- 1

WHO/BS/2014.2234 2

ENGLISH ONLY 3

4

5

6

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION 7

Geneva, 13 to 17 October 2014 8

9

A WHO collaborative study to evaluate a candidate International 10

Standard for anti-Typhoid capsular Vi polysaccharide IgG (Human) 11

12

Sjoerd Rijpkema1*, Jason Hockley

2, Victoria Last

1, Anita Marwaha

1 and Peter Rigsby

2 13

14 1Division of Bacteriology and

2Biostatistics Section, National Institute for Biological Standards and 15

Control, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom;*(corresponding author)

16

* Email address: Sjoerd.Rijpkema@nibsc.org 17

18 NOTE: 19

This document has been prepared for the purpose of inviting comments and suggestions on the 20

proposals contained therein, which will then be considered by the Expert Committee on Biological 21

Standardization (ECBS). Comments MUST be received by 4 October 2014 and should be 22

addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Technologies, 23

Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible 24

Officer: Dr Jongwon Kim at email: kimjon@who.int. 25

26

© World Health Organization 2014 27

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 28 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for 29 permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to 30 WHO Press, at the above address (fax: +41 22 791 4806; e-mail: permissions@who.int). 31

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever 32 on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or 33 concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may 34 not yet be full agreement. 35 36 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the 37 World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names 38 of proprietary products are distinguished by initial capital letters. 39

All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. 40 However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the 41 interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising 42 from its use. The named authors alone are responsible for the views expressed in this publication. 43

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WHO/BS/2014.2234

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Summary 1

Candidate International Standard (IS) 10/126 is a freeze dried preparation of 0.5 mL pooled sera, 2

taken from 9 volunteers immunised with a Vi polysaccharide (Vi) tetanus toxoid conjugate vaccine 3

and contains IgG directed against Vi and lipopolysaccharide of Salmonella enterica subspecies 4

enterica serovar Typhi. A collaborative study comprising ten laboratories from seven countries 5

assessed the suitability of candidate IS 10/126 for Vi ELISAs, alongside reference reagent for 6

human anti-Vi IgG, Vi-IgGR1, 2011 provided by the Center for Biologics Evaluation & Research 7

(CBER; Szu et al., Vaccine 2013;31:1970–1974). Both reference materials were tested in a 8

commercially available Vi ELISA kit (n=3), an ELISA based on CBER reference reagent Vi Lot 9

no. 05 (Vi 05 ELISA; n=7) and in-house ELISAs based on locally prepared Vi (n=4). Of the latter 10

two ELISAs, 9 out of 11 assays were based on slight modifications of the procedure by Szu et al. 11

(2013). Candidate IS 10/126 is suitable for use in Vi ELISAs. The relative potency (RP) of 12

candidate IS 10/126 compared to Vi-IgGR1, 2011 depended on the ELISA format. The GM of the RP 13

based on the kit ELISA was calculated as 0.65 (geometric coefficients of variation (GCV) 15%; 14

n=3), for the Vi 05 ELISA as 1.40 (GCV 67%; n=6) and for the in-house ELISAs as 0.41 (GCV 15

77%; n=4). The overall geometric mean (GM) of the RP was calculated as 0.80 (95% Confidence 16

Interval 0.52 - 1.24; GCV 105%; n=13). Based on the RP of all ELISA data, we recommend that 17

10/126 be established as the 1st IS for anti-Typhoid capsular Vi polysaccharide IgG (human), with 18

an assigned potency of 0.5 IU per ampoule or 1 IU mL-1

. 19

20

WHO/BS/2014.2234

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Introduction 1

Typhoid is caused by an infection with Salmonella enterica subspecies enterica serovar Typhi (S. 2

Typhi). S. typhi expresses a Vi capsular polysaccharide (Vi), which is considered the main virulence 3

factor and a protective antigen (4, 14). The disease is responsible for considerable morbidity and 4

mortality in developing countries, although the true incidence of typhoid fever remains unknown. A 5

study published in 2004 estimated 22 million cases occur each year, causing 216 000 deaths (2). 6

Typhoid affects infants, school-age children and young adults in particular. 7

8

Vaccination is the most cost effective preventative strategy to control the disease. Licensed typhoid 9

vaccines, the oral live attenuated Ty21a vaccine and the injectable Vi polysaccharide vaccine are not 10

suitable for use in children under five and two years of age respectively. It has been known for some 11

time that Vi conjugate vaccines are highly immunogenic and provide a booster response (15, 16). A 12

‘proof of principle’ clinical trial followed, which showed that Vi recombinant Exoprotein A 13

conjugate vaccine (Vi-rEPA) was safe and efficacious in 2-5 year olds (10). The development of Vi 14

conjugate vaccines accelerated as subsequent clinical trials established that Vi-rEPA could be used 15

as part of the Expanded Programme on Immunization and that vaccination induced a long term 16

protection against typhoid in infants and pre-school age children (9, 20). These studies also 17

confirmed that protection against typhoid correlated with a serum anti Vi-IgG level of 3.5 ELISA 18

Units (EU) mL-1

at year 4 (20). 19

20

In 2012, the World Health Organization (WHO) organised a meeting to develop guidelines on 21

quality, safety and efficacy of typhoid conjugate vaccines in Jeju (Republic of Korea). The aim of 22

the meeting was to ‘review the scientific basis for regulatory evaluation of typhoid conjugate 23

vaccines under clinical development with emphasis on manufacturing and quality control, non-24

clinical and clinical evaluation and standardisation’ (6). The meeting concluded that reference 25

materials are needed for Vi and anti-Vi antibodies to calibrate immunoassays assays, for comparison 26

of clinical trial studies, and for the analysis of typhoid conjugate vaccines by immunoassays and 27

physicochemical assays. The guidelines were published in 2013 (21). 28

29

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WHO/BS/2014.2234

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The Jeju meeting was timely: the Vi tetanus toxoid conjugate vaccine (Vi-TT) produced by Bharat 1

Biotech International Ltd (BBIL) of India has received a marketing licence, five Vi conjugate 2

vaccines using different carrier proteins are in clinical trials, and reference reagents for Vi and 3

human anti-Vi IgG have since become available (6, 17). The reference reagent for human anti-Vi 4

IgG, Vi-IgGR1, 2011 contains 33 µg of purified anti-Vi IgG mL-1

equivalent to 26.6 EU mL-1

(17). The 5

aim of Vi-IgGR1, 2011 is to facilitate the evaluation of the immunogenicity of Vi conjugate vaccines. 6

The protective level of anti-Vi IgG in clinical trial volunteers, relative to Vi-IgGR1, 2011 was 7

established as 4.3 µg mL-1

at year 4 and as 10 µg mL-1

at month 6 after vaccination (17, 18, 20). 8

9

The 1st International Reference Preparation of anti-typhoid serum (equine; TYS) was developed by 10

Dr A Felix in 1935. In 2009, the Expert Committee on Biological Standardization of WHO endorsed 11

the replacement of TYS and for this purpose, candidate international standard (IS) 10/126 for anti-12

Vi IgG (human) was developed at the National Institute for Biological Standards and Control 13

(NIBSC). Sera from 9 volunteers of a Phase II clinical trial of the aforementioned Vi-TT were 14

selected as the source for candidate IS 10/126 and pooled, filled and freeze dried. 15

16

At the Jeju meeting, it was decided that ‘a serum standard containing defined antibody contents, 17

either in a mass unit or in an arbitrary international unit (IU) that is inter-convertible with mass unit 18

is required to support cross calibration’ (6). Reference preparation Vi-IgGR1, 2011 and candidate IS 19

10/126 differ in immunization schedules, formulation of the vaccine, matrix, and geographical 20

origin of the source material (see Table 1). The intended use of candidate IS 10/126 is to act as a 21

benchmark for the immunogenicity of Vi conjugate vaccines in clinical trial studies. Comparisons 22

between studies that use either one or the other reference material are likely to lead to confusion. 23

Therefore the current collaborative study (CS) has been designed to provide a head to head 24

comparison between candidate IS 10/126 and Vi-IgGR1, 2011. 25

The primary aims of the CS were: 26

1. To assess the suitability of candidate IS 10/126 as an IS for human anti-Vi antibody 27

2. To assess the potency of candidate IS 10/126 relative to Vi-IgGR1, 2011. 28

3. To assess the reactivity of both reference preparations in Vi ELISAs, currently in use. 29

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WHO/BS/2014.2234

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Materials and Methods 1

2

Participating laboratories and assay codification 3

Ten participating laboratories from seven countries, including vaccine manufacturers, national 4

control laboratories and research laboratories, tested the samples and supplied the test data (see 5

appendix 2 for details). Data were collected and analysed at NIBSC. Throughout the study, 6

participating laboratories and assays were identified by a randomly assigned code number to 7

maintain confidentiality. Each participant received two sets of samples comprising coded ampoules 8

(A-C) and one ampoule of Vi-IgGR1, 2011 (coded NIH). The shipment also included one vial of S. 9

Typhi Vi Lot no. 05. 10

11

Samples 12

A brief characterisation of the study samples, study codes, NIBSC codes and the reactivity in Vi 13

ELISA are given in Table 1. Reference reagents Vi Lot no. 05 and Vi-IgGR1, 2011 were established at 14

the National Institutes of Health and are distributed by CBER (Food & Drug Administration of the 15

Department of Health and Human Services). CBER donated 22 vials of freeze dried Vi-IgGR1, 2011 16

and 10 vials of freeze dried Vi Lot no. 05 for distribution among participants of the CS. All samples 17

tested negative for antibodies to HIV 1/2 and Hepatitis C and Hepatitis B surface antigen. All 18

samples were distributed as lyophilized preparations at room temperature by courier. Samples that 19

were used to analyse the stability of candidate IS 10/126 were distributed on dry ice. NIBSC 20

samples A-C were reconstituted as described in the ‘instructions for use’. 21

Samples donated by CBER were reconstituted as described in the accompanying ‘product circular’. 22

Details of the production and characterisation of Vi-IgGR1, 2011 are given in Szu et al. (2013) and Vi 23

Lot no. 05 was produced as described by Bystrycki et al. (1994). 24

25

Characterization of the candidate international standard 10/126 26

Approximately one litre of human serum was collected, with consent, from a total of 9 volunteers 27

(24-30 years old) who had received two doses of Vi-TT conjugate vaccine (25 µg Vi; BBIL) as part 28

of a phase II clinical trial (see Table 1). The use of source material for candidate IS 10/126 was 29

approved by the Human Materials Advisory Committee of NIBSC (10_002SR). 30

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At NIBSC, samples were thawed and pooled. Individual sera tested positive for antibodies to Vi and 1

lipopolysaccharide (LPS) of S. Typhi and tetanus toxin (results not shown). 2

The serum pool tested positive for antibodies to Vi and LPS of S. Typhi (Figure 1A and 1B). The 3

serum pool was dispensed in 0.5 mL aliquots into glass ampoules coded 10/126. The mean fill 4

weight for 78 ampoules was 0.5181 g (CV of 0.19 %). On the same day, freeze-drying under 5

vacuum started and was completed after four days. Ampoules were back filled with pure N2 6

(moisture content <10 ppm). Residual moisture, measured by the Karl-Fischer method, for 6 7

ampoules was 0.23 % (CV of 17.60 %). An integrity test was not performed; however the oxygen 8

level in the headspace of 12 ampoules was checked and found to be 0.59% (CV 29.55%). This 9

implies these ampoules passed a test for integrity because the presence of cracks would be 10

associated with an oxygen level of around 20%, similar to that found in the atmosphere. Twenty-six 11

ampoules were rejected during the production process, 44 ampoules were held for accelerated 12

degradation studies and 1681 ampoules were stored at -20 oC. These are available for distribution by 13

NIBSC. 14

The effect of freeze drying on levels of specific IgG titres was determined in samples of 10/126 15

taken before and after freeze drying. ELISAs for Vi and LPS of S. Typhi were carried out as 16

described previously (13). The results show that freeze drying of 10/126 did not affect the titre of 17

IgG directed against Vi or LPS (Figures 1A and 1B). 18

19

Test methods 20

A description of ELISA procedures accompanied the raw data, which were returned to NIBSC for 21

analysis. Three different ELISA formats were used: in-house Vi ELISAs, the VaccZyme™ S. typhi 22

Vi IgG ELISA (VaccZyme ELISA, Binding Site, UK) and the ELISA is based on Vi lot 05 (Vi 05 23

ELISA, see Table 2). 24

25

Most participating laboratories that used the Vi 05 ELISA followed the published procedure with 26

some modifications (17). In brief, Nunc Maxisorp flat bottom ELISA plates were used as a solid 27

phase, phosphate buffered saline (PBS; pH 7.4) was used as coating buffer, Brij35 or BrijL23 28

(Sigma) as detergent, bovine serum albumin was used as a blocking agent, bound antibody was 29

WHO/BS/2014.2234

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detected by a combination of a mouse anti-human IgG monoclonal antibody and an alkaline 1

phosphatase labelled rat anti-mouse IgG antibody and 4-nitrophenyl phosphate (Sigma) as substrate. 2

The main change to the published procedure was the use of alkaline phosphatase labelled goat anti 3

human IgG for detection in all Vi 05 ELISAs. Lab code 2.1 used round bottom Nunc Maxisorp 4

ELISA plates, Tween-20 as detergent and fat-free milk was used as blocking agent. Lab codes 5 and 5

10 used Costar ELISA plates. 6

7

The in-house ELISAs differed from the Vi 05 ELISA in few aspects. Most participating laboratories 8

used locally produced Vi from S. Typhi and followed the procedure, buffers and reagents as 9

described (17), with the exception of lab codes 2.2 and 9. Lab code 2.2 used Vi from Citrobacter 10

freundii as antigen, Tween-20 as detergent and fat-free milk as blocking agent. Lab code 9 used 11

streptavidin coated wells to bind biotinylated Vi, Tween-20 was used as detergent, fat-free milk as a 12

blocking agent, horseradish peroxidase conjugated goat anti-human IgG and TMB was used as 13

substrate. 14

Participating laboratories which used the Vacczyme ELISA followed procedures described by the 15

manufacturer. Briefly, this ELISA is based on Vi of S. typhi and has a sensitivity of 7.4 EU mL-1

16

and a range of 7.4 to 600 EU mL-1

. Five calibrators, which cover the assay range, and a high and a 17

low positive control were included in each run. 18

19

Data analysis 20

ELISA data were analysed using parallel line bioassay comparing log10-transformed assay response 21

to log10 concentration in a four-parameter logistic model using Vi- IgGR1, 2011 as a reference. All 22

analysis was performed using version 5.0 of EDQM’s CombiStats software (3). Linearity was 23

assessed visually, and parallelism was assessed by the ratio of slopes of the test and reference 24

samples, with ratios outside of 0.80 to 1.25 considered as invalid. The final estimate in each assay 25

for candidate IS 10/126 was taken as the geometric mean (GM) of the two coded duplicates. 26

All mean estimates shown in this report are unweighted GM estimates. Variability between 27

laboratories has been expressed using geometric coefficients of variation (GCV = {10s-1}×100% 28

where s is the standard deviation of the log10-transformed estimates). 29

The results from lab codes 2.1 and 2.2 were analysed by the two tailed Student-t-test. 30

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Results and Discussion 1

All ten laboratories reported results for samples Vi-IgGR1, 2011, A, B and C. The results are 2

summarised in Figure 2 and Tables 3 and 4. Individual results of the samples are given in Appendix 3

1: Tables A1, A2 and A3. Duplicates A and C and negative control B were identified correctly by all 4

participating laboratories, with the exception of participant 3. Following analysis, the results for lab 5

code 3 were found to be non-parallel for sample A and a high GCV was recorded for sample C 6

(Tables 3, 4, A1 and A2). This prevented the identification of samples A and C as duplicates. 7

Sample B had a high value relative to Vi-IgGR1, 2011 and could not be identified as negative or 8

borderline sample (Table A3). 9

10

It is evident from Figure 2 and Table 3, that the Vacczyme ELISA displayed the least variation, 11

followed by the in-house ELISA and the Vi 05 ELISA. The Vacczyme ELISA results gave a GM 12

value of 0.65 for the potency of candidate IS 10/126 relative to reference Vi-IgGR1, 2011, which is 13

equal to 17.3 EU mL-1

, taken from the published value of 26.6 EU mL-1

for Vi-IgGR1, 2011 (17). This 14

ELISA had a GCV of 15%, indicating that this method is robust and easily reproduced by the three 15

participating laboratories. 16

17

The Vi 05 ELISA, based on modifications of the procedure published by Szu et al. (2013) had a 18

GCV of 82% (Table 3). Analysis of the ELISA data revealed that lab code 3 contributed 19

significantly to this GCV. As mentioned above, this was caused by a non-parallel result for sample 20

A and a high GCV for the analysis of sample C (Tables 4, A1 and A2). Because of these spurious 21

data, results from lab code 3 were excluded from the analysis of Vi 05 ELISA data and the overall 22

ELISA data. This improved the GCV of the Vi 05 ELISA to 67% and gave a GM value of 1.40 for 23

the potency of candidate IS 10/126 relative to Vi-IgGR1, 2011 or 37.2 EU mL-1

(Table 3). 24

25

The relative potency (RP) for candidate IS 10/126 by in-house ELISAs was calculated at 0.41 or 26

10.9 EU mL-1

with the highest GCV (77%). This may well reflect a greater variety in procedures 27

and reagents, such as locally produced Vi which originated from S. Typhi (lab codes 1.1 and 6.1) or 28

C. freundii (lab code 2.2) and the use of biotinylated Vi (lab code 9). 29

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The RP of candidate IS 10/126 based on all ELISA data, with exception of lab code 3, was 0.80 with 1

a 95% Confidence Interval (CI) of 0.52 - 1.24 and a GCV of 105% (Table 3). 2

3

The results of the in-house ELISAs and the Vi 05 ELISA showed considerable variation among 4

participating laboratories and this contributed to the wide range of RPs for candidate IS 10/126. The 5

cause of this variation is unclear. Several factors may have contributed to the variability of the 6

ELISA. 7

Analysis of reagent use and procedures did not reveal specific factors associated with a high or a 8

low RP of candidate IS 10/126. Poor binding of Vi to ELISA plates has been reported. In-house Vi 9

preparations are likely to vary in their physicochemical characteristics, for example the number of 10

repeating units or the level of O-acetylation and these may well influence the capacity of Vi to bind 11

to the solid phase. 12

13

Alternatives to improve binding of Vi include a pre-coating step (e.g. poly-L-lysine), the use of anti-14

Vi capture antibodies and the use of modified Vi, for example by tyramination or biotinylation (5, 15

11, 12). These modifications resulted in superior coating of ELISA plates and increased specificity 16

and sensitivity of the assay. Indeed in this study, the in-house ELISA based on biotinylated Vi had a 17

very low GCV and the signal to noise ratio improved compared to a conventional Vi ELISA (lab 18

code 9, personal communication). Pre-coated ELISA plates or the use of anti-Vi capture antibodies 19

were not reported in this study. 20

21

Lastly, there may be qualitative differences of the anti-Vi IgG response generated by immunisation 22

with Vi-rEPA or Vi-TT. The presentation of candidate IS 10/126 as a pool of sera could result in 23

increased non-specific binding compared to purified IgG of Vi-IgGR1, 2011. Candidate IS 10/126 24

contains a high titre of anti-LPS IgG, either as a result of vaccination or as a result of natural 25

infection. The presence of safe levels of endotoxin in unconjugated Vi vaccines has been associated 26

with anti-LPS IgG in sera of the recipients of these vaccines (19). This observation was confirmed 27

by analysis of B cell populations from recipients of unconjugated Vi vaccine using an enzyme-28

linked immunospot assay, which distinguished between B-cells that produced IgG directed against 29

Vi and B-cells that produced IgG directed against LPS of S. Typhi (7). Low levels of LPS are likely 30

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to be present in Vi preparations used for ELISAs in this study. LPS from S. Typhi may also be 1

present in Vi conjugate vaccines. 2

Therefore the anti-LPS antibodies may have contributed to the RP of candidate IS 10/126 measured 3

by several Vi ELISAs. Indeed analysis of the results of lab code 2.1 showed these were significantly 4

higher than those of lab code 2.2 (p<0.001, Table 3). Both ELISAs use the same procedure and 5

reagents but differ in the origin of Vi, namely Vi 05 from S. Typhi and Vi from C. freundii, 6

respectively. The difference in RP for candidate IS 10/126 can thus be explained by serological 7

differences between LPS of S. Typhi and LPS of C. freundii and additional reactivity of anti-LPS 8

IgG in the Vi 05 ELISA will be lacking in the ELISA based on Vi from C. freundii (19). It is 9

conceivable that sera from recipients of a Vi conjugate vaccine, who reside in areas that are endemic 10

for typhoid, will contain anti-LPS IgG and produce results similar to candidate IS 10/126 in ELISAs 11

based on Vi from S. Typhi. The presence of LPS in Vi preparations could also explain the variation 12

in antibody levels detected in the negative control (sample B). 13

14

Taken together, these observations emphasise the need for reference reagents such as Vi and the 15

need for further standardization of Vi ELISAs, in particular if these are used to analyse the 16

immunogenicity of experimental typhoid vaccines in clinical trials. 17

18

Stability studies 19

Participants 1 and 8 reported results for the stability of candidate IS 10/126 tested by lab code 1.2 20

and 8. Samples had been stored for 1418 days (appr. 3.9 years) at elevated temperatures +4oC, 21

+20oC and +37

oC and at -20

oC. Two samples from each temperature were tested in duplicate in 22

ELISA. The relative contents of the accelerated thermal degradation samples determined by 23

labcodes 1.2 and 8 were used to fit Arrhenius equations relating degradation rate to absolute 24

temperature assuming first-order decay and hence predict the degradation rates when stored at -20°C 25

(8). 26

27

The data from lab code 1.2 showed no significant loss of potency (results not shown). These data 28

were not used for further analysis. Data from lab code 8 showed estimated losses of potency of 29

0.14%, 0.60% and 2.27% per month at storage temperatures of +4oC, +20

oC and +37

oC respectively 30

WHO/BS/2014.2234

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(Table 5). Analysis of the dataset from lab code 8 gave a predicted loss of potency of 0.15% per year 1

when stored at -20oC. 2

3

Commutability studies 4

The fitness of candidate IS 10/126 for use in determining the anti-Vi IgG content of clinical sera will 5

need to be assessed using sera from clinical trials of Vi conjugate vaccines. Negotiations with 6

manufacturers are currently underway to exchange materials. 7

8

Recommendations 9

Candidate IS 10/126 consists of 0.5 mL freeze dried serum and is suitable for use in various Vi 10

ELISA formats. The overall GM of the RP of 10/126, excluding lab code 3, was calculated as 0.80 11

with a 95% CI of 0.52 - 1.24 (GCV 105%; n=13). The GM of the RP differed depending on ELISA 12

formats: the GM was calculated as 0.65 (GCV 15%; n=3) from the kit ELISA, as 1.40 (GCV 67%; 13

n=6) from the Vi 05 ELISA and as 0.41 (GCV 77%; n=4) from in-house ELISAs. We recommended 14

that 10/126 is established as the 1st

IS for anti-Typhoid capsular Vi polysaccharide IgG (human) 15

with an assigned potency of 0.5 IU per ampoule (equivalent to 1 IU mL-1

). The corresponding 16

unitage for Vi-IgGR1, 2011 is approximately 1.25 IU per ampoule, based on the GM of the overall 17

ELISA data, which showed an unexpected level of variability. 18

19

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Replies from participants 1

Replies were received from 7 out of 9 participants. Comments are in italics and actions are 2

indicated. All but one participant agreed that the discussion and interpretation are a fair 3

representation of the overall data presented. This participant pointed out that the unitage assignment 4

was based on variable ELISA results and a statement to this effect has been included in the 5

Recommendation section. The variation in RP of candidate IS 10/126 is an important outcome of 6

the CS and highlights the effect of different matrices of the reference material, the variation in 7

ELISA procedures and the lack of a ‘gold standard’ asay for the detection of anti-Vi. 8

Participant 2 remarked on the inconsistent use of the abbreviation S. Typhi asked for the affiliation 9

to be amended. Both have been addressed. Relevant comments and our replies are given below. 10

- ‘Suggest using US FDA or CBER or NIH Reference Reagent rather than US Reference 11

Reagent which does not provide any specific ownership of material. CEBR is sighted as the 12

supplier later in the document’ CBER reference reagent is used instead of U.S. reference 13

reagent throughout the manuscript. 14

- In the Recommendation section ‘... it would be advisable to also convert the anti‐Vi 15

protective level into IU/mL or alternatively, provide the μg/mL correlation of IS 10/126. In 16

the absence of an approved translation of units, each individual lab will be left to estimate 17

their own estimation of protection derived from the IS 10/126 data and the data presented by 18

Szu et al and could add additional level of confusion to the field.’ As pointed out above, the 19

variation of individual ELISA results was considerable and there are also differences in the 20

matrices to consider, namely pooled sera for 10/126 versus purified IgG for Vi-IgGR1, 2011. 21

Thus based on the average RP of 10/126, a general value in µg IgG mL-1

per IU mL-1

would 22

have no meaning, because the link between unitage and µg IgG mL-1

depends on the ELISA 23

of choice. This request was not introduced. 24

- ‘Table 1: No justification is provided for the difference in EU presented for the NIH 25

reactivity in Vi ELISA of NIH and VaccZyme.’ This is addressed by referring to the use of 26

two types of ELISA in the heading of the table. 27

- ‘Table 4: Was the statistical analysis of lab code 2.1 and 2.2 done to compare Citrobacter 28

with Typhi Vi? Why was this comparison not carried out between other ELISAs’ This 29

comparison was specifically carried out for 2.1 and 2.2 which use identical procedures, to 30

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tease out the effect of S. Typhi LPS that may be present in Vi of S. Typhi and which is 1

lacking from C. freundii Vi. This set of conditions does not apply to the other ELISAs which 2

differ in procedures and use Vi from S. Typhi. 3

- Stability studies. Table 5 only presents the results from Lab code 8 although the text 4

indicates information also derived from lab code 1.2.Suggest rewording. The reader is left 5

asking how the “data not shown” for lab code 1.2 contributed, did it corroborate that of lab 6

code 8? Where any biochemical, physiochemical testing done on the antibodies at various 7

temperatures or only ELISA? This has been clarified by adding the sentence ’These data 8

were not used for further analysis.’ Lab code 1.2 and 8 implies that only ELISAs were used 9

for the analysis. 10

- Need to define the criterion on which Non‐parallel and response range of reference were set. 11

The complete dose response curve is analysed. Non-parallelism is determined at 1% level. 12

Participant 3 asked for the affiliation to be amended. 13

Participant 4 remarked on ‘the inconsistent use of the prefix for 10/126 and the abbreviation S. 14

Typhi’ in the Summary. Both issues have been addressed. The apparent inconsistency on the number 15

of tests for the in-house ELISA and the Vi05 ELISA and the number of participants as given in the 16

have been addressed. The cut off for the Vacczyme ELISA was requested and has been given. The 17

affiliation was amended. 18

Participant 5 requested more information on the Vacczyme ELISA, which was added. The 19

affiliation was amended. 20

Participant 6 had no comments. 21

Participant 7 had no comments. 22

23

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Participant 9 noted typos and ambiguities which were acted on. Several comments were 1

regarding data analysis, the assignment of the IU and the variability of the ELISA results were 2

made. Other relevant comments and our replies are given below. 3

- In Recommendations ‘…present the relative potency as per assay, not the average. Or state 4

clearly the caveat that it represents an equivalence calculated from an average from very 5

different results.’ A statement to this effect has been included. 6

- In Summary and Recommendations‘… considering leaving out the equivalence of 1 IU/m 7

IS10/126 corresponding to 1.25 IU/ml Vi-IgGR1, 2011, which seems to have been calculated 8

from the GM of all ELISAs, correct? 1/0.80 (looking at Table 3) and the reason is that the 9

equivalence is totally unrealistic. For us, the equivalence is 1 IU/ml of IS 10/126= 4 IU/ml of 10

NIH Vi-IgGR1, 2011.’ We accept that the RPs varied considerably based on various ELISA 11

data. We qualified the assignment of IU to Vi-IgGR1, 2011 in ‘Recommendations’ and removed 12

the statement from ‘Summary’. 13

- In the Results and Discussion‘… such high level of IgG anti-LPS in the pool. These subjects 14

were not vaccinated with an LPS containing vaccine. I would not expect such high levels 15

(~104 EU/ml) of anti-LPS because of traces of LPS in the Vi vaccine. This may well explain 16

the high background in the negative control (sample B) in several of the assays. Perhaps an 17

important recommendation is for a thorough check up of LPS in the Vi preparations used for 18

coating.’ We agree with this hypothesis and have included two statements in the discussion 19

section. However a check on the presence of LPS in Vi preparations is not mentioned. 20

Instead we state ‘Taken together, these observations emphasise the need for reference 21

reagents such as Vi and the need for further standardization of Vi ELISAs, in particular if 22

these are used to analyse the immunogenicity of experimental typhoid vaccines in clinical 23

trials. ‘ 24

- ‘A link between IU mL-1

and µg mL-1

was thought to be ‘very useful to the field since 1) 25

many publications refer to micrograms per ml of anti-Vi; 2) the NIH standard is calibrated 26

in micrograms per ml.’ This point was also raised by participant 2. This association has not 27

been made because of differences in the matrices, namely pooled sera for 10/126 versus 28

purified IgG for Vi-IgGR1, 2011 and the variation between ELISA data as stated above. We 29

WHO/BS/2014.2234

Page 15

qualified the IU assigned to Vi-IgGR1, 2011 and to confirm an association between IU and µg 1

would contradict the ‘Recommendations’. 2

- It was noted that ‘The different relative potency for IS10/126 over Vi-IgGR1, 2011 reported by 3

the different assays may also derive from the fact that IS 10/126 is a pool of sera as opposed 4

to purified Vi and more likely to produce non-specific binding.’ This hypothesis has been 5

included in the ‘Discussion’ section to explain the variation in RP in different assays. 6

- ‘..plotted or tabulated the potency or titers of the positive samples over background 7

(negative sample B) for each of the labs. The reason is that if the background for the 8

negative sample is too high, then the values reported for the positive samples have to be 9

taken with caution.’ This analysis was not done for all lab codes, however the observation of 10

an uncharacteristically low A/B and C/B ratio provided a reason to exclude results from lab 11

code 3 from the analysis as stated in the Results and Discussion section. 12

13

WHO/BS/2014.2234

Page 16

Acknowledgements 1

We gratefully acknowledge the staff of the participating laboratories for their important 2

contributions, time, expertise and effort, which were indispensable for the completion of this CS 3

(see Appendix 2, Table A4). We are grateful to Mr R Venkatesan of BBIL for organising the 4

transfer and documentation of the source material for 10/126, to Dr SC Szu of National Institutes of 5

Health and to Drs J Kenney and J Cipollo of the Division of Biological Standards and Quality 6

Control, the Office of Compliance and Biologics Quality, CBER, the Federal Drug Administration 7

for making the reference reagents Vi-IgGR1, 2011 and Vi Lot 05 available. We would also like to 8

thank Dr G Meller Program Officer of Enteric and Diarrheal Diseases Global Health Program for 9

her support. Finally we thank Mr M Harris and Mr G Divall and their colleagues at the Centre for 10

Biological Reference Materials for processing 10/126, coding and packaging of samples and 11

organising the distribution of sample packs for the CS. 12

13

WHO/BS/2014.2234

Page 17

References 1

1. Bystricky S, Szu SC. O-acetylation affects the binding properties of the carboxyl groups on the Vi 2

bacterial polysaccharide. Biophys Chem 1994;51:1-7. 3

2. Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ 4

2004;82:346-353. 5

3. EDQM – Council of Europe. CombiStats v5.0. www.combistats.eu. 6

4. Felix A, Pitt RM. A new antigen of B. typhosus. Its relation to virulence and to active and passive 7

immunisation. Lancet 1934;227:186-191. 8

5. Ferry BL, Misbah SA, Stephens P, Sherrell Z, Lythgoe H, Bateman E, Banner C, Jones J, 9

Groome N, Chapel HM. Development of an anti-Salmonella typhi Vi ELISA: 10

assessment of immunocompetence in healthy donors. Clin Exp Immunol 2004;136:297- 11

303. 12

6. Jones C, Lee CK, Ahn C, Shin J, Knezevic I. Working Group on quality, safety and efficacy of 13

typhoid Vi capsular polysaccharide conjugate vaccines, Jeju, Republic of Korea, 5-7 September 14

2012. Vaccine 2013;31:4466– 4469 15

7. Kantele A, Pakkanen SH, Karttunen R, Kantele JM. Head-to-head comparison of humoral 16

immune responses to Vi capsular polysaccharide and Salmonella Typhi Ty21a typhoid vaccines-a 17

randomized trial. PLoS ONE 2013;8:e60583. 18

8. Kirkwood TBL. Predicting the stability of biological standards and products. Biometrics, 19

33:1977:736-742. 20

9. Lanh MN, Phan VB, Vo AH, Tran CT, Lin FY, Bryla DA, Chu C, Schiloach J, Robbins JB, 21

Schneerson R, Szu SC. Persistent efficacy of Vi conjugate vaccine against typhoid fever in young 22

children. N Engl J Med 2003;349:1390-1391. 23

10. Lin FY, Ho VA, Khiem HB, Trach DD, Bay PV, Thanh TC, Kossaczka Z, Bryla DA, 24

Shiloach J, Robbins JB, Schneerson R, Szu SC, Lanh MN, Hunt S, Trinh L, Kaufman JB. 25

The efficacy of a Salmonella typhi Vi conjugate vaccine in two-to-five- year-old Children. N Engl 26

J Med 2001;344:1263-1269. 27

28

WHO/BS/2014.2234

Page 18

1

11. Losonsky GA, Ferreccio C, Kotloff KL, Kaintuck S, Robbins JB, Levine MM. Development 2

and evaluation of an enzyme-linked immunosorbent assay for serum Vi antibodies for detection of 3

chronic Salmonella typhi carriers. J Clin Microbiol 1987;25: 2266-2269 4

12. Pickard D, Kingsley RA, Hale C, Turner K, Sivaraman K, Wetter M, Langridge G, Dougan 5

G. Genomewide mutagenesis screen identifies multiple genes contributing to Vi capsular 6

expression in Salmonella enterica Serovar Typhi. J Bacteriol 2013;195:1320–1326. 7

13. Rijpkema S, Durrani Z, Lemercinier X, Jones C. Detection of O-acetylated Vi polysaccharide 8

of Salmonella enterica subspecies typhi by enzyme immunoassay. Biologicals 2004;32:11-16. 9

14. Robbins JD, Robbins JB. Re-examination of the protective role of the capsular polysaccharide 10

(Vi antigen) of Salmonella typhi. J Infect Dis 1984;150:436-449. 11

15. Szu SC, Stone AL, Robbins JD, Schneerson R, Robbins JB. Vi capsular polysaccharide-protein 12

conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in 13

laboratory animals. J Exp Med 1987;166:1510-1524. 14

16. Szu SC, Taylor DN, Trofa AC, Clements JD, Shiloach J, Sadoff JC, Bryla DA, Robbins JB. 15

Laboratory and preliminary clinical characterization of Vi capsular polysaccharide-protein 16

conjugate vaccines. Infect Immun 1994, 62(10):4440-4404. 17

17. Szu SC, Hunt S, Xie G, Robbins JB, Schneerson R, Gupta RK, Zhao Z, Tan X. A human IgG 18

anti-Vi reference for Salmonella typhi with weight-based antibody units assigned. Vaccine 19

2013;31:1970–1974. 20

18. Szu SC, Klugman K, Hunt S. Re-examination of immune response and estimation of anti-Vi IgG 21

protective threshold against typhoid fever-based on the efficacy trial of Vi conjugate in young 22

children. Vaccine 2014;32:2359–2363. 23

19. Tacket CO, Ferreccio C, Robbins JB, Tsai C-M, Schulz D, Cadoz M, Goudeau A, Levine 24

MM. Safety and immunogenicity of two Salmonella typhi Vi capsular polysaccharide vaccines. J 25

Infect Dis 1986;154:342–345. 26

20. Thiem VD, Lin FY, Canh DG, Son NH, Anh DD, Mao ND, Chu C, Hunt SW, Robbins JB, 27

Schneerson R, Szu SC. The Vi conjugate typhoid vaccine is safe, elicits protective levels of IgG 28

anti-Vi, and is compatible with routine infant vaccines. Clin Vaccine Immunol 2011;18:730-735. 29

WHO/BS/2014.2234

Page 19

21. WHO Guidelines on the quality, safety and efficacy of typhoid conjugate vaccines. 64th

1

Meeting of the WHO Expert Committee on Biological Standardization, 21-25 October 2013. 2

WHO Typhoid BS2215 doc v1.14_Web_Version. 3

WHO/BS/2014.2234

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Table 1: Characteristics of samples used in this study

Study

code

NIBSC code Description of source material and vaccine

formulation

Average reactivity in Vi

ELISA in EU1,2

± SD (CV)

NIH Vi-IgGR1, 2011 Purified anti-Vi IgG from sera of five

volunteers who received 24 µg Vi-rEPA

(Lanzhou Institute of Biological Products,

China).

26.6 ± 1.2 (5%)1 and

1279.8 ± 128.3 (10%)2

A,C 10/126 Pooled anti-Vi sera from 9 volunteers who

received two doses of 25 µg Vi-TT (Bharat

Biotech International Ltd, India).

831.0 ± 109.4 (13%)2

B 01/576 Pool of seven normal human sera. 32.0 ± 1.4 (4%)2

1: ELISA units based on data from Szu et al. (2013).

2: ELISA units based on four tests by VaccZyme ELISA

Table 2: Assays used in this study

ELISA method Participant codes Lab code Total assays

In-house Vi ELISA

Vi 05 ELISA

VaccZyme ELISA

1, 2, 6, 9

1, 2, 3, 4, 5, 6, 10

4, 7, 8

1.1, 2.2, 6.1, 9

1.2, 2.1, 3, 4.2, 5, 6.2, 10

4.1, 7, 8

4

7

3

Table 3: Summary of relative potency of candidate IS 10/126 to Vi-IgGR1, 2011 by ELISA

method

In-house Vi ELISA VaccZyme ELISA Vi 05 ELISA All ELISAs

Lab

Code

GM Lab

code

GM Lab

code

GM GM excl.

Lab code 3

GM excl.

Lab code 3 1.1 0.24 4.1 0.60 1.2 0.65

2.2 0.73 7 0.59 2.1 1.011

6.1 0.61 8 0.77 3 3.78

9 0.25 4.2 2.68

5 1.55

6.2 1.32

10 2.13

GM

95%

CI

GCV

N

0.41

(0.16 – 1.01)

77%

4

0.65

(0.46 – 0.93)

15%

3

1.62

(0.93 – 2.81)

82%

7

1.40

(0.82 – 2.40)

67%

6

0.80

(0.52-1.24)

105%

13

1: p<0.001 compared to lab code 2.2.

WHO/BS/2014.2234

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Table 4: Potency estimates of 10/126 calculated from the geometric mean of coded duplicates

A and C

Lab

code ELISA

method Day 1 Day 2 Day

3 Day

4 GM GCV

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

1 1.1 In-house 0.25 0.27 0.27 0.28 0.24 0.22 0.22 0.21 0.24 12.6%

1.2 Vi 05 0.64 0.64 0.65 0.63 0.65 0.68 0.67 0.65 0.65 2.9%

2.1 Vi 05 0.95 0.67 0.98 0.94 1.12 1.17 1.19 1.21 1.011 21.9%

2.2 In-house 0.65 0.67 0.72 0.68 0.80 0.85 0.73 0.78 0.73 9.8%

3 Vi 05 NP 6.05 3.50 2.55 3.78 54.7%

4.1 VaccZyme 0.62 0.61 0.55 0.64 0.60 6.4%

4.2 Vi 05 2.76 2.80 2.59 2.56 2.68 4.6%

5 Vi 05 1.39 1.68 1.54 1.62 1.55 8.4%

6.1 In-house 0.43 0.45 0.56 0.54 0.69 0.83 0.68 0.82 0.61 28.9%

6.2 Vi 05 1.22 1.34 1.20 1.46 1.14 1.61 1.32 14.0%

7 VaccZyme 0.50 0.70 0.59 26.1%

8 VaccZyme 0.84 0.76 0.73 0.75 0.77 5.9%

9 In-house 0.26 0.24 0.26 0.26 0.25 4.3% 10 Vi 05 1.37 2.51 2.30 2.60 2.13 35.0%

NP = Non-parallel 1: p<0.001 compared to lab code 2.2.

Table 5: Results from candidate IS 10/126 stored at elevated temperatures

for 3.9 years

Lab code Temperature GM Relative Potency

1 95% CI

8 +4oC 0.901 0.866 – 0.938

+20oC 0.766 0.744 – 0.788

+37oC 0.339 0.324 – 0.355

1: Relative to 10/126 stored at -20

oC

WHO/BS/2014.2234

Page 22

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

100 1000 10000 100000

O.D

(4

50

nm

)

Dilution

A

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

100 1000 10000 100000

O.D

(4

50

nm

)

Dilution

B

Figure 1. Anti-Vi and LPS IgG titres of candidate IS 10/126, pre- and post-freeze drying.

1A: Anti- Vi IgG in liquid 10/126 ( ) and in freeze dried 10/126 ( ). 1B: anti-S. Typhi LPS IgG in

liquid 10/126 ( ) and in freeze dried 10/126 ( ) .

WHO/BS/2014.2234

Page 23

Figure 2. Relative potency of candidate IS 10/126 to Vi-IgGR1, 2011.

WHO/BS/2014.2234

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Appendices

Pg

Appendix 1:

Table A1: ELISA results for sample A as relative potency…………………………………………25

Table A2: ELISA results for sample C as relative potency…………………………………………26

Table A3: ELISA results for sample B as relative potency…………………………………………27

Appendix 2:

Table A4: Participants of the collaborative study………………………………………………...…28

Appendix 3:

Instructions for Use and Material safety Data Sheet for 10/126…………………………………….29

WHO/BS/2014.2234

Page 25

Appendix 1

Table A1: ELISA results for sample A as relative potency

Lab

code

Day 1 Day 2

Day

3

Day

4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

1 GM GCV

1.1 0.26 0.28 0.28 0.27 0.24 0.21 0.21 0.20

0.24 13.2%

1.2 0.63 0.63 0.65 0.62 0.66 0.69 0.67 0.66

0.65 4.0%

2.1 0.92 0.65 0.96 0.93 1.09 1.15 1.17 1.23

1.00 22.8%

2.2 0.61 0.65 0.69 0.72 0.75 0.85 0.78 0.78

0.72 11.5%

3 NP

6.24

NP NP 6.24 -

4.1 0.62 0.62 0.54 0.58

0.59 7.2%

4.2 2.68 2.78 2.55 2.77

2.69 4.1%

5 1.26 NP 1.43 1.68

1.45 15.6%

6.1 0.50 0.51 0.61 0.68 0.81 0.98 0.74 0.96

0.70 29.8%

6.2 1.19 1.48 1.17 1.72 1.25 2.05

1.45 25.2%

7 0.52 0.69

0.59 22.4%

8 0.80

0.73

0.68 0.77 0.75 7.2%

9 0.27 0.24 0.26 0.25 0.25 4.8%

10 1.06 2.62 2.45 3.37

2.19 65.2%

GM

95% CI

GCV

N

0.81

(0.52- 1.25)

106%

13

Empty cell: no assay performed

WHO/BS/2014.2234

Page 26

Appendix 1 continued

Table A2: ELISA results for sample C as relative potency

Lab

code

Day 1 Day 2 Day 3 Day 4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

1 GM GCV

1.1 0.25 0.27 0.27 0.30 0.25 0.22 0.22 0.22

0.25 12.7%

1.2 0.64 0.65 0.66 0.63 0.64 0.67 0.67 0.64

0.65 2.3%

2.1 0.98 0.68 0.99 0.95 1.15 1.18 1.21 1.19

1.03 21.1%

2.2 0.70 0.68 0.74 0.65 0.84 0.85 0.69 0.77

0.74 10.6%

3 NP

5.87

3.50 2.55 3.74 52.2%

4.1 0.61 0.60 0.57 0.71

0.62 10.0%

4.2 2.84 2.82 2.63 2.37

2.66 8.8%

5 1.54 1.68 1.66 1.55

1.61 4.5%

6.1 0.37 0.39 0.51 0.43 0.59 0.71 0.62 0.70

0.52 28.9%

6.2 1.24 1.22 1.22 1.24 1.04 1.26

1.20 7.2%

7 0.49 0.71

0.59 29.8%

8 0.87

0.78

0.79 0.73 0.79 7.2%

9 0.25 0.24 0.25 0.27 0.25 5.8%

10 1.76 2.40 2.17 2.00

2.07 14.1%

GM

95% CI

GCV

N

0.79

(0.51- 1.22)

105%

13

WHO/BS/2014.2234

Page 27

Appendix 1 continued

Table A3: ELISA results for sample B as relative potency

Lab

code

Day 1 Day 2

Day

3

Day

4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

2

Plate

3

Plate

4

Plate

1

Plate

1 GM GCV

1.1 0.02 0.03 0.03 0.03 0.02 NP 0.01 NP

0.02 42.2%

1.2 0.04 0.04 NP 0.04 NP NP NP NP

0.04 4.2%

2.1 0.02 NP 0.01 0.01 0.01 0.01 0.01 0.01

0.01 27.5%

2.2 NP NP NP NP NP NP NP NP

-

3 NP

1.61

0.81 NP 1.14 -

4.1 0.02 NP 0.02 0.01

0.02 5.0%

4.2 NP NP 0.41 0.43

0.42 3.7%

5 0.09 NP 0.10 NP

0.10 13.1%

6.1 0.22 0.26 0.25 0.26 0.10 0.11 0.10 0.10

0.16 59.3%

6.2 NP NP NP NP 0.13 0.17

0.15 20.8%

7 0.01 0.00

0.01 70.0%

8 RR

RR

RR RR

-

9 0.004 0.004 0.005 0.004 0.004 9.4%

10 NP NP NP NP

-

GM

95% CI

GCV

N

0.04

(0.01- 0.11)

359%

10

NP = Non-Parallel

RR = Outside of the response range of the reference

WHO/BS/2014.2234

Page 28

Appendix 2

Table A4: Participants of the collaborative study

Name Laboratory Country

Dr. D Chandran, Dr S

Prasad Chittineni

Shantha Biotechnics Limited – EOU -1, Athvelli

Village, Medchal Mandal, Ranga Reddy, Andhra

Pradesh

India

Head Dr A Bhardwaj,

Dr S Goel

Central Drugs Laboratory, Central Research

Institute Campus, Kasauli, District Solan H.P.

India

Mr K. Gopinathan Head of Quality Control, Bharat Biotech

International Ltd, Genome Valley, Hyderabad,

Andhra Pradesh

India

Dr LB Martin,

S Sanzone

Novartis Vaccines Institute for Global Health, Via

Fiorentina 1, Siena

Italy

Professor M Zeng

Dr Bin Wang

Institute for Biological Product Control, National

Institute for Food and Drug Control, No.2 Tiantan

Xili, Beijing

Peoples Republic

of China

Dr LY Ping The first research department, Lanzhou Institute of

Biological Products, 888 Yanchang Road Lanzhou,

Gansu

Peoples Republic

of China

Dr JS Yang Clinical Immunology, International Vaccine

Institute, SNU Research Park, San 4-8, Bongcheon-

7-dong, Kwanak-gu, Seoul

Republic of

Korea

Dr S Rijpkema, Ms A

Marwaha, Mr A Logan

Division of Bacteriology, National Institute for

Biological Standards and Control, Potters Bar

United Kingdom

Professor M Pasetti,

Mrs K Bowser, Mrs M

Reymann

Center for Vaccine Development, University of

Maryland Baltimore, 685 West Baltimore Street,

Room 480, Baltimore, MD

United States

Dr Nguyen T H Linh,

Dr L A Thu

National Institute for Control of Vaccine and

Biologicals/ Vien kiem dinh quoc gia vac xin va

sinh pham y te, Hoang Mai District, Ha Noi City

Viet Nam

WHO/BS/2014.2234

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Appendix 3

WHO International Standard

1st International Standard for anti-Typhoid capsular Vi polysaccharide IgG (human) NIBSC code: 10/126 Instructions for use

(Version 2.00, Dated 20/06/2014)

N/A 1. INTENDED USE Candidate International Standard (IS) 10/126 consists of a freeze dried serum pool taken from nine volunteers who received two doses of a Vi polysaccharide (Vi) tetanus toxoid conjugate vaccine as part of a Phase II trial. The Vi was derived from Salmonella enterica subspecies enterica serovar Typhi. Besides anti-Vi IgG, candidate IS 10/126 contains IgG directed against tetanus toxin and lipopolysaccharide of S. Typhi [1]. A collaborative study with 10 participating laboratories was designed to evaluate candidate IS 10/126 in Vi ELISAs and to compare its potency relative to the reference reagent for anti-Vi IgG: Vi-IgG R1, 2011 from the Center for Biologics Evaluation & Research (CBER), which contains 33 ug anti-Vi IgG/mL [2]. The collaborative study confirmed that candidate IS 10/26 is suitable for use in a Vi ELISA kit, in-house Vi ELISAs and the Vi 05 ELISA [1] The Vi 05 ELISA uses CBER reference reagent Vi lot 05 as coating agent [2]. For all ELISAs, the geometric mean of the potency of candidate IS 10/126 relative to Vi-IgG R1, 2011 was calculated as 0.80 (95% CI 0.52 – 1.25; geometric coefficient of variation 105%; n=13). The potency of candidate IS 10/126 relative to Vi-IgG R1, 2011 varied depending on the ELISA format and the participating laboratory [1] 2. CAUTION This preparation is not for administration to humans Human source material As with all materials of biological origin, this preparation should be regarded as potentially hazardous to health. It should be used and discarded according to your own laboratory's safety procedures. Such safety procedures should include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to avoid cuts. 3. UNITAGE

A provisional unitage of 0.5 IU per vial (1 IU/mL if reconstituted with 0.5 mL) has been assigned to candidate IS 10/126, PENDING FINAL CONFIRMATION by ECBS 4. CONTENTS Country of origin of biological material: India One vial contains the freeze dried residue of 0.5mL serum. The mean fill weight was 0.5181 g (CV of 0.19 %, n=78). Ampoules were back filled with pure nitrogen (moisture content <10 ppm). Residual moisture measured by the Karl-Fischer method for 6 ampoules was 0.23% (CV of 17.60 %, n=6) 5. STORAGE Vials should be stored at -20 C or below on receipt. Restored material should be stored at 4 C if used within 7 days. Unused contents should be aliquotted and stored at -20 C or lower. Please note: because of the inherent stability of lyophilized material, NIBSC may ship these materials at ambient temperature. 6. DIRECTIONS FOR OPENING Din Ampoule

WHO/BS/2014.2234

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7. USE OF MATERIAL No attempt should be made to weigh out any portion of the freeze-dried material prior to reconstitution The contents of the vial should be reconstituted with 0.5 ml distilled water using safety precautions as described above. It is recommended that the material be used in ELISA. A sequential dilution range (e.g. 1/100 to 1/800) of the material is prepared and tested in duplicate [1]. 8. STABILITY Reference materials are held at NIBSC within assured, temperature-controlled storage facilities. Reference Materials should be stored on receipt as indicated on the label. NIBSC follows the policy of WHO with respect to its reference materials. 9. REFERENCES 1 Rijpkema S, Hockley J, Last V, Marwaha A, Rigsby P. A WHO collaborative study to evaluate a candidate International Standard for anti-Typhoid capsular Vi polysaccharide IgG (human). Submitted 2 Szu SC, Hunt S, Xie G, Robbins JB, Schneerson R, Gupta RK, Zhao Z, Tan X. A human IgG anti-Vi reference for Salmonella typhi with weight-based antibody units assigned. Vaccine 2013;31:1970-4: 10. ACKNOWLEDGEMENTS We are grateful to Dr R Venkatesan of Bharat Biotech International Limited for his assistance in providing the source material for candidate IS 10/126 and to Dr Szu of the National Institutes of Health and Drs J Cipollo and J Kenney of CBER, Food & Drug Administration, Department of Health and Human Services for donation of reference reagents Vi-IgG R1, 2011 and Vi lot 05. 11. FURTHER INFORMATION Further information can be obtained as follows; This material: enquiries@nibsc.org WHO Biological Standards: http://www.who.int/biologicals/en/ JCTLM Higher order reference materials: http://www.bipm.org/en/committees/jc/jctlm/ Derivation of International Units: http://www.nibsc.org/products/biological_reference_materials/frequently_asked_questions/how_are_international_units.aspx Ordering standards from NIBSC: http://www.nibsc.org/products/ordering_information/frequently_asked_questions.aspx NIBSC Terms & Conditions: http://www.nibsc.org/terms_and_conditions.aspx 12. CUSTOMER FEEDBACK Customers are encouraged to provide feedback on the suitability or use of the material provided or other aspects of our service. Please send any comments to enquiries@nibsc.org 13. CITATION In all publications, including data sheets, in which this material is referenced, it is important that the preparation's title, its status, the NIBSC code number, and the name and address of NIBSC are cited and cited correctly.

WHO/BS/2014.2234

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14. MATERIAL SAFETY SHEET

Physical and Chemical properties

Physical appearance: Off White cake Corrosive: No

Stable: Yes Oxidising: No

Hygroscopic: Yes Irritant: No

Flammable: No Handling:See caution, Section 2

Other (specify): Material for human origin

Toxicological properties

Effects of inhalation: Not established, avoid inhalation

Effects of ingestion: Not established, avoid ingestion

Effects of skin absorption: Not established, avoid contact with skin

Suggested First Aid

Inhalation: Seek medical advice

Ingestion: Seek medical advice

Contact with eyes: Wash with copious amounts of water. Seek medical advice

Contact with skin: Wash thoroughly with water

Action on Spillage and Method of Disposal

Spillage of ampoule contents should be taken up with absorbent material wetted with an appropriate disinfectant. Rinse area with an appropriate disinfectant followed by water. Absorbent materials used to treat spillage should be treated as biological waste.

15. LIABILITY AND LOSS

In the event that this document is translated into another language, the English language version shall

prevail in the event of any inconsistencies between the documents.

Unless expressly stated otherwise by NIBSC, NIBSC’s Standard Terms and Conditions for the Supply of

Materials (available at http://www.nibsc.org/About_Us/Terms_and_Conditions.aspx or upon request by the Recipient) (“Conditions”) apply to the exclusion of all other terms and are hereby incorporated into this document by reference. The Recipient's attention is drawn in particular to the provisions of clause 11 of the Conditions. 16. INFORMATION FOR CUSTOMS USE ONLY

Country of origin for customs purposes*: United Kingdom * Defined as the country where the goods have been produced and/or sufficiently processed to be classed as originating from the country of supply, for example a change of state such as freeze-drying.

Net weight: 0.045 g

Toxicity Statement: Non-toxic

Veterinary certificate or other statement if applicable. Attached: No

17. CERTIFICATE OF ANALYSIS

NIBSC does not provide a Certificate of Analysis for WHO Biological Reference Materials because they are internationally recognised primary reference materials fully described in the instructions for use. The reference materials are established according to the WHO Recommendations for the preparation, characterization and establishment of international and other biological reference standards http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biolefstandardsrev2004.pdf (revised 2004). They are officially endorsed by the WHO Expert Committee on Biological Standardization (ECBS) based on the report of the international collaborative study which established their suitability for the intended use.