease 37

(53, 117). Therefore, the primary endpoint for nonclinical immunogenicity studies of Vi 38

conjugate vaccine should be the level of anti-Vi elicited. 39

40

Such studies may include an evaluation of seroconversion rates and/or geometric mean antibody 41

titres. Nonclinical studies may, where possible, be designed to assess relevant immune 42

responses, including functional immune response (for instance, serum bactericidal antibodies, 43

opsonophagocytic activity, and serum-dependent opsonophagocytic killing). These studies may 44

also be designed to address interference between antigens in the case of multi-antigen vaccine 45

formats. The response to each antigen should be evaluated. 46

47

WHO/BS/2013. 2215

Although there have been advances (see section General considerations), no ideal animal model 1

currently exists that establishes direct serological or immunological correlates of clinical 2

protection. In the absence of such a model, the difficulty is to ensure that the production batches 3

have the same protective efficacy as those shown to be protective in clinical trials. Therefore, 4

emphasis is increasingly placed on assuring consistency in manufacture through the use of 5

modern physical, chemical and immunological methods. 6 7

B.4 Nonclinical toxicity and safety 8

9

WHO nonclinical guidelines (118) should apply to nonclinical toxicity and safety assessment of 10

vaccines. Toxicity studies for Vi polysaccharide conjugate typhoid vaccines may be performed 11

in an appropriate animal model. Such studies should entail the careful analysis of all major 12

organs, as well as tissues near to and distal from the site of administration, to detect 13

unanticipated direct toxic effects over a wide range of doses, including those sufficiently 14

exceeding the intended clinically relevant dose or amount. If any novel proteins are used to 15

manufacture conjugate vaccines, toxicity studies should be performed on these proteins first. 16

Nonclinical safety studies should be done in accordance with good laboratory practices as 17

described elsewhere (119, 120). 18

19

20

WHO/BS/2013. 2215

Part C. Clinical evaluation of new typhoid conjugate vaccines 1

2

C.1 General principles 3

4

C.1.1 General considerations for clinical studies 5

6

In general, clinical trials should adhere to the principles described in the WHO guidelines on 7

good clinical practice (121). 8

9

General principles described in the WHO guideline on regulatory expectations for clinical 10

evaluation of vaccines apply to Vi polysaccharide conjugate vaccines and should be followed 11

(122). Some of the issues that are specific to conjugate vaccines and/or particularly to the clinical 12

development program for Vi conjugate vaccines are discussed in the following sections and 13

should be read in conjunction with the general guidance mentioned above. 14

15

In particular, the methodological and statistical considerations described in sections B2 and B3 16

of the WHO guideline on regulatory expectations for clinical evaluation of vaccines (122) should 17

be taken into account. 18

19

The suggestions for clinical development programs that are provided in this section should be 20

viewed in the light of further data on the safety, immunogenicity, efficacy and effectiveness of 21

Vi conjugate vaccines that may emerge, including insight into immunological correlate(s) of 22

protection. This section recognizes that the content of the clinical program is expected to change 23

once licensed Vi polysaccharide conjugate vaccines become widely available for use in various 24

age groups. 25

26

C.1.2 Outline of the clinical development program 27

28 In accordance with WHO clinical guidelines (122), the early clinical development program 29

should serve to identify an appropriate dose of conjugated Vi antigen and suitable immunization 30

schedule(s) for the target age group(s). These initial studies should also provide a preliminary 31

assessment of vaccine safety. Adequate dose and regimen-finding studies are necessary for each 32

candidate Vi conjugate vaccine that is developed since it is not possible to extrapolate the 33

antigen content and schedule identified for one conjugate vaccine to another. This consideration 34

applies even if the carrier protein is the same between two Vi conjugate vaccines since 35

experience with other conjugated polysaccharide vaccines has indicated that differences in the 36

conjugation chemistry can affect immunogenicity. 37

38

It is recommended that the major part of the pre-licensure clinical development program is 39

conducted in subjects who are representative of the intended target population. 40

41

The minimum acceptable content of the pre-licensure clinical program for each candidate 42

conjugate vaccine and the expectations for data to be generated in the post-licensure period 43

should be discussed between sponsors and the relevant NRAs. Factors expected to have an 44

important influence on the pre-licensure program include the intended target age range and the 45

availability of licensed unconjugated and/or conjugated Vi vaccines in each age group. 46

47

WHO/BS/2013. 2215

Although data on antibody persistence and responses to booster doses are considered to be very 1

important, the collection and submission of much of these data would usually occur at various 2

times in the post-approval period. Therefore sponsors and NRAs should agree the minimum 3

duration of follow-up that would be required before submitting an initial application dossier. It is 4

highly recommended that at least 2 post immunization serum antibody level be reported and 5

compared with the control group receiving the unconjugated Vi polysaccharide to establish 6

superiority and persistence of protection of Vi conjugate vaccine. 7

8

C.1.3 Evidence to support efficacy 9

10

Subjects aged at least 2 years 11

12

Protective efficacy studies against typhoid can only be conducted in endemic areas with 13

relatively high rates of disease. In endemic areas a prospective comparison of a Vi conjugate 14

with an unvaccinated control group in subjects aged from 2 years upwards is not considered to 15

be appropriate because there are licensed vaccines that have documented efficacy against 16

typhoid in certain age sub-groups. A relative protective efficacy study (e.g. comparison of a 17

candidate Vi conjugate vaccine with an unconjugated Vi vaccine) is not likely to be feasible due 18

to the study size that would be required to derive robust statistical conclusions. 19

20

Taking these issues into account, as well as the evidence supporting the role of anti-Vi IgG 21

antibody in protection against Vi-expressing S. Typhi, it is not considered necessary to estimate 22

the protective efficacy of candidate Vi conjugate vaccines against typhoid in subjects aged at 23

least 2 years. In this age group the pre-licensure assessment of the likely protective efficacy of 24

conjugated Vi vaccines could be based on appropriate comparative immunogenicity studies (see 25

section C.3). 26

27

Nevertheless, successful human typhoid challenge studies in healthy adults using an appropriate 28

and validated model (i.e. in which some protective efficacy of unconjugated Vi vaccines is 29

detectable) could provide considerable supportive evidence of the efficacy of a Vi conjugate 30

vaccine. Human challenge studies could also provide at least limited information on the 31

relationship between the immune response and various efficacy parameters. If, in consultation 32

with NRAs, sponsors decide to conduct human typhoid challenge studies it is recommended that 33

they should be undertaken only by physicians with appropriate expertise and in a carefully 34

controlled setting to ensure the safety of volunteers. Healthy adults should be pre-screened to 35

detect underlying pre-existing conditions and to exclude risk factors for complications (including 36

gall bladder disease). The challenge strain should be well characterized with full information on 37

its susceptibility to antibacterial agents. 38

39

Subjects aged < 2 years 40

41

Currently, there is no information on the protective efficacy or effectiveness against typhoid of 42

any Vi conjugate vaccine in children aged < 2 years when first vaccinated. Therefore there is a 43

need to carefully consider the potential value and the feasibility of conducting a prospective 44

randomized protective efficacy study in a region with documented background rates of proven 45

typhoid disease in this age group. Whether such a study is required or whether it can be replaced 46

by an appropriate assessment of immunogenicity followed by post-approval effectiveness studies 47

can only be decided on a case by case basis following discussions between sponsors and NRAs. 48

WHO/BS/2013. 2215

Since the conduct of a pre-licensure protective efficacy study would be expected to prolong the 1

time to approval, the decision regarding the requirement for such a study should take into factors 2

such as the regional burden of typhoid disease in this age group. 3

4

If a pre-licensure protective efficacy study is conducted, it should compare rates of febrile 5

illnesses associated with a positive blood culture for S. Typhi between a group that receives the 6

candidate Vi conjugate vaccine and an appropriate control group. A double-blind design is 7

recommended but this would require that the control group is randomized to a non-typhoid 8

vaccine from which they may derive some benefit that is indistinguishable in appearance from 9

the candidate conjugate vaccine and is administered in the same way (i.e. route and schedule). If 10

a suitable non-typhoid vaccine cannot be identified then the control subjects could be 11

unvaccinated (i.e. avoiding the use of a placebo injection). In this case a double-blind design 12

would not be possible but it would be important to make every effort to ensure that investigators 13

are unaware of the treatment assignment. 14

15

Further information on the design and conduct of protective efficacy studies and the assessment 16

of vaccine effectiveness is provided in the WHO guidelines on clinical evaluation of vaccines: 17

regulatory expectations (122). 18

19

Vaccine effectiveness 20

21

Whether or not a pre-licensure study of protective efficacy against typhoid is performed, it is 22

recommended that efforts are made to estimate vaccine effectiveness in the post-licensure period. 23

See section C.5. 24

25

C.2 Assessment of the immune response 26

27

C.2.1 Anti-Vi total IgG in sera 28

29

It is usually preferred that the primary parameter for assessing the humoral immune response to a 30

vaccine is based on a measure of functional antibody. However, there are no well-established or 31

standardised assays for assessing functional antibody responses to Vi-containing vaccines and it 32

is not known how the results of such assays correlate with vaccine efficacy. A correlation 33

between total serum antibody (59) or total anti-Vi IgG in sera (61, 65, 67, 68, 123) and 34

protection against typhoid has been described although there is no well-established cut-off value 35

that clearly predicts prevention of clinical disease. On this basis, it could be acceptable to base 36

the primary assessment of the immunogenicity of candidate Vi conjugate vaccines on total anti-37

Vi IgG levels. 38

39

In recent years, the assessment of immune response to licensed unconjugated Vi vaccines has 40

predominantly been based on measurement of total anti-Vi IgG in sera determined by an 41

enzyme-linked immunosorbent assay (ELISA) (62, 66, 124). Older assays such as RIA (53) and 42

passive haemagglutination (55) are now rarely used (57). However, several ELISAs have been 43

used in various studies with different vaccines (69, 125). At the time of preparing these 44

guidelines, there is no international standard available. However, reagents and a software 45

analysis tool for Vi antibody ELISA assay are available from the US FDA and US CDC free of 46

charge and contact information listed in below: 47

48

WHO/BS/2013. 2215

Table 1. Contact details for materials for Vi antibody ELISA assays 1

2

It is essential that the assays used to report data from clinical studies that are considered to be 3

pivotal for an application dossier should be fully validated. Once an international standard 4

becomes available all sponsors should use this standard to calibrate the assays they are using to 5

determine anti-Vi IgG concentrations. 6

7

C.2.2 Other immune response parameters 8

9

As part of the overall characterisation of the immune response to candidate Vi conjugate 10

vaccines sponsors may consider evaluation of one or more of the following at least in subsets of 11

sera obtained from different age groups and at different time points: 12

13

Serum bactericidal antibody (SBA) 14

Opsonophagocytic antibody (OPA) 15

Antibody avidity - following an initial T-cell-dependent immune response in Vi antigen-16

naïve individuals it would be expected that antibody avidity should increase over time 17

and should also demonstrate differences between post-primary and post-booster doses. 18

IgG sub-class responses 19

Evidence of T-cell-dependent immune response with memory B-cell recruitment (for 20

instance, an anamestic response to a booster dose of vaccine or detection of memory B 21

cells using an in-vitro cultured enzyme-linked immunosorbent spot assay (ELISPOT) 22

23

C.2.3 Characterisation of the immune response 24

25

Antibody kinetics 26

27

The anti-Vi antibody kinetic should be assessed in recipients of the candidate Vi conjugate 28

vaccine group and in subjects who receive any control Vi-containing vaccine (licensed 29

unconjugated or conjugated) after the primary series and following booster doses. 30

31

Following the primary series (which may consist of one or several doses) sera may be collected 32

at approximately Day 14 and 28, 6 months and then as a minimum at one year and three years. 33

Following a booster dose a rapid rise in anti-Vi would be expected if there has been efficient 34

priming of the immune system. Therefore it is suggested that sera should be obtained at 35

approximately 6, 10 and 28 days post-dose and then at pre-planned intervals. 36

Name Provider address E-mail/web site

S. typhi anti-Vi

(Human) &

S. typhi Vi

polysaccharide,

lot 05

Dr. Willie Vann, Center for Biologics

Evaluation and Research, US Food &

Drug Administration, 10903 New

Hampshire Avenue, Silver Spring, MD

20903 USA

[email protected]

http://www.fda.gov/Biologics

BloodVaccines/ScienceResea

rch/BiologicsResearchAreas/

default.htm

ELISA

Calculation

program

Mr. Brian Plikaytis, Center for Disease

Control and Prevention, Atlanta, GA

USA

http://www.cdc.gov/ncidod/d

bmd/bimb/elisa.htm

mailto:[email protected]

http://www.cdc.gov/ncidod/dbmd/bimb/elisa.htm

http://www.cdc.gov/ncidod/dbmd/bimb/elisa.htm

WHO/BS/2013. 2215

1

To reduce the number of samplings per subject, groups could be sub-randomised to be bled at 2

different time points. It is suggested that all subjects should at least provide pre- and Day 28 3

post-vaccination samples. Longer-term assessments of post-primary and post-boost levels should 4

be planned at least in subgroups of vaccinated subjects. 5 6

Immune memory 7

8

Due to concerns that vaccination with unconjugated Vi polysaccharide can lead to 9

hyporesponsiveness to sequential doses and may potentially blunt the immune response to a 10

conjugated Vi vaccine, unconjugated Vi polysaccharide should not be administered to subjects 11

primed with a candidate Vi conjugate vaccine in order to demonstrate that the initial dose(s) of 12

the conjugate elicited a T-cell-dependent immune response. 13

14

Elicitation of a T-cell-dependent initial immune response by the initial dose(s) can be assessed 15

by administering a further dose of the Vi conjugate vaccine after and interval of approximately 16

6-12 months has elapsed. The immune response observed (ideally by measuring not only anti-Vi 17

IgG but also functional antibody, antibody avidity and using ELISPOT) following this single 18

dose of Vi conjugate to subjects who completed a primary series with the same vaccine can be 19

compared with the response to a first dose in previously unvaccinated subjects of the same age. 20

The immune response to a single dose of the Vi conjugate vaccine in primed subjects should be 21

superior to that in Vi-naïve subjects. See further in section C.3.4 regarding the administration of 22

booster doses, including the administration of Vi conjugate vaccine to subjects who previously 23

received conjugated or unconjugated Vi vaccines. 24

25

C.2.4 Analyses of immune responses 26

27

Although elicitation of anti-Vi IgG by vaccination has been shown to correlate with protection, 28

the minimum concentration of anti-Vi IgG required for protection against typhoid remains 29

uncertain (61, 65, 67, 68, 123). A conservative estimation from an efficacy trial in pre-school 30

children showed 4.2 ug/ml can give >90% protection or a response. 31

32

It is recommended that the assessment of anti-Vi IgG concentrations should take into account all 33

of the following: 34

35

Proportions of vaccinees who achieve levels above one or more pre-defined threshold 36

concentrations. Analyses of protective efficacy observed over time in a prospective 37

randomized and placebo-controlled study with one Vi conjugate vaccine in children aged 38

2-5 years when first vaccinated have suggested a benchmark (or “threshold” value) that 39

could be applied to the interpretation of anti-Vi IgG concentrations (65, 67, 68, 83, 123). 40

Based on the assay recently applied by these workers to stored sera a threshold value of 41

4.3 µg/ml appeared to be associated with a high level of sustained protection. 42

43

In order to compare and to predict the efficacy of the newly manufacture Vi conjugate 44

with those studied in the efficacy, the anti-Vi IgG level at the period of bleeding should 45

be compared and used as a surrogate for efficacy (67). An example is the antibody level 46

at 28 days and 6 months after injection should be about 87 µg/ml and 28 µg/ml calibrated 47

with the US reference standard (65, 67). 48

49

WHO/BS/2013. 2215

Until such time as an international standard becomes available, sponsors who wish to 1

apply this threshold value to the results of their own assays would need to perform a 2

calibration against the assay used in the abovementioned studies of efficacy. . 3

4

Seroconversion rates. For example, seroconversion may be defined as change from 5

seronegative pre-vaccination to seropositive (e.g. based on the assay cut-off or based on 6

achieving a defined threshold value) post-vaccination or at least a 4-fold increment from 7

pre- to post-vaccination in subjects seropositive at baseline. 8

Reverse cumulative distributions (RCDs). 9

Geometric mean concentrations (GMCs). 10

11

The selection of the most appropriate immune response parameter for the primary assessment of 12

immunogenicity in any one study should take into account the population selected for 13

investigation, the anticipated pre-existing antibody concentrations that may reflect prior 14

vaccinations and/or natural exposure and whether the assessment relates to post-primary series or 15

post-boosting. Whichever parameter is selected for the pre-defined primary analysis (see section 16

C3), between-group comparisons based on the other parameters should be presented. 17

18

C.3 Clinical study designs 19

20

C.3.1 Studies that compare conjugated with unconjugated Vi vaccines 21

22

Studies that compare candidate Vi conjugate vaccines with licensed unconjugated Vi vaccines 23

can only be conducted in subjects aged at least 2 years. Data should be generated across the 24

entire age range for which a claim for use will be sought. Studies should employ stratification by 25

appropriate age sub-groups or separate studies should be conducted in different age groups. 26

27

It is recommended that these studies are randomized and double-blind. If the sponsor proposes to 28

administer more than one dose of Vi conjugate in any age sub-group there will be a need to 29

consider matching of the schedule in the unconjugated Vi vaccine control group. Sponsors 30

should identify suitable non-typhoid vaccines that could be administered to the control group in 31

order to avoid or at least to minimise the need for placebo injections. The selection of the 32

unconjugated Vi control vaccine for each study should take into account the available evidence 33

on safety and immunogenicity and should be discussed with the relevant NRA(s). 34

35

The primary comparison of immune responses could be based on: 36

37

Percentages that achieve anti-Vi IgG levels above a pre-defined threshold value (e.g. as 38

suggested in section C.2.4) 39

Seroconversion rates 40

41

measured in samples collected at Day 28 and 6 months after initial vaccination is completed (i.e. 42

after the single or after the last assigned dose of the primary series) or in samples collected at an 43

alternative time point if justified by data on the antibody kinetic. 44

45

The primary analysis should be based on demonstrating that the immune response to the Vi 46

conjugate is at least non-inferior vs. the immune response to the control vaccine. The pre-defined 47

margin of non-inferiority should be carefully justified. Protocols may also plan for a sequential 48

WHO/BS/2013. 2215

analysis to assess whether there is superiority for immune responses to the Vi conjugate vaccine 1

if the pre-defined criterion for concluding non-inferiority has been met. 2

3

C.3.2 Studies that compare vaccinated and unvaccinated groups 4

5

These studies should employ random allocation to the Vi conjugate candidate vaccine (i.e. 6

vaccinated group) or to a licensed non-typhoid vaccine from which study subjects may derive 7

some benefit (i.e. unvaccinated group). 8

9

This study design is most likely to be employed in subjects aged < 2 years. At the present time 10

there are no Vi-containing vaccines known to be efficacious in this age group, which means that 11

the immune response data cannot provide a direct bridge to vaccine efficacy. Therefore there is a 12

need to consider the options for interpretation of the anti-Vi IgG immune response to a candidate 13

Vi conjugate vaccine. 14

15

The immune response in the candidate Vi conjugate vaccine group should be superior to that in 16

the unvaccinated group. In addition, the immune response observed after the last assigned dose 17

could be compared to: 18

19

The immune response to unconjugated Vi vaccine in one or more older age groups and/or 20

The immune response to the same candidate Vi conjugate vaccine in one or more older 21

age groups. 22

23

The comparative data could be derived from subjects (e.g. in children aged 2–5 years) enrolled 24

into a randomised study of candidate Vi conjugate vaccine vs. unconjugated Vi vaccine that has 25

successfully demonstrated non-inferiority as described in section C.3.1. 26

27

The primary analysis should be based on demonstrating that the immune response to the 28

candidate Vi conjugate vaccine is at least non-inferior vs. the immune response to the control 29

vaccine in another age group. However, comparing immune responses between age groups (and 30

likely between regimens) is not straightforward. For example, seroconversion rates may be 31

impacted by pre-existing antibody and final GMCs may vary by age. Therefore it may be 32

appropriate to place more weight on comparing the proportions that achieve post-vaccination 33

anti-Vi IgG at least above a threshold value (e.g. as discussed in section C.2.4). 34

35

C.3.3 Studies that compare conjugated Vi vaccines 36

37

The availability of licensed Vi conjugate vaccines will have implications for clinical study 38

designs in all age groups. Some of the issues to take into account include: 39

40

Whether the protective efficacy of any licensed Vi conjugate vaccine has been documented in 41 a pre-licensure protective efficacy study and/or from post-approval effectiveness data. If so, 42 then comparative immunogenicity studies against such a vaccine would allow for a direct 43 bridging between anti-Vi IgG and protection. 44

Whether efficacy or effectiveness data point to a specific anti-Vi antibody concentration that 45 strongly correlates with efficacy. 46

Whether and where Vi conjugate vaccines have been introduced into routine immunization 47 programs and in which age groups. 48

WHO/BS/2013. 2215

Investigator and subject willingness to participate in studies that employ an unconjugated Vi 1 vaccine control group or an unvaccinated control group. 2

3

It is to be expected that there will be a transition over time so that the likely protective efficacy 4

of candidate Vi conjugate vaccines will be assessed in comparative immunogenicity studies vs. 5

licensed Vi conjugates. The selection of the most appropriate licensed Vi conjugate vaccines for 6

these comparative studies must be agreed between sponsors and NRAs. However, the optimal 7

candidate would be a Vi conjugate vaccine for which protective efficacy has been demonstrated. 8

If there are no licensed conjugates of documented efficacy, then the extent of the comparative 9

immunogenicity data in each age sub-group of interest for the available licensed Vi conjugate 10

vaccines should be taken into account. 11

12

The aim of these studies would be to demonstrate non-inferiority of the immune response to the 13

candidate vs. licensed Vi conjugate vaccine. If efficacy data have supported derivation of an 14

anti-Vi antibody concentration that strongly correlates with protection then the proportions that 15

achieve at least this concentration after vaccination should be compared. 16

17

C.3.4 Antibody persistence and booster doses 18

19 Longer-term assessment of antibody persistence is considered to be essential. It is suggested that at 20 the time of initial approval of a Vi conjugate vaccine there should be adequate documentation of anti-21 Vi concentrations for at least one year after administration of the initial dose(s). The collection of 22 further data on antibody persistence should be planned but, subject to agreement with NRAs, could 23 be reported at intervals after initial approval. 24 25 In studies of conjugated versus unconjugated Vi vaccines, the antibody persistence data should be 26 compared between the randomized groups. Based on antibody decay curves observed following 27 unconjugated Vi vaccines, data up to one year can indicate whether there is any difference between 28

vaccines in the initial rate of decrease in anti-Vi antibody. While there is no established 29

immunological correlate of protection, it is suggested that antibody persistence data are viewed 30

in terms of percentages of vaccinees that still have levels above a pre-defined anti-Vi IgG 31

threshold concentration. 32 33 Determining the need for and the appropriate timing of a Vi conjugate booster dose is not 34 straightforward and may differ between age groups and between populations (for instance, there may 35 be a considerable natural boosting effect in highly endemic regions). Bacteraemia can be detected 36 very shortly after oral inoculation with S. Typhi and several days before the onset of clinical 37 symptoms. This suggests that it may not be appropriate to rely on immune memory responses to 38 achieve a sufficiently rapid rise in anti-Vi antibody to protect individual subjects. In addition, 39 available data with one Vi conjugate vaccine suggest that protection against typhoid depends on 40 maintaining a certain level of anti-Vi antibody (65, 67, 68, 83, 123). 41 42 Extensive post-approval data on antibody persistence and vaccine effectiveness would be needed to 43 support decisions on the need for boosting. Nevertheless, in order to facilitate decisions regarding 44 introduction of booster doses, it is recommended that studies in all age groups should plan to 45 document the immune responses to booster doses at pre-determined intervals. Subjects could be sub-46 randomized to be boosted at different time points after the initial dose(s). As mentioned in section 47 C.2.3, by including an unvaccinated control group, these data can also be used to demonstrate that 48 the initial dose(s) elicited a T-cell-dependent immune response. 49 50 In studies of a candidate Vi conjugate vaccine versus an unconjugated Vi vaccine, it is important to 51 compare the immune responses to a sequential (booster) dose of the Vi conjugate vaccine between 52

WHO/BS/2013. 2215

groups. These data can be used to determine whether prior exposure to unconjugated Vi 1 polysaccharide may blunt the immune response to a conjugate vaccine as a result of induction of 2 hypo-responsiveness. The data may indicate whether more than one dose of the conjugate is needed 3 in these subjects, which would be important information for the introduction of Vi conjugate 4 vaccines into regions where there has been extensive use of unconjugated Vi vaccines in the past. 5 6

The assessment of immune responses to a booster dose should be based on the immediate pre-7

boost vs. post boost antibody levels. The post-dose rate of change in immune parameters as well 8

as the magnitude of the response observed should be compared between groups primed with the 9

same Vi conjugate, unprimed subjects and subjects that previously received unconjugated Vi 10

vaccine. If the Vi conjugate vaccine efficiently primed the immune system then the onset of the 11

post-boost response should be faster and the antibody levels achieved should be higher than in 12

the other groups (see section C.2.3). 13

14

C.3.5 Immune responses to and effects of the carrier protein 15

16

To date, proteins such as CRM197, DT, TT and rEPA have been used in the production of 17

various Vi conjugate vaccines. Based on experience with other types of conjugate vaccines that 18

use CRM197, DT or TT as the carrier protein, there is some potential that the immune response 19

to the conjugated antigen may be reduced in subjects with high pre-vaccination levels of tetanus 20

or diphtheria antitoxin. This phenomenon should be explored during the development of Vi 21

conjugate vaccines by analyzing post-vaccination responses according to pre-vaccination 22

antibody levels. The potential clinical significance of any effect that is observed will require 23

careful consideration. 24

25

C.3.6 Co-administration with other vaccines 26

27

Concomitant administration of some types of conjugates with other vaccines in routine use, 28

including other conjugated vaccines, may give rise to detectable immune interference (which 29

may be a depression or an enhancement of antibody levels) although the magnitude of the effect 30

observed may not necessarily be of potential clinical significance. The possible effects of co-31

administration on immune responses cannot be predicted simply from consideration of the 32

vaccine content. Therefore clinical studies are needed in which candidate Vi conjugate vaccines 33

are co-administered with other vaccines that are representative of types that, for convenience and 34

vaccine program-related reasons, are very likely to be given at the same clinic visits. These 35

studies could range from co-administration with routine vaccines in infants and toddlers in 36

endemic areas to co-administration with commonly used travelers’ vaccines in residents of non-37

endemic areas. 38

39

Co-administration studies may be conducted before and/or after initial licensure depending on 40

the perceived importance of being able to recommend co-administration with specific types of 41

vaccines to facilitate use within existing routine vaccination programs in specific age groups. 42

43

In co-administration studies the immune response to the Vi conjugate and to all other co-44

administered antigens should be evaluated. The approach to these studies is based primarily on 45

demonstrating non-inferiority of immune responses to antigens when vaccines are co-46

administered compared to each vaccine given alone, with careful justification of pre-defined 47

non-inferiority margins. 48

49

WHO/BS/2013. 2215

C.4 Pre-licensure assessment of safety 1

2

There is no evidence currently available that points to anticipation of specific safety issues for Vi 3

conjugate vaccines. At present it is only possible to recommend that the assessment of safety in 4

pre-licensure studies should follow the usual approaches to ensure comprehensive monitoring 5

and data collection. 6

7

C.5 Post-marketing studies and surveillance 8

9

The information in the application dossier is likely to be restricted to studies of safety and 10

immunogenicity that have been conducted in certain geographical areas and in populations with 11

particular demographic features. In addition, the total population evaluated for safety in pre-12

licensure clinical studies may be limited such that only those adverse events that occur at a 13

frequency of at least 1/1000 persons vaccinated can be described with any degree of confidence 14

(section B.7.4 in WHO clinical guidelines (122). Therefore, it is considered critically important 15

that well-developed plans are in place prior to licensure for the assessment of vaccine safety and 16

effectiveness during routine use in the post-approval period. In particular: 17

18

Vaccine effectiveness studies and vaccine impact studies should include a careful 19

evaluation of any herd immunity effect of Vi conjugates. It may not be possible to collect 20

vaccine-specific effectiveness data if more than one Vi conjugate is introduced 21

concurrently in the same region(s) but the overall effectiveness of a program (whether 22

routine or an outbreak intervention) that includes specific vaccines is still informative. 23

Further attempts should be made to identify an immunological correlate of protection. 24

This requires additional considerations to the usual issues surrounding the selected 25

approach to assessing effectiveness. 26

If the pre-licensure safety database is limited in size or if any particular safety issues are 27

observed (in clinical studies and/or after approval) a dedicated post-authorization safety 28

study may be necessary in addition to routine passive surveillance. 29

30

Sound and comprehensive safety and effectiveness data cannot be collected by the sponsors 31

alone. Therefore, there should be planned collaborations between sponsors and public health 32

bodies to assure adequate collection of reliable data in areas where there is routine and 33

widespread use of a Vi conjugate vaccine. Protocols should be developed before initial approval 34

and should form an element of the application dossier. These can then be refined once it is 35

known where and how an individual vaccine will actually be used. 36

37

Other issues to be addressed after initial licensure include: 38

39

Assessment of longer-term antibody levels in selected cohorts, including post-boost 40

antibody levels (see section C.3.4). 41

Safety and immunogenicity studies in populations not included in pre-licensure studies in 42

which there is good reason to expect that immune responses may differ (e.g. 43

immunosuppressed subjects, age groups not previously studied). Additional safety and 44

immunogenicity studies may also be considered if there is a good scientific rationale to 45

anticipate that the immune response to the Vi conjugate in the pre-licensure study 46

population (e.g. residents of endemic areas) may not predict that in some other 47

populations (e.g. residents of non-endemic areas traveling to endemic areas) that have not 48

been studied. 49

50

WHO/BS/2013. 2215

Assessment of the possibility that widespread usage and high immunization coverage in a 1

population where typhoid fever is endemic selects for the emergence of otherwise rare Vi-2

negative variants of S. Typhi (126-129), recognizing that such variants exist and can cause 3

typhoid fever, albeit with lower attack rates (130, 131). 4

5

All data collected should be submitted to the responsible regulatory authorities at regular 6

intervals so that any implications for the marketing authorization can be assessed and appropriate 7

actions can be taken. 8

WHO/BS/2013. 2215

Part D. Guidelines for national regulatory authorities 1

D.1 General guidelines 2

3

The general recommendations for NRAs and national control laboratories given in the 4

Guidelines for national authorities on quality assurance for biological products (132) and 5

Guidelines for independent lot release of vaccines by regulatory authorities (133) should apply. 6

7

These guidelines specify that no new biological substance should be released until consistency of 8

manufacturing and quality as demonstrated by a consistent release of batches has been 9

established. 10

11

The detailed procedures of production and control and any significant changes in them that may 12

affect quality, safety and efficacy of a Vi polysaccharide conjugate typhoid vaccine should be 13

discussed with and approved by the NRA. The NRA may obtain the product-specific working 14

reference from the manufacturer to be used for lot release until an international or national 15

standard preparation is established. 16

17

Consistency of production has been recognized as an essential component in the quality 18

assurance of vaccines. In particular, the NRA should carefully monitor production records and 19

quality control test results for clinical lots as well as a series of consecutive lots of the final bulk 20

and final product. 21

22

D.2 Official release and certification 23

24

A vaccine lot should be released only if it fulfils the national requirements and/or Part A of the 25

present guidelines. 26

27

A summary lot release protocol in Appendix 1, signed by the responsible official of the 28

manufacturing establishment, should be prepared and submitted to the NRA in support of a 29

request for release of the vaccine for use. 30

31

A certificate signed by the appropriate official of the NRA should be provided to the 32

manufacturing establishment and should certify that the lot of vaccine in question meets all 33

national requirements as well as Part A of the present guidelines. The certificate should also state 34

the lot number, the number under which the lot was released, and the number appearing on the 35

labels of the containers. In addition, the date of the last satisfactory determination of critical 36

quality parameters (such as a ratio of free and bound Vi polysaccharide concentrations) as well 37

as assigned expiry date on the basis of shelf life should be stated. A model certificate is given in 38

Appendix 2. A copy of the official national release document should be attached. The purpose of 39

the certificate is to facilitate the exchange of typhoid conjugate vaccines between countries. 40

41

Authors & acknowledgments 42

43

The first draft of these guidelines was prepared by: Dr J. Mathew, Advanced Pediatrics Centre, 44

Post Graduate Institute of Medical Education and Research, Chandigarh, India and Dr M. 45

Levine, University of Maryland School of Medicine, Baltimore, MD, USA (General 46

considerations); Dr S. Rijpkema, National Institute for Biological Standards & Control, Potters 47

Bar, Herts., UK (Part A); Dr J. Cipollo, Center for Biologics Evaluation and Research, Food and 48

WHO/BS/2013. 2215

Drug Administration, Bethesda, MD, USA (Part B); Dr M. Powell, Medicines and Healthcare 1

Regulatory Agency, London, UK (Part C); and Dr J. Shin, Department of Essential Medicines 2

and Health Products, World Health Organization, Geneva, Switzerland (consolidation of each 3

part) following two expert consultations: i) joint KFDA/WHO working group held in Jeju, 4

Republic of Korea, 5–7 September 2012; ii) WHO clinical working group for which financial 5

and technical support from Dr C. Nelson, Coalition against Typhoid (CaT) Secretariat, Sabin 6

Vaccine Institute, Washington DC, USA held in London, 7–8 January 2013; and iii) WHO 7

informal consultation held in Geneva, 29–30 April 2013. Acknowledgments are due to the 8

following experts who contributed to improving earlier versions by providing written comments 9

and proposed changes: Dr C. Nelson, Coalition against Typhoid (CaT) Secretariat, Sabin 10

Vaccine Institute, Washington DC, USA; Dr I. Feavers, National Institute for Biological 11

Standards & Control, Potters Bar, Herts, UK; Dr M. Levine, University of Maryland School of 12

Medicine, Baltimore, MD, USA; Dr A. Pollard, Department of Paediatrics, Oxford University, 13

Oxford, UK. Acknowledgments are extended to the following participants dedicated to 14

discussion at the joint KFDA/WHO working group held in Jeju, Republic of Korea, 5–7 15

September 2012: Dr C. Ahn, Biologics Research Division, National Institute of Food & Drug 16

Safety Evaluation, Osong, Republic of Korea; Dr M. Bonnet, Sanofi Pasteur, Lyon, France 17

(IFPMA Representative); Dr J. Cipollo, Center for Biologics Evaluation and Research, Food and 18

Drug Administration, Bethesda, MD, USA; Dr R. Carbis, International Vaccine Institute, SNU 19

Research Park, Seoul, Republic of Korea; Dr D. Cardoso Gonzalez, Finlay Institute, Ciudad 20

Habana, Cuba (DCVMN Representative); Dr D. Garcia, National Drug and Health Products 21

Safety Agency, Lyon, France; Dr H. Izumiya, National Institute of Infectious Diseases, Tokyo, 22

Japan; Ms W. Jaroenkunathum, Institute of Biological Products, Ministry of Public Health, 23

Nonthaburi, Thailand; Dr C. Jones, National Institute for Biological Standards and Control, 24

Potters Bar, Herts., UK (Rapporteur); Dr B.-G. Kim, National Center for Lot Release, Korea 25

Food and Drug Administration, Osong, Republic of Korea; Dr C.-K. Lee, Korea Food & Drug 26

Administration, Osong, Republic of Korea (Chair); Dr T. King, Jr., Food and Drug 27

Administration, Filinvest Corporate City, Philippines; Dr I. Knezevic, Department of Essential 28

Medicines and Health Products, World Health Organization, Geneva, Switzerland; Ms D 29

Kusmiaty, National Quality Control Laboratory of Drug and Food, National Agency of Drug and 30

Food Control, Jakarta, Indonesia; Dr M. Levine, University of Maryland School of Medicine, 31

Baltimore, MD, USA; Dr L. Martin, Novartis Vaccines Institute for Global Health, Siena, Italy; 32

Dr J. Mathew, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and 33

Research, Chandigarh, India; Dr G. Meller, Bill and Melinda Gates Foundation, Seattle, 34

Washington, USA (Observer); Dr. M. Morita, Department of Bacteriology I, National Institute 35

of Infectious Diseases, Tokyo, Japan; Dr P. Namgyal, Initiative for Vaccine Research, 36

Immunization, Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland; Dr 37

C. Nelson, Coalition against Typhoid Secretariat, Sabin Vaccine Institute, Washington DC, 38

USA; Ms N. Nurainy, Biofarma, Bandung, Indonesia (DCVMN Representative); Dr H.-J. Oh, 39

Korea Food & Drug Administration, Osong, Republic of Korea; Dr M. Paste; GSK Vaccines, 40

Wavre, Belgium (IFPMA Representative); Dr. A. Ramkishan, Central Drugs Standard Control 41

Organization, Ministry of Health and Family Welfare, New Dehli, India; Dr S. Rijpkema, 42

National Institute for Biological Standards & Control, Potters Bar, Herts., UK; Dr S. 43

Sahastrabuddhe, International Vaccine Institute, SNU Research Park, Seoul, Korea; Dr J. Shin, 44

Department of Essential Medicines and Health Products, World Health Organization, Geneva, 45

Switzerland; Dr S. Szu, National Institutes of Health, Bethesda, MD, USA; Ms J. Tresnabudi, 46

Biofarma, Bandung, Indonesia (Observer); Dr M. Zeng, National Institutes of Food and Drug 47

Control, Beijing, China; and the following senior managers and staff members from the Korea 48

Food and Drug Administration: Ms Y. Choi, Dr S.-T. Chung, Ms S.-Y. Eum,; Dr S.-H. Hong, Dr 49

S.-J. Kang, Dr S.-Y. Kang, Dr K.-H. Lee, Dr K.-T. Nam, Dr I.-S. Shin, and Dr Y. Sohn. 50

Acknowledgments are due to the following experts who attended a WHO clinical working 51

group held in London, 7-8 January 2013 for review of clinical data and agreement on key points 52

WHO/BS/2013. 2215

to be considered in clinical guidelines: Dr I. Feavers, National Institute for Biological Standards 1

and Control, Potters Bar, Hert., UK; Dr E. Griffiths, Kingston-upon-Thames, Surrey, UK 2

(Chair); Dr I. Knezevic, Department of Essential Medicines and Health Products, World Health 3

Organization, Geneva, Switzerland; Dr M. Levine, University of Maryland School of Medicine, 4

Center for Vaccine Development, Baltimore, MD, USA; Dr C. Nelson, Coalition against 5

Typhoid (CaT) Secretariat, Sabin Vaccine Institute, Washington DC, USA (Rapporteur); Dr A. 6

Pollard, Department of Paediatrics, Oxford University, Headington, Oxford, UK; Dr M. Powell, 7

Medicines and Healthcare Regulatory Agency, London, UK; and Dr S. Rijpkema, National 8

Institute for Biological Standards & Control, Potters Bar, Herts., UK. 9

10

The second draft of these guidelines was prepared taking into consideration i) written comments 11

during the first public consultation through website production of draft document and ii) 12

discussion in the process of WHO informal consultation held in Geneva, 29–30 April 2013 by: 13

Dr J. Mathew, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and 14

Research, Chandigarh, India (General considerations); Dr S. Rijpkema, National Institute for 15

Biological Standards & Control, Potters Bar, Herts., UK (Part A); Dr J. Cipollo, Center for 16

Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA (Part 17

B); Dr M. Powell, Medicines and Healthcare Regulatory Agency, London, UK (Part C); and Dr 18

J. Shin, Department of Essential Medicines and Health Products, World Health Organization, 19

Geneva, Switzerland. Acknowledgments are due to the following experts who provided written 20

comments to the first draft posted in a WHO website in March-April 2013 for public 21

consultation: Dr M. Bonnet, Sanofi Pasteur, Lyon, France; Dr P. Chagnaud, National Drug and 22

Health Products Safety Agency (ANSM), Lyon, France; Dr J. Cipollo, Centre for Biologics 23

Evaluation and Research, Bethesda, MD, USA; Dr S. Dutta, National Institute of Cholera and 24

Enteric Diseases, Beliaghata, Kolkata, West Bengal, India; Ms M.-J. Escoto-López, Center for 25

State Control on the Quality of Drugs (CECMED), Habana, Cuba; Dr E. Griffiths, Kingston-26

upon-Thames, Surrey, UK; Dr A. Goel, Biological E. Ltd., Hyderabad, India; Ms W. 27

Jaroenkunathum, Ministry of Public Health, Nonthaburi, Thailand; Dr O. Le Doledec, National 28

Drug and Health Products Safety Agency (ANSM), Lyon, France; Professor M. Levine, 29

University of Maryland School of Medicine, Baltimore, MD, USA; Dr L. Martin, Novartis 30

Vaccines Institute for Global Health, Siena, Italy; Dr A. Merkle, Paul-Ehrlich-Institut, Langen, 31

Germany; Dr K. Meunier, National Drug and Health Products Safety Agency (ANSM), Lyon, 32

France; Dr T. Mongeau, Centre for Biologics Evaluation and Research, Bethesda, MD, USA; Dr 33

S. Morgeaux, National Drug and Health Products Safety Agency (ANSM), Lyon, France; Dr T. 34

Morris, United States Pharmacopoeial Convention, Rockville, MD, USA; Dr C. Nelson, Sabin 35

Vaccine Institute, Washington DC, USA; Dr H.-J. Oh, Ministry of Food & Drug Safety, Osong, 36

Republic of Korea; Dr S. Park, Seoul Regional Office of Ministry of Food & Drug Safety, Seoul, 37

Republic of Korea; Dr M. Paste, GlaxoSmithKline Biologicals, Wavre, Belgium; Dr B. Patnaik, 38

Bharat Biotech, Hyderabad, India; Dr S. Rijpkema, National Institute for Biological Standards & 39

Control, Potters Bar, Herts., UK; Dr J. Robbins, New York, USA; Dr R. Schneerson, Bethesda, 40

MD, USA; Dr S. Sontakke, BGTD, Health Canada, Ottawa, Canada; Dr S. Szu, National 41

Institute of Child Health & Human Development, Bethesda, MD, USA; Dr W. Van Molle, 42

Scientific Institute of Public health, Brussels, Belgium; Dr A. Worobec, Centre for Biologics 43

Evaluation and Research, Bethesda, MD, USA; Ms D. Kusmiaty, National Quality Control 44

Laboratory of Drug and Food, Jakarta, Indonesia; Dr G. Xie, China National Biotec Group, 45

Beijing, China; Dr M. Zeng, National Institutes of Food and Drug Control, Beijing, China; Japan 46

Paediatric Society; State Food and Drug Administration and Center for Drug Evaluation of 47

China. Acknowledgments are due to the following experts who participated in an April 2013 48

meeting in Geneva: Dr A. Bentsi-Enchill, Initiative for Vaccine Research, Immunization, 49

Vaccines and Biologicals, World Health Organization, Geneva, Switzerland; Dr M. Bonnet, 50

Sanofi Pasteur, Lyon, France (IFPMA Representative); Dr J. Cipollo, Center for Biologics 51

Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA; Dr J. Cirunay, 52

WHO/BS/2013. 2215

Food and Drug Administration, Filinvest Corporate City, Philippines; Dr R. Carbis, International 1

Vaccine Institute, SNU Research Park, Seoul, Republic of Korea; Dr D. Cardoso-Gonzalez, 2

Finlay Institute, Ciudad Habana, Cuba (DCVMN Representative); Ms J. Dahlan, National 3

Quality Control Laboratory of Drug and Food, National Agency of Drug and Food Control, 4

Jakarta, Indonesia; Ms M.-J. Escoto-López, Center for State Control on the Quality of Drugs 5

(CECMED), Habana, Cuba; Dr I. Feavers, National Institute for Biological Standards and 6

Control, Potters Bar, Hert., UK; Dr D. Garcia, National Drug and Health Products Safety 7

Agency (ANSM), Lyon, France; Mr K. Gopinathan, Bharat Biotech International, Hyderabad, 8

India; Dr E. Griffiths, Kingston-upon-Thames, Surrey, UK (Chair); Ms W. Jaroenkunathum, 9

Institute of Biological Products, Ministry of Public Health, Nonthaburi, Thailand; Dr C. Jones, 10

National Institute for Biological Standards and Control, Potters Bar, Herts., UK; Dr C.-K. Lee, 11

Korea Food & Drug Administration, Osong, Republic of Korea (Co-Chair); Dr I. Knezevic, 12


Switzerland; Dr H. Langar, Essential Vaccines and Biological Policy, Eastern Mediterannean 14

Regional Office, World Health Organization, Cairo, Egypt; Dr M. Levine, University of MD 15

School of Medicine, Baltimore, MD, USA (Rapporteur); Dr L.B. Martin, Novartis Vaccines 16

Institute for Global Health, Siena, Italy; Dr J. Mathew, Advanced Pediatrics Centre, Post 17

Graduate Institute of Medical Education and Research, Chandigarh, India; Dr E. Mohamed, The 18

Biovac Institute, Cape Town, South Africa (DCVMN Representative); Dr M. Morita, Department 19

of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan; Dr C. Nelson, 20

Coalition against Typhoid Secretariat, Sabin Vaccine Institute, Washington DC, USA; Dr V.C. 21

Nguyen, National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam; Dr S. Nishioka, 22


Switzerland; Dr H.-J. Oh, Korea Food & Drug Administration, Osong, Republic of Korea; Dr M. 24

Paste; GSK Vaccines, Wavre, Belgium (IFPMA Representative); Dr A. Podda, Novartis 25

Vaccines Institute for Global Health, Siena, Italy; Dr A. Pollard, Department of Paediatrics, 26

Oxford University, Headington, Oxford, UK; Dr M. Powell, Medicines and Healthcare 27

Regulatory Agency, London, UK; Dr A. Ramkishan, Central Drugs Standard Control 28

Organization, Ministry of Health and Family Welfare, New Dehli, India; Dr S. Rijpkema, 29

National Institute for Biological Standards & Control, Potters Bar, Herts., UK; Dr J. Robbins, 30

New York, USA (via teleconference); Dr S. Sahastrabuddhe, International Vaccine Institute, 31

SNU Research Park, Seoul, Korea; Dr J. Shin, Department of Essential Medicines and Health 32

Products, World Health Organization, Geneva, Switzerland; Dr S. Szu, National Institutes of 33

Health, Bethesda, MD, USA (via teleconference); Ms J. Tresnabudi, Biofarma, Bandung, 34

Indonesia; Ms G. Trisnasari, Biofarma, Bandung, Indonesia (DCVMN Representative); Ms A. 35

Visala, Central Drugs Standard Control Organization, Food & Drugs Administration, New Delhi, 36

India; and Dr M. Zeng, National Institutes of Food and Drug Control, Beijing, China. A further 37

improved draft was prepared considering written comments to the second draft and the following 38

experts are acknowledged for their additional contributions: Ms J. Dahlan, National Quality 39

Control Laboratory of Drug and Food, National Agency of Drug and Food Control, Jakarta, 40

Indonesia; Ms W. Jaroenkunathum, Institute of Biological Products, Ministry of Public Health, 41

Nonthaburi, Thailand; Dr M. Levine, University of Maryland School of Medicine, Center for 42

Vaccine Development, Baltimore, MD, USA; Dr L. Martin, Novartis Vaccines Institute for 43

Global Health, Siena, Italy; Dr S. Szu, National Institutes of Health, Bethesda, MD, USA; and 44

Ms G. Trisnasari, Biofarma, Bandung, Indonesia. Special thanks go to Mr K. Gopinathan, Bharat 45

Biotech International, Hyderabad, India for provision of draft model summary protocol and lot 46

release certificate. 47

48

49

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Clin Microbiol, 2005, 43:4418-25. 33

129. Wain J et al. Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates 34

from Pakistan. J Clin Microbiol, 2005, 43:1158-65. 35

130. Hornick RB et al. Typhoid fever: pathogenesis and immunologic control. 2. N Engl J 36

Med, 1970, 283:739-46. 37

131. Hornick RB et al. Typhoid fever: pathogenesis and immunologic control. N Engl J Med, 38

1970, 283:686-91. 39

132. Guidelines for national authorities on quality assurance for biological products. In: WHO 40

Expert Committee on Biological Standardization. Forty-second report. Geneva, World 41

Health Organization, 1992, Annex 2 (WHO Technical Report Series No. 822). 42

http://www.who.int/biologicals/publications/trs/areas/vaccines/regulatory/WHO_TRS_8243

2_A2.pdf. 44

133. Guidelines for independent lot release of vaccines by regulatory authorities (Adopted, 45

2010). In: WHO Expert Committee on Biological Standardization. Geneva, World Health 46

Organization, 47

http://www.who.int/biologicals/areas/vaccines/lot_release_of_vaccines/en/index.html. 48

49

50

51

http://www.who.int/biologicals/publications/trs/areas/vaccines/nonclinical_evaluation/ANNEX%201Nonclinical.P31-63.pdf

http://www.who.int/biologicals/publications/trs/areas/vaccines/nonclinical_evaluation/ANNEX%201Nonclinical.P31-63.pdf

http://dx.doi.org/10.1787/9789264078536-en

http://apps.who.int/medicinedocs/pdf/whozip13e/whozip13e.pdf

http://www.who.int/biologicals/publications/trs/areas/vaccines/clinical_evaluation/035-101.pdf

http://www.who.int/biologicals/publications/trs/areas/vaccines/clinical_evaluation/035-101.pdf

http://www.who.int/biologicals/publications/trs/areas/vaccines/regulatory/WHO_TRS_822_A2.pdf

http://www.who.int/biologicals/publications/trs/areas/vaccines/regulatory/WHO_TRS_822_A2.pdf

http://www.who.int/biologicals/areas/vaccines/lot_release_of_vaccines/en/index.html

WHO/BS/2013. 2215

Appendix 1: Model summary protocol for manufacture and control 1

of typhoid conjugate vaccines 2

3

The following protocol is intended for guidance, and indicates the information that should be 4

provided as a minimum by the manufacturer to the NRA. Information and tests may be added or 5

deleted as required by the NRA, if applicable. 6

7

It is thus possible that a protocol for a specific product may differ in detail from the model 8

provided. The essential point is that all relevant details demonstrating compliance with the 9

license and with the relevant WHO recommendations of a particular product should be given 10

in the protocol submitted. 11

12

The section concerning the final product must be accompanied by a sample of the label and 13

a copy of the leaflet that accompanies the vaccine container. If the protocol is being submitted 14

in support of a request to permit importation, it must also be accompanied by a lot release 15

certificate from the NRA of the country in which the vaccine was produced stating that the 16

product meets national requirements as well as Part A recommendations of this document 17

published by WHO. 18

19

Summary information on final lots 20

21

International name of product ___________________________ 22

Commercial name ___________________________ 23

Product license (Marketing Authorization) No. ___________________________ 24

Country ___________________________ 25

Name and address of manufacturer ___________________________ 26

Final packing lot number ___________________________ 27

Type of containers ___________________________ 28

Number of containers in this packing lot ___________________________ 29

Final container lot number ___________________________ 30

Number of filler containers in this final lot ___________________________ 31

Date of manufacturing ___________________________ 32

Nature of final product (absorbed) ___________________________ 33

Preservative and nominal concentration ___________________________ 34

Volume of each recommended single human dose ___________________________ 35

Number of doses per final container ___________________________ 36

Summary of the composition: ___________________________ 37

(Include a summary of the qualitative and quantitative composition of the vaccine per human 38

dose including the conjugate, any adjuvant used and other excipients) 39

40

Shelf-life approved (months) ___________________________ 41

Expiry date ___________________________ 42

Storage conditions ___________________________ 43

44

WHO/BS/2013. 2215

The following sections are intended for the reporting of the results of the tests performed 1

during the production of the vaccine, so that the complete document will provide evidence of 2

consistency of production; thus if any test has to be repeated, this must be indicated. Any 3

abnormal results should be recorded on a separate sheet. 4

5

Detailed information on manufacture and control 6

7

SUMMARY OF STARTING MATERIALS 8

9

It is possible that a number of bulk lots are used to produce a single final lot. A summary of 10

the bulk polysaccharide, activated saccharide, bulk carrier protein and bulk conjugate lots that 11

contribute to the final lot should be provided. 12

13

CONTROL OF TYPHOID Vi POLYSACCHARIDE 14

15

Bacterial strain 16

Identity of S. Typhi Ty2 / Citrobacter freundii ___________________________ 17

Origin and short history ___________________________ 18

Authority that approved the strain ___________________________ 19

Date approved ___________________________ 20

21

Bacterial culture media for seed lot preparation 22

and Vi production 23

Free from forming precipitate with addition of CTAB ___________________________ 24

Free from toxic or allergic reactions ___________________________ 25

Any components of animal origin ___________________________ 26

Certificate of TSE-free ___________________________ 27

28

Master seed lot 29

Lot no. ___________________________ 30

Date of master seed lot established ___________________________ 31

32

Working seed lot 33

Lot no. ___________________________ 34

Date of working seed lot established ___________________________ 35

Control tests on working seed lot ___________________________ 36

Date of reconstitution of seed lot ___________________________ 37

38

WHO/BS/2013. 2215

Control of single harvests 1

2

List the single harvests and indicate the medium, dates of inoculation, temperature of 3

incubation, dates of harvests, volumes, results of tests for bacterial growth rate, pH, purity 4

and identity, the method and date of inactivation, the method of purification, and the yield of 5

purified polysaccharide. 6

7

Control of purified typhoid Vi polysaccharide 8

9

Lot no. ___________________________ ___________________________ 10


Volume ___________________________ ___________________________ 12

Identity 13

Date tested ___________________________ 14

Method ___________________________ 15

Specification ___________________________ 16

Result ___________________________ 17

Purity 18

Date tested ___________________________ 19

Method ___________________________ 20

Specification ___________________________ 21

Result ___________________________ 22

Molecular size/mass distribution 23

Date tested ___________________________ 24

Method ___________________________ 25

Specification ___________________________ 26

Result ___________________________ 27

Polysaccharide content 28

Date tested ___________________________ 29

Method ___________________________ 30

Specification ___________________________ 31

Result ___________________________ 32

O-acetyl content 33

Date tested ___________________________ 34

Method ___________________________ 35

Specification ___________________________ 36

Result ___________________________ 37

Moisture content 38

WHO/BS/2013. 2215

Date tested ___________________________ 1

Method ___________________________ 2

Specification ___________________________ 3

Result ___________________________ 4

Protein impurity 5

Date tested ___________________________ 6

Method ___________________________ 7

Specification ___________________________ 8

Result ___________________________ 9

Nucleic acid impurity 10

Date tested ___________________________ 11

Method ___________________________ 12

Specification ___________________________ 13

Result ___________________________ 14

Phenol content 15

Date tested ___________________________ 16

Method ___________________________ 17

Specification ___________________________ 18

Result ___________________________ 19

Endotoxin/pyrogen content 20

Date tested ___________________________ 21

Method ___________________________ 22

Specification ___________________________ 23

Result ___________________________ 24

Residues of process related contaminants 25

Date tested ___________________________ 26

Method ___________________________ 27

Specification ___________________________ 28

Result 29

30

Control of modified polysaccharide 31

32

Lot no. ___________________________ 33

Method of chemical modification ___________________________ 34

Extent of activation for conjugation 35

Date tested ___________________________ 36

Method ___________________________ 37

Specification ___________________________ 38

Result ___________________________ 39

WHO/BS/2013. 2215


Date tested ___________________________ 2

Method ___________________________ 3

Specification ___________________________ 4

Result ___________________________ 5

6

CONTROL OF CARRIER PROTEIN (e.g. DT/TT/CRM197/OMP/rEPA) 7

8

Microorganisms used 9

Identity of strain used in carrier protein production ___________________________ 10

Origin and short history ___________________________ 11

Authority that approved the strain ___________________________ 12

Date approved ___________________________ 13

14

Bacterial culture media for seed lot preparation 15

and carrier protein production 16

Free from forming precipitate with addition of CTAB ___________________________ 17

Free from toxic or allergic reactions ___________________________ 18

Any components of animal origin ___________________________ 19

Certificate of TSE-free ___________________________ 20

21

Master seed lot 22

Lot no. ___________________________ 23

Date established ___________________________ 24

25

Working seed lot 26

Lot no. ___________________________ 27

Date established ___________________________ 28

Control tests on working seed lot ___________________________ 29

Date of reconstitution of seed lot ___________________________ 30

31

Control of carrier protein production 32

33

List the lot numbers of harvests and indicate the medium, dates of inoculation, temperature of 34

incubation, dates of harvests, volumes, results of tests for bacterial growth rate, pH, purity 35

and identity, the method and date of inactivation, the method of purification, and the yield of 36

purified carrier protein. Provide documented evidence that carrier protein is non-toxic. 37

38

Purified carrier protein 39

WHO/BS/2013. 2215

Lot no. ___________________________ 1

Date produced ___________________________ 2

Identity 3

Date tested ___________________________ 4

Method ___________________________ 5

Specification ___________________________ 6

Result ___________________________ 7

Purity 8

Date tested ___________________________ 9

Method ___________________________ 10

Specification ___________________________ 11

Result ___________________________ 12

13

Modified carrier protein 14

Lot no. ___________________________ 15

Date produced ___________________________ 16

Method of modification ___________________________ 17

Extent of activation 18

Date tested ___________________________ 19

Method ___________________________ 20

Specification ___________________________ 21

Result ___________________________ 22

23

CONTROL OF PURIFIED BULK CONJUGATE 24

25

Production details of bulk conjugate 26

27

List the lot numbers of the saccharine and carrier protein used in the manufacture of the 28

conjugate vaccines, the production procedure, date of manufacture and yield. 29

30

Tests on purified bulk conjugate 31

32

Identity 33

Date tested ___________________________ 34

Method ___________________________ 35

Specification ___________________________ 36

Result ___________________________ 37

WHO/BS/2013. 2215


Date tested ___________________________ 2

Method ___________________________ 3

Specification ___________________________ 4

Result ___________________________ 5

O-acetyl content 6

Date tested ___________________________ 7

Method ___________________________ 8

Specification ___________________________ 9

Result ___________________________ 10

Residual reagents 11

Date tested ___________________________ 12

Method ___________________________ 13

Specification ___________________________ 14

Result ___________________________ 15

Vi polysaccharide content 16

Date tested ___________________________ 17

Method ___________________________ 18

Specification ___________________________ 19

Result ___________________________ 20

Conjugated and unbound (free) polysaccharide 21

Date tested ___________________________ 22

Method ___________________________ 23

Specification ___________________________ 24

Result ___________________________ 25

Protein content 26

Date tested ___________________________ 27

Method ___________________________ 28

Specification ___________________________ 29

Result ___________________________ 30

Conjugation markers 31

Date tested ___________________________ 32

Method ___________________________ 33

Specification ___________________________ 34

Result ___________________________ 35

Absence of reactive functional groups (capping markers) 36

Date tested ___________________________ 37

Method ___________________________ 38

WHO/BS/2013. 2215

Specification ___________________________ 1

Result ___________________________ 2

Polysaccharide to protein ratio 3

Date tested ___________________________ 4

Method ___________________________ 5

Specification ___________________________ 6

Result ___________________________ 7


Date tested ___________________________ 9

Method ___________________________ 10

Specification ___________________________ 11

Result ___________________________ 12

Bacterial & mycotic bioburden 13

Method ___________________________ 14

Media ___________________________ 15

Volume tested ___________________________ 16

Date of inoculation ___________________________ 17

Date of end of test ___________________________ 18

Specification ___________________________ 19

Result ___________________________ 20

Specific toxicity of carrier protein (where appropriate) 21

Method ___________________________ 22

Strain and type of animals ___________________________ 23

Number of animals ___________________________ 24

Route of injection ___________________________ 25

Volume of injection ___________________________ 26

Quantity of protein injected ___________________________ 27

Date of start of test ___________________________ 28


Specification ___________________________ 30

Result ___________________________ 31

pH 32

Date tested ___________________________ 33

Method ___________________________ 34

Specification ___________________________ 35

Result ___________________________ 36

Appearance 37

Date tested ___________________________ 38

Method ___________________________ 39

WHO/BS/2013. 2215

Specification ___________________________ 1

Result ___________________________ 2

3

Depending upon the conjugation chemistry used to produce the vaccine, suitable tests should 4

also be included demonstrating that residual reagents and reaction by-products are below a 5

specified level. 6

7

8

CONTROL OF FINAL BULK 9

Lot no. ___________________________ 10

Date prepared ___________________________ 11

Preservative (if used) 12

Name and nature ___________________________ 13

Lot no. ___________________________ 14

Final concentration in the final bulk ___________________________ 15

16

Stabilizer (if used) 17

Name and nature ___________________________ 18

Lot no. ___________________________ 19


21

Adjuvant (if used) 22

Name and nature ___________________________ 23

Lot no. ___________________________ 24


26

Test on final bulk 27

Bacterial & mycotic sterility 28

Method ___________________________ 29

Media ___________________________ 30

Volume tested ___________________________ 31



Specification ___________________________ 34

Result ___________________________ 35

36

FILLING AND CONTAINERS 37

Lot no. ___________________________ 38

Date of sterile filtration ___________________________ 39

WHO/BS/2013. 2215

Date of filling ___________________________ 1

Volume of final bulk filled filling ___________________________ 2

Volume per container number of ___________________________ 3

Containers filled (gross) ___________________________ 4

Date of Lyophilization (if applicable) ___________________________ 5

Number if containers rejected during inspection ___________________________ 6

Number of containers sampled ___________________________ 7

Total number of containers (net) ___________________________ 8

Maximum period of storage approved ___________________________ 9

Storage temperature and period ___________________________ 10

11

CONTROL TESTS ON FINAL LOT 12

Inspection of final containers 13

Date tested ___________________________ 14

Method ___________________________ 15

Specification ___________________________ 16

Results ___________________________ 17

Appearance before & after reconstitution2 ___________________________ 18

Diluent used ___________________________ 19

Lot number of diluent used ___________________________ 20

Tests on final lot 21

Identity 22

Date tested ___________________________ 23

Method ___________________________ 24

Specification ___________________________ 25

Result ___________________________ 26

Sterility 27

Method ___________________________ 28

Media ___________________________ 29

No. of containers tested ___________________________ 30



Specification ___________________________ 33

Result ___________________________ 34

Polysaccharide content 35

Date tested ___________________________ 36

Method ___________________________ 37

Specification ___________________________ 38

2 Only applies to lyophilized vaccines

WHO/BS/2013. 2215

Result ___________________________ 1

Unbound (free) polysaccharide 2

Date tested ___________________________ 3

Method ___________________________ 4

Specification ___________________________ 5

Result ___________________________ 6

O-acetyl content 7

Date tested ___________________________ 8

Method ___________________________ 9

Specification ___________________________ 10

Result ___________________________ 11


Date tested ___________________________ 13

Method ___________________________ 14

Specification ___________________________ 15

Result ___________________________ 16


Date tested ___________________________ 18

Method ___________________________ 19

Specification ___________________________ 20

Result ___________________________ 21

Adjuvant content (if applicable) 3 22

Date tested ___________________________ 23

Nature and concentration of adjuvant per human dose ___________________________ 24

Method ___________________________ 25

Specification ___________________________ 26

Result ___________________________ 27

Preservative content (if applicable) 28

Date tested ___________________________ 29

Method ___________________________ 30

Specification ___________________________ 31

Result ___________________________ 32

General safety 33

Date tested ___________________________ 34

Method ___________________________ 35

Specification ___________________________ 36

3 Only applies when adjuvant is present in the final container

WHO/BS/2013. 2215

Result ___________________________ 1

pH 2

Date tested ___________________________ 3

Method ___________________________ 4

Specification ___________________________ 5

Result ___________________________ 6

Osmolality 7

Date tested ___________________________ 8

Method ___________________________ 9

Specification ___________________________ 10

Result ___________________________ 11

Residual moisture 4 12

Date tested ___________________________ 13

Method ___________________________ 14

Specification ___________________________ 15

Result ___________________________ 16

17

Control of diluent (if applicable) 18

Name and composition of diluent: ___________________________ 19

Lot number: ___________________________ 20

Date of filling: ___________________________ 21

Type of diluent container: ___________________________ 22

Appearance: ___________________________ 23

Filling volume per container: ___________________________ 24

Maximum period of storage approved: ___________________________ 25

Storage temperature and period: ___________________________ 26

Other specifications: ___________________________ 27

28

29

STABILITY EVALUATION 5 30

31

Describe separately all relevant details including, but not limited to, changes in the proportion 32

of free saccharide, molecular size/mass distribution, pH, residual moisture in accelerated 33

degradation tests, and after storage for the maximum period claimed for the product at the 34

recommended temperature As described in section A.11. 35

36

37

4 Only applies to lyophilized vaccines

5 Needed only for sufficient batches to validate production method and proposed shelf-life

WHO/BS/2013. 2215

CONTROL OF ADJUVANT 6 1

2

Summary of production details of adjuvant 3

4

When an adjuvant suspension is provided for the reconstitution of lyophilized vaccine a 5

summary protocol of production and control details should be provided. The information 6

provided and tests performed will depend on the adjuvant used. 7

8

Summary information on adjuvant 9

10

Name and address of manufacturer ___________________________ 11

Nature of the adjuvant ___________________________ 12

Lot no. ___________________________ 13


Expiry date ___________________________ 15

16

Tests on adjuvant 17

Adjuvant content 18

Date tested ___________________________ 19

Method ___________________________ 20

Specification ___________________________ 21

Result ___________________________ 22

Appearance 23

Date tested ___________________________ 24

Method ___________________________ 25

Specification ___________________________ 26

Result ___________________________ 27

Purity/impurity 28

Date tested ___________________________ 29

Method ___________________________ 30

Specification ___________________________ 31

Result 32

pH 33

Date tested ___________________________ 34

Method ___________________________ 35

Specification ___________________________ 36

6 This section is only required when adjuvant is provided separately for the suspension of a

lyophilized vaccine.

WHO/BS/2013. 2215

Result ___________________________ 1

Pyrogenicity 7 2

Date tested ___________________________ 3

Method ___________________________ 4

Specification ___________________________ 5

Result ___________________________ 6

Sterility 7

Method ___________________________ 8

Media ___________________________ 9

No. of containers used ___________________________ 10



Specification ___________________________ 13

Result ___________________________ 14

15

16

CERTIFICATION BY THE MANUFACTURER 17

18

Name of head of control of the manufacturer ______________________________________ 19

20

Certification by person from the control laboratory of the manufacturing company taking 21

overall responsibility for the production and control of the vaccine. 22

23

I certify that lot No. ___________________ of Typhoid Conjugate Vaccine, whose number 24

appears on the label of the final containers, meets national requirements and satisfies Part A of 25

the WHO Guidelines on the quality, safety and efficacy of typhoid conjugate vaccine (WHO 26

TRS ________________). 27

28

Signature ___________________________________ 29

Name (typed) _______________________________ 30

Date _______________________________________ 31

32

33

34

7 A pyrogen test of the adjuvant is not needed if a pyrogen test was performed on the

adjuvant reconstituted vaccine.

WHO/BS/2013. 2215

Appendix 2: Model certificate for the release of typhoid conjugate 1

vaccines 2

3

This certificate is to be provided by the National Regulatory Authority of the country where the 4

vaccines have been manufactured, upon request by the manufacturer 5

6

Certificate number _____________________ 7

8

9

LOT RELEASE CERTIFICATE 10

11

The following lot(s) of Typhoid Conjugate Vaccine produced by __________________1 in 12

___________________2, whose numbers appear on the labels of the final containers, meet all 13

national requirements3 and Part A

4 of the WHO Guidelines on the quality, safety and efficacy 14

of typhoid conjugate vaccine (WHO TRS _____________), adopted ____________5, and 15

comply with Good Manufacturing Practices for Pharmaceutical Products 6 and Good 16

Manufacturing Practices for Biological Products 7. As a minimum, this certificate is based on 17

examination of the summary protocol of manufacturing and control. 18

19

Final Lot no. ___________________________ 20

No. of released human doses in this final lot ___________________________ 21

Expiry date ___________________________ 22

23

The Director of the National Regulatory Authority (or Authority as appropriate): 24

Name (Typed) ___________________________ 25

Signature ___________________________ 26

Date ___________________________ 27

28

__________________________ 29 1. Name of manufacturer 30 2. Country of origin 31 3. If any national requirements are not met, specify which one(s) and indicate why 32

release of the lot(s) has nevertheless been authorized by the national regulatory authority 33 4. With the exception of provisions on distribution and shipping, which the national 34

regulatory authority may not be in a position to assess. 35 5. WHO Technical Report Series, No. , YYYY, Annex . 36 6. WHO Technical Report Series, No. 840, 1992, Annex 1. 37 7. WHO Technical Report Series, No. 800, 1992, Annex 1. 38

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