WHO/BS/10.2134

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 18 to 22 October 2010

Report on a Collaborative Study to Assess the Suitability of a First

International Standard for Anti-Vaccinia plasma, Human

Maureen Bentley*, Peter Christian, Michelle Hamill and Alan Heath

National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South

Mimms, Potters Bar, Herts, EN6 3QG, UK

* Study co-ordinator: Email: mbentley@nibsc.hpa.org.uk, Tel:+44 (0) 1707 641302; Fax: +44 (0) 1707 641050

© World Health Organization 2010

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

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

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

WHO/BS/10.2134

Page 2

SUMMARY A candidate for the 1st International Standard (IS) for anti-vaccinia (plasma) was produced

from a pool of defibrinated human plasma. This candidate (NIBSC Code 05/124) was filled, lyophilized and sealed into ampoules in June 2005 and approximately 2700 ampoules are

available to WHO. Stability studies were carried out on the candidate using the Plaque

Reduction Neutralization Test (PRNT) using the 1st British Standard for anti-smallpox serum (assigned the value of 1000 units) as an interim standard. Stability studies over the course of

40 months indicated good stability, with no detectable loss in potency relative to the -20°C

reference at +4°C and only a 5% loss at +20°C after 1.4 years storage. The usual Arrhenius model for accelerated degradation studies gave an estimated percentage loss per year of

0.002% when stored at -20°C. A collaborative study was carried out to further characterize

the candidate standard and 9 laboratories from 7 countries participated in, and completed the study. The study comprised the candidate (in coded duplicate) and five other lyophilized

sera/plasma including the 1st International Standard for anti-smallpox and the homologous 1st

British Standard for anti-smallpox serum. Laboratories were invited to carry out assays by the methods that would normally be employed in that laboratory and in total six laboratories

returned data from PRNT and six from Enzyme-Linked Immunosorbent Assay (ELISA) (3

laboratories returned data from both methods). When calibrations were performed against the 1st British Standard for anti-smallpox serum highly significant differences were found across

all samples between the PRNT and ELISA results. Calibration against the candidate 1st

International Standard for anti-vaccinia reduced the differences between the assay methods and indicated that it would be an acceptable reference for the calibration of anti-vaccinia

plasma/serum. It is recommended that the candidate 1st International Standard (IS) for anti-

vaccinia (plasma) (05/124) be assigned the potency of 55 IU/ampoule. The data indicated that the candidate 1st International Standard for anti-vaccinia would not necessarily be suitable for

the standardization of anti-variola sera/plasma.

INTRODUCTION After the eradication of smallpox in 1977 interest in the standardization of anti-poxvirus titres

in human serum waned significantly until the beginning of the current century. Renewed fears

of the re-emergence of smallpox as a biological weapon and the consequent development, production, stockpiling and use of smallpox vaccines for the first time in over 30 years also

led to renewed interest in standardization of serum anti-poxvirus titres.

Prior to this resurgence in interest, standardization of anti-poxvirus titres had centered around

the First International Standard for anti-smallpox serum which was established in 1966. This

Standard was produced from a serum pool collected from smallpox-convalescent patients during the early 1960’s (WHO Committee on Biological Standardization; Eighteenth Report.

WHO Tech. Rep. Series No. 329 p18). As implied in the report of the Collaborative Study

used to establish this Standard (Anderson and Skegg, 1970) the International Standard was only generated from part of this pool. The remainder of the pool was used to make the First

British Standard for anti-smallpox serum (established in 1965) – which was assigned the same

unitage as the International Standard i.e. 1000 IU/ampoule.

The First International Standard for anti-smallpox serum was originally stored at the Statens

Serum Institute in Copenhagen and in 1997 a large number of standards and reference preparations that had been stored there were moved to NIBSC. At this time all transferred

material was checked for blood-borne pathogens, and in the case of the First International

WHO/BS/10.2134

Page 3

Standard for anti-smallpox serum it was found to be heavily contaminated with Hepatitis B surface antigen (HBSAg). As interest in this reference was considered to be minimal all

stocks were subsequently destroyed. Somewhat fortuitously, although stocks of the First

International Reference Preparation were destroyed it was not realized at the time that the 1st British Standard for anti-smallpox serum was essentially the same material and stocks of it

had not been destroyed (see Figure 1).

In 2002 the issue was raised whether there were any existing stocks of the anti-smallpox sera

and if it would be possible to produce a replacement and/or alternative reagent. The only

reagents that could be identified were the 1st British Standard for anti-smallpox serum and two ampoules of the 1st International Standard for anti-variola serum. Furthermore, both of

these were strongly positive for HBSAg, indicating that the existing reagents were not

suitable for future use and an alternative should be sought for standardization of anti-vaccinia reagents.

An approach was therefore decided upon in which we would first ascertain if available samples with suitable activity already existed and, failing this, if it was possible to identify a

suitable source of raw materials for a new reagent.

BULK MATERIALS AND PROCESSING Preliminary Studies Study Design In the absence of an existing anti-vaccinia standard the preliminary studies to identify/produce a suitable candidate were designed using the 1st British Standard for anti-smallpox serum

(equivalent to the 1st International Standard for anti-smallpox) as a surrogate reference for anti

vaccinia activity. In the original study to establish this reference (see Anderson and Skegg, 1970) the collaborating laboratories had used neutralization of vaccinia virus as the method

for calibration of the standard and so a methodological link to this material clearly existed.

However, although a link may well exist, where the unitage of the 1st British Standard for anti-smallpox serum was used to calculate potency in the current study these are referred to as

variola-units (VUs) rather than IUs to clearly distinguish them from the anti-variola activity of

the 1st British Standard for anti-smallpox serum.

The study was designed in two phases:

1) analysis of a number of existing filled and lyophilized human sera to ascertain if any of these had suitable activity for use as the intended reference. This study was carried out

using a Plaque Reduction Neutralization Test (PRNT) and a single challenge virus (Lister

Strain) 2) further characterization of materials identified in 1), or subsequently obtained from

alternate sources, against a broader range of challenge viruses in PRNT.

Results At the commencement of the study a number of filled and lyophilized human sera produced

prior to 1980 were available at NIBSC. There was a reasonable expectation that many of these products may have suitable levels of anti-vaccinia activity as the serum pools had been

derived from patients that had a high likelihood of having been vaccinated against smallpox

using vaccinia virus. Several of these sera were assessed for anti-vaccinia activity using a Plaque Reduction Neutralization Test (PRNT). In this assay vaccinia virus produces plaques

in sensitive cell cultures. Specific anti-vaccinia antibodies neutralize the virus and prevent

plaque formation. Equal volumes of dilutions of the test sera are mixed with a standard dose

WHO/BS/10.2134

Page 4

of challenge virus and after pre-incubation (1 hour, 37°C) the mixture added to the media overlay of a Vero cell monolayer. After incubation (4 days, 37°C) the cell monolayers are

stained and the plaques counted. Standard conditions for the assay were a target of 30 plaque-

forming units (pfu) added in a volume of 70 µl added to a cell overlay volume of 500µl. The final dilution of the serum in the pre-incubation sample that reduced the number of plaques by

50% (from a control pre-incubated in the absence of serum) was taken as the assay end-point

i.e. Effective Dilution 50% (ED50). The ED50 is used to calculate the potency of the sample in Variola Units (VUs) by comparison of the sample ED50 with the ED50 of the International

Standard (in this case the 1st British Standard for anti-smallpox serum).

Using the PRNT the sera tested in the preliminary studies were found to have detectable but

low levels of anti-vaccinia activity. Results are shown in Table 1. As these levels were much

lower than those in the First British Standard for anti-smallpox serum they were therefore not considered suitable for use as a candidate International Standard for anti-vaccinia.

Samples for suitable replacements were then sought from commercial plasma fractionators – and in particular those known to produce Vaccinia Immunoglobulins (VIGs). One

manufacturer agreed to provide three litres of plasma that had been collected from volunteers

specifically immunized with vaccinia (New York City Board of Health – NYCBH – strain) for the purposes of producing VIGs. This plasma was tested in PRNT at NIBSC (as described

above) to ascertain its suitability for production of the candidate. Tests on the candidate

plasma were carried out against four challenge viruses.

• Strain Lister. Produced on the flanks of sheep.

• Strain Lister. Cell adapted isolate produced in Chick Embryo fibroblasts. • Strain New York City Board of Health (NYCBH). Produced on the flanks of calves.

• Strain NYCBH. Clonal isolate produced in MRC-5 cells.

Results from this study are shown in Table 2. The plasma was considered suitable for the

intended purpose and consequently used for the production of the candidate IS.

Preparation of the Candidate IS The candidate NIBSC code 05/124 was produced from a pool of defibrinated plasma collected

from volunteers that had been immunized with the NYCBH strain of vaccinia virus (produced

in cell culture) specifically for the purpose of producing plasma to be used in the production of anti-vaccinia antibodies (VIGs). The plasma was filled, lyophilized and sealed into

ampoules at the Centre for Biological Reference Materials (CBRM) at NIBSC on 23/06/05.

Approximately 2700 ampoules are available to WHO.

The pool, in a single container, was received by CBRM on 23/06/05. The ampoules were ink

jet labelled before filling and the total number of ampoules filled on 23/06/05 was 3157. The mean weight of the fill was 1.0063gms (taken from a mean of 42 with check weights being

taken at approximately two minute intervals) with a coefficient of variation of 0.97%. Freeze

drying commenced 23/06/05 and was completed 28/06/05. Results for testing for vacuum, cracks or pin-holes showed nil failures. The mean dry weight of the fill measured by

coulometric Karl Fischer was 0.0722g (taken from a mean of 6) and the residual moisture

content 0.56%. The candidate material was filled in 2005 when testing for residual oxygen content was not

routinely carried out at NIBSC. Stability studies demonstrate that the material is very stable at

WHO/BS/10.2134

Page 5

normal storage temperatures and indicates that retrospective testing for residual oxygen content is unnecessary.

The plasma pool provided for preparation of 05/124 was tested and found negative for HBsAg, HCV antibody, HIV antibody and HCV RNA by PCR. The ampoules have been stored since

production at -20oC at NIBSC. A summary of the product characteristics are shown in the

table below.

Product Summary for the Candidate 1st International Standard for Anti-Vaccinia

(Plasma) (05/124)

Presentation Ampoule

Number of ampoules available 2700

Excipients/additives None

Coefficient of variation of the liquid fill 0.97%

Residual Moisture 0.56%

STABILITY STUDIES Accelerated Degradation Methods Samples of the candidate 1st International Standard for anti-vaccinia (05/124), were removed

from NIBSC storage at -20oC and maintained at -70oC, +4oC, +20oC and +37oC and +56oC for various periods. The overall accelerated degradation/stability study plan allows for the

assessment to be carried on into the foreseeable future.

Samples from a given temperature were tested by PRNT as briefly described above alongside

the candidate material that had been kept at -20oC. Each test comprised at least three

ampoules of the treated samples, 3 ampoules of the -20oC baseline and three ampoules of the 1st British Standard for anti-smallpox serum (stored at -70 oC). The test was completed over

three assays each of which comprised one ampoule of reference, one ampoule of -20oC

baseline and one ampoule of each sample being tested. Values for the ED50 for each ampoule from each time/temperature point were calculated along with geometric means of these values.

Relative potency as a percentage against the -20oC baseline were also calculated.

Results The geometric Mean ED50

and relative potencies of each time/temperature point relative to -

20°C sample (expressed as percentage) are shown in Table 3. The results reported in Table 3 cover a span of 1187 days and were generated between December 2006 and March 2010; a

total of approximately 40 months. These data show good stability for the reference with no

detectable loss in potency relative to the -20°C reference at +4°C and only a 5% loss at +20°C after 1.4 years storage. The Arrhenius-equation, which assumes a first-order model of

degradation, was applied to the data to estimate the long term stability. Predicted percentage

losses per month or year when stored at different temperatures are shown in table 4. The estimated percentage loss per year at -20°C is 0.002% and at +4°C it is 0.18%. The estimated

percentage loss per month is 0.2% at +20°C and 2.7% at +37°C. These figures indicate very

good stability for long term storage at -20°C. The figures for the higher temperatures also indicate that stability during transportation will not be a problem.

WHO/BS/10.2134

Page 6

Stability on Reconstitution Methods Samples were reconstituted in 1ml of distilled water and stored at +4 ºC before being assayed

alongside a freshly reconstituted sample. A PRNT as described above was used to estimate the ED50 for the samples.

Results After 4 and 5 weeks at +4 ºC, the reconstituted samples had ED50s of 347 and 309

respectively, compared with an ED50 of 339 for the untreated control; this indicates no

substantive loss over the 4-5 week time span at +4 ºC.

Effect of Heat Inactivation Methods Three ampoules of the candidate were reconstituted in 1ml of distilled water and then heat

treated at 56ºC for 30 minutes before assaying in PRNT against a reconstituted ampoule of the

candidate that had been kept at room temperature.

Results The heat inactivated samples had ED50s of 338, 331 and 309 respectively compared with the untreated control having an ED50 of 457. While this is a relatively limited study it does

indicate that there is a significant effect of heat inactivation on the candidate and that this

should be taken into consideration when the recommendations for its use are drawn up.

COLLABORATIVE STUDY Study Plan Aim of the study The aim of the study was to assess the suitability of a candidate International Standard (IS) for

the assay of anti-vaccinia antibodies and to assign a potency for the Standard in International

Units (IU).

Participants Nine Laboratories from 7 countries agreed to participate in the collaborative study, (2 National Control Laboratories, 3 manufacturers, 3 Public Health Laboratories and 1

University). Data were received from all laboratories.

Laboratories are referred to by a code number throughout this report. Code numbers were

allocated at random, and are not related to the order of listing in Appendix 1.

Materials and Methods Study samples A total of seven samples were included in the study and were selected to provide as wide a range of anti-vaccinia titres as possible. Initial analysis of the samples was carried out at

NIBSC using a PRNT with the Lister strain of the virus on Vero cells. The following were the

samples - with their relevant study codes – included in the study:

A. 1st British Standard for anti-smallpox. Derived from a pool of convalescent smallpox

patients. B. NIBSC 05/124 the candidate 1st International Standard for anti-vaccinia plasma.

Derived from plasma collected from volunteers vaccinated with cell-culture derived

vaccinia.

WHO/BS/10.2134

Page 7

C. NIBSC 82/528 a low anti-vaccinia titre serum originally filled as an anti-D.

pteronyssinus (dust-mite) human serum. Serum donors probably vaccinated with 1st

generation (animal-derived) smallpox vaccine.

D. Commercially produced vaccinia immunoglobulin diluted in defibrinated human plasma to produce a sample with intermediate anti-vaccinia titre. Derived from plasma

collected from volunteers vaccinated with cell-culture derived vaccinia.

E. NIBSC 05/124 the candidate 1st International Standard for anti-vaccinia plasma (a coded duplicate of sample B).

F. NIBSC 82/585 a low anti-vaccinia titre serum originally filled as a high concentration

cortisone human serum. Serum donors probably vaccinated with 1st generation smallpox vaccine.

G. 1st International Standard for anti-smallpox serum; 1 ampoule reconstituted in 1ml of

sterile distilled water and then diluted 1 in 10 in foetal calf serum. Derived from a pool of convalescent smallpox patients.

All of the samples with the exception of G were provided as lyophilized powders to be

reconstituted in 1ml of sterile distilled water. Sample G was provided as a frozen sample and comprised one ampoule of the 1st International Standard for anti-smallpox serum reconstituted

in 1ml of sterile distilled water and then diluted 1 in 10 in foetal calf serum. This sample was

provided in this way as only a single ampoule of the 1st International Standard for anti-smallpox serum was available and we wished to try and provide some link between the 1st

International Standard for anti-smallpox serum and the 1st British Standard for anti-smallpox

serum in the current study. The dilution was made in foetal calf serum (FCS) rather than human plasma as studies at NIBSC had indicated that FCS has no residual anti-vaccinia

activity. All samples were shipped to participants on dry ice and participants were requested

to store samples at or below -70oC.

Study design Participating laboratories were sent panels of coded samples, and requested to perform three independent assays on separate occasions, testing all samples in the panel concurrently.

Participants were requested to perform Enzyme Linked Immunosorbent Assay (ELISA)

and/or PRNT using the usual assay methodology employed in their laboratory.

Where a laboratory repeated assays using a variation in assay technique, these results were

analyzed separately and referred to by a separate code (e.g. 5A).

Analysis of Data Each of the assay methods employed in the study (ELISA or PRNT) were analyzed separately.

For ELISA methods the data were analyzed as multiple parallel line analysis on the linear section of the dose-response curve. If necessary, the response was also log transformed to

meet the required assumptions of linearity and parallelism. Where the data failed to fit these

models then a 4-parameter logistic regression dose-response model was used.

For the PRNT assays, the data were analyzed using a PROBIT method. Potencies of samples

relative to Sample A were then combined over the laboratories for each assay method using the un-weighted geometric means. Potencies relative to Sample B were also calculated in the

same manner. Estimates of ED50’s for all samples were also obtained using a Spearman

Karber method.

WHO/BS/10.2134

Page 8

Analysis of PRNT assays was carried out in Combistats (European Directorate for the Quality of Medicines http://combistats.edqm.eu/) whereas analysis of ELISA assays was carried out

using specially designed in-house statistical software.

The variation between assays within laboratories was assessed by calculating geometric

coefficients of variation (%GCV) (Kirkwood 1979) between assays of potency estimates for

each sample. An overall figure for each laboratory and method was obtained by taking a pooled estimate of the %GCV across the study samples. An estimate of the between

laboratory variability was obtained by calculating the %GCV of the individual laboratory

means for each method and sample.

The variation within assays between the coded duplicate samples B and E was assessed by

calculating the difference in log potency estimates of B and E within each assay, and calculating the root mean square (%RMS) average of this difference across assays (square

root of the average squared difference). This was then expressed in percentage terms as the

average fold difference (lowest to highest). For example, a two-fold difference between potency estimates of B and E in an assay would represent a 100% difference.

Assay Methods and Data Received Three laboratories returned data from PRNT alone, 3 laboratories from ELISA alone and 3 returned data from both PRNT and ELISA. Therefore a total of 6 laboratories returned data

for PRNT and 6 for ELISA.

Each laboratory was asked to carry out three assays using either the PRNT, ELISA or both

methods. They were requested to test all samples concurrently in each assay, and to perform

the three assays on separate occasions. Laboratories were asked to return all raw assay data for analysis by NIBSC. This was to ensure a uniform approach to the method of analysis.

PRNT

A range of conditions were employed by the participating laboratories using PRNT and these

are summarized in Table 5a.

Laboratories 1, 4, 6, 7 and 9 returned data from three assays and utilized a single challenge

virus strain across the three assays.

Laboratory 2 returned data from three assays for each of two challenge viruses. For the assays

involving one of the challenge strains (Laboratory 2A) the neutralization failed to reach 50%

in two of the assays of sample A. An ED50 could not be calculated for this sample in these assays. However, potency estimates relative to samples A and B could still be obtained using

the Probit model, which does not require all samples to attain the ED50.

ELISA

Assay conditions employed for the ELISAs are shown in Table 5b.

Laboratory 1 returned results where data from 2 assays with sample F could not be included

due to lack of linearity and parallelism.

Laboratory 5 used an in-house ELISA in which the plates were first coated with either

purified vaccinia B5 antigen, purified vaccinia A27 antigen or vaccinia-infected cell lysate.

The results from these are reported as 5A, 5B and 5C respectively. Data from 5A and 5C were

WHO/BS/10.2134

Page 9

analyzed using a 4-parameter logistic model. As the assays from 5A and 5B are substantially different from the assays otherwise employed in the study these data were not used in the

calculation of overall mean potencies for the study.

Laboratory 8 returned data which was analyzed using the 4-parameter logistic regression

dose-response model.

Laboratory 9 reported data from only 1 or 2 dose levels per sample and only 1 replicate per

dose level. These data were not used in the calculation of overall mean potencies for the study.

RESULTS PRNT Neutralization end-points Individual estimates of the ED50 from the PRNTs are provided in Appendix 2 (Table A1).

Potency relative to 1

st British Standard for anti-smallpox serum

The individual estimates of potency relative to the 1st British Standard for anti-smallpox

serum (assigned potency 1000 IU/ampoule) are given in Appendix 2 for both PRNT and ELISA. The laboratory geometric mean estimates of potency are presented in Table 6 for

PRNT and Table 7 for ELISA. The potency estimates are also shown in histogram form in

Figures 2a, 3a, 4a, 5a, 6a and 7a (PRNT – white boxes; ELISA shaded boxes).

The overall potency estimates for each of the samples included in the study were found to be

higher for the PRNT than for the ELISA (Table 8) with overall estimates from ELISA for any given sample ranging between 30.2% (Sample C) and 83.5% (Sample G) of the estimate from

PRNT.

While the number of samples and the number of laboratories included in the study is

relatively limited a number of general observations were made about the calibration relative to

1st British Standard for anti-smallpox serum. First with the PRNT, when a challenge virus produced on the flanks of animals was used (Laboratories 4 and 6) this appeared to produce

higher estimates of potency than challenge viruses produced in cells (Laboratories 1, 2A, 2B,

7, 9). For instance, the geometric mean estimates for Samples B, C, D and F were 80.34, 41.89, 151.42 and 27.61 with challenge virus from the flanks of animals whereas the

estimates were 43.08, 19.72, 121.77 and 12.76 with cell culture produced challenge virus.

Whether this is a real effect of the source of challenge virus or just laboratory to laboratory variation cannot be ascertained at the present time – although notably the preliminary data

generated on the candidate plasma (Table 2) indicates very little effect due to different

challenge viruses. Notwithstanding the latter, attention should be drawn to the possible effects of challenge virus in the Instructions for Use.

Similarly with ELISA it was also noted that the antigen used to coat the plates in indirect ELISA (see Table 5B) may also have an effect on the estimate of potency. For instance, the

geometric mean estimates of potency for samples of the candidate 1st International Standard

for anti-vaccinia (Samples B and E) were 35.23 and 35.72 if a coating antigen inactivated with β-propiolactone (BPL) was used (Laboratories 1 and 6 respectively) whereas untreated

cell lysates (Laboratories 3, 5C, and 8) produced estimates of 22.73 and 20.98. Differences

were less pronounced with other samples e.g. for geometric mean estimates of potency for C, F were 7.97 and 7.06 and 8.78 and 4.71 for BPL inactivated antigen and cell lysate

respectively. Again, such factors as coating antigen may affect the results obtained with

ELISA and this should be borne in mind when designing an assay.

WHO/BS/10.2134

Page 10

Within laboratories-between assay variability was assessed by calculating geometric

coefficients of variation (%GCV) between assays of potency estimates for each sample. An

overall figure for each laboratory and method was obtained by taking a pooled estimate of the %GCV across all of the study samples. These data are presented in Table 9 and show overall

estimates of within laboratories-between assay variability of 23.4% and 40.0% for ELISA and

PRNT respectively. These estimates are relatively high but in the case of the ELISA are driven by the very high value from one laboratory (65.7% from laboratory 9). Excluding this

laboratory the estimate for the ELISA is much lower i.e. 13.0% with a range of 4.7%-24.0%.

Estimates from the PRNT are slightly more uniform with a range of 20.8% -57.8% and no obvious outliers.

The variation within assays between the coded duplicate samples B and E was assessed by calculating the difference in log potency estimates of B and E within each assay, and

calculating the root mean square average of this difference across assays (square root of the

average squared difference). These data (expressed as a percentage) are shown in Table 10 and show overall estimates of within assay – between duplicate variability of 11.5% and

27.8% for ELISA and PRNT respectively. For the ELISA these estimates show relatively

good agreement between laboratories with a range of 4.2% to 19.5%. Again, the estimates from the PRNT are higher and show a range of 9.1%-47.3%.

Duplicate samples (B and E) were included in the current study and two distinct assay methods were employed in the study namely PRNT and ELISA. To calculate an overall

potency for the candidate 1st International Standard for Anti-vaccinia (05/124) the potency

data from the PRNT against the 1st British Standard for anti-smallpox serum for samples B and E were pooled as the mean potency for B and E from these assays were not significantly

different (paired t-test, untransformed data in Table 5, p=0.059). The geometric mean of the

pooled potency data was 55 VU/ampoule (%GCV = 56.7%).

Next the data from samples B and E were pooled from the ELISA studies as the mean potency

for B and E from these assays were not found to be significantly different (paired t-test, log transformed data in Table 6, p=0.937). The geometric mean of the pooled potency data was

27 VU/ampoule (%GCV = 50.5%).

While the potency estimates for the candidate duplicates B and E, relative to the 1st British

Standard for anti-smallpox serum, were not found to differ significantly for a given

methodology, the mean potency estimates for the two methodologies were significantly different (t-test, P<0.001). On this basis we next calculated potencies against the proposed

candidate rather than against the 1st British Standard for anti-smallpox serum to see if the

estimates between assay methods were improved.

Potency relative to Candidate 1st International Standard for anti-vaccinia

Potencies of the study samples relative to Candidate 1st International Standard for anti-

vaccinia were calculated using Sample B as the reference and taking an assigned value of 55 VU/ampoule. This value was used rather than an arbitrary assignment (as might normally be

made for a new standard) to allow comparison back to the assigned value of Sample A (and

also the related Sample G).

The individual estimates of potency from the PRNT relative to the Sample B are provided in

Appendix 2 (Table A4). The laboratory geometric mean estimates of potency are presented in

WHO/BS/10.2134

Page 11

Table 11. The potency estimates are also shown in histogram form in Figures 2b, 3b, 4b, 5b, 6b and 7b (PRNT – white boxes; ELISA - shaded boxes).

The “back estimate” for sample A is in good agreement with the assigned value of 1000 VU/ampoule and the estimate for sample G is in good agreement with that of sample A i.e. G

is a 1/10 dilution of the 1st International Standard for anti-variola and therefore directly related

to Sample A i.e. effectively a 1/10 dilution of Sample A. The overall estimate for the duplicate of the candidate, Sample E (62.9 VU/ampoule), is in agreement with the assigned

value of Sample B i.e. 55 VU/ampoule.

In the analysis of the data from calibration against 1st British Standard for anti-smallpox

serum it was noted that challenge virus may have an effect on the calibration of samples. In

particular, when challenge virus produced on the flanks of animals was used (Laboratories 4 and 6) this appeared to produce higher estimates of potency than challenge viruses produced

in cells (Laboratories 1, 2A, 2B, 7, 9). However, when this analysis was repeated with

potencies calculated relative to the candidate 1st International Standard for anti-vaccinia the differences for some of the samples was much reduced. For instance the geometric mean

estimates for Samples E, C, D and F were 54.65, 30.53, 103.66 and 18.86 with challenge virus

from the flanks of animals whereas the estimates were 66.47, 25.19, 155.50 and 16.30 with cell culture produced challenge virus. In contrast, for Samples A and G i.e. anti-smallpox sera

rather than anti-vaccinia sera/immunoglobulin, geometric mean estimates were 684.53 and

40.60 respectively with challenge virus from the flanks of animals and 1276.88 and 180.32 with cell-culture derived virus.

Calibration from the PRNT against Sample B produced within laboratories-between assay variability estimates (%GCV) that ranged between 15.1% (Sample E) and 128.2% (Sample G)

(Table 11 and 13). It is noticeable however that when within laboratories-between assay

variability estimates are compared between the calibrations against the 1st British Standard for anti-smallpox serum (Table 8) and against the candidate 1st International Standard for anti-

vaccinia that the overall estimates are generally lower for the latter and that the highest

estimates are obtained for the calibration of the anti-variola sera i.e. samples A and G.

The individual estimates of potency from the ELISA relative to the Sample B are provided in

Appendix 2 (Table A5) and the geometric mean estimates of potency in Table 12 and are summarized in Table 13. The potency estimates are also shown in histogram form in Figures

2b, 3b, 4b, 5b, 6b and 7b (PRNT – white boxes; ELISA - shaded boxes). Taking into account

that the original potency estimate for Sample B from the ELISA was approximately half of that from the PRNT (Table 8), the “back estimate” for Sample A (2030.7 VU/ampoule) is in

good agreement with the expected value i.e. 2 times the assigned value = 2000 VU/ampoule.

Again, for the ELISA alone there is good agreement between Sample A and G and the estimate for the duplicate candidate sample E (52.7 VU/ampoule) is in good agreement with

the assigned value for the candidate (55 VU/ampoule).

As with the estimates from the PRNT, the calibration from the ELISA relative the candidate

produced generally lower estimates of the within laboratories-between assay variability

(Table 13) than did the calibration against 1st British Standard for anti-smallpox serum, with the potency estimates for the two anti-variola sera (sample A and G) showing the highest

variability. These differences are shown in Table 14B and are summarized as the percentage

difference i.e. difference between the two methods divided by the mean of the two and

WHO/BS/10.2134

Page 12

expressed as a percentage. These data are shown alongside the differences from the initial calibration relative to the 1st British Standard for anti-smallpox serum (Table 14A).

The commutability of the reference has been demonstrated in the current study by its ability to produce broadly reproducible results in the two assay methods when used across a range of

samples. Notably, the low potency samples (C and F) originally identified in PRNT by

reference to the 1st British Standard for anti-smallpox serum proved to be low potency samples in the PRNT and the ELISA when calibrated against either the 1st British Standard

for anti-smallpox serum or the candidate 1st International Standard for anti-vaccinia. The

sample derived from purified VIG (sample D) also performed comparably in the two assays – relative to the other anti-vaccinia samples (B, C, E and F) – demonstrating that the candidate

was suitable for calibration of not only sera but also purified anti-vaccinia IgGs.

DISCUSSION The primary aim of the collaborative study was to assess the suitability of a candidate 1st

International Standard (IS) for the assay of anti-vaccinia antibodies and to assign a potency

for the IS in International Units. While there was no IS for anti-vaccinia at the commencement of the study an IS for anti-variola (smallpox) had previously been established.

These related orthopoxviruses show high levels of serological cross-reactivity and vaccinia

virus is routinely used as the vaccine for smallpox and it is the cross-protection between these two viruses that led to the eventual success of the WHO’s smallpox eradication campaign. On

that basis and given that preliminary studies demonstrated that the 1st British Standard for

anti-smallpox serum performed satisfactorily as an anti-vaccinia standard (e.g. see Table 2), we considered that the 1st IS for anti-variola and its parallel reagent, the 1st British Standard

for anti-smallpox serum, were able to act as surrogate IS to the current studies and hence

could provide a link between the 1st IS for anti-variola and the 1st IS for anti-vaccinia. It was on this basis that the collaborative study to characterise the candidate 1st IS for anti-vaccinia

was designed.

Potency relative to 1

st British Standard for anti-smallpox serum

Nine laboratories participated in the study with three performing the Plaque Reduction

Neutralization Test (PRNT), three the Enzyme Linked Immunosorbent Assay (ELISA) and three using both methods. Within this framework, the laboratories carrying out PRNT used a

relatively limited range of challenge viruses (see Table 5a) with 6 out of the 7 assays reported

using a Lister strain (albeit derived from a range of sources). Despite this limited variability in challenge virus used for the PRNT, the overall estimates for individual samples of variability

in potency against the 1st British Standard for anti-smallpox serum ranged between 34.5%

and 91.4% (Table 6 and 8).

For the ELISA assay a range of strains were used to produce the virus lysates used in primary

coating of the assay plates. In the case of Laboratory 5, data were also provided on the use of purified vaccinia antigens rather than cell lysates as primary coating antigens (see Table 5b

and A3 – Laboratories 5A and 5B). In these cases it was decided that the assays were

significantly different from the other ELISA assays used and so data from these assays were not used to calculate final potencies. However, it is of interest that the use of the purified

antigens produced results that were generally outside of the range of the results from the other

laboratories for most of the samples1 - with the B5 antigen tending to produce underestimates

1 Data from Laboratory 9 are excluded from this and other analyses as they did not meet the requirements for the number of dose levels and replicates needed to produce robust estimates of the potency.

WHO/BS/10.2134

Page 13

of potency and the A27 overestimates. Even though more virus strains were used in the ELISAs than in the PRNT it is noticeable that the overall estimates of potency variability

against the 1st British Standard for anti-smallpox serum for individual samples are generally

lower for the ELISA (range 12.6% to 60.5%) (Tables 7 and 8).

In a similar vein, both within laboratory-between assay variability (Table 9) and within assay-

between duplicate variability (Table 10) for the ELISA are lower than for the PRNT. Similar results have been noted previously for anti-measles serum when assayed in PRNT and ELISA

(WHO, 2006).

Overall, both assays employed in the current study gave internally consistent results against

the duplicated samples (B and E) and against similar samples (A and G). In the case of the

duplicated samples of the candidate each assay produced mean estimates of potency that were not significantly different. In the case of sample G, which was 1/10 dilution of the 1st

International Standard for anti-variola (the parallel fill of the 1st British Standard for anti-

smallpox serum) the PRNT and ELISA produced potency estimates of 110.22 and 92.1 VU/ampoule respectively – both of which are in good agreement with the predicted 100

VU/ampoule.

As duplicate samples of the candidate 1st International Standard for anti-vaccinia were

included in the study we attempted to calculate an overall potency against the 1st British

Standard for anti-smallpox serum. However, a significant difference was found between the mean potency estimated by PRNT and ELISA for the candidate 1st International Standard for

anti-vaccinia. This may not be surprising as the majority of the ELISAs used in the current

study measure the amount of anti-vaccinia IgG and not the amount of neutralizing anti-body. A very similar situation has been previously noted for anti-measles sera (WHO, 2006)

Potency relative to Candidate 1st International Standard for anti-vaccinia

The potency of the other samples relative to the candidate 1st International Standard for anti-vaccinia were calculated to ascertain if this provided greater consistency both within and

between testing methodologies than did the use of the 1st British Standard for anti-smallpox

serum. A value of 55 units per ampoule was assigned to the candidate 1st International Standard for anti-vaccinia as this provided a link to the 1st British Standard for anti-smallpox

serum.

Calibrations of samples relative to the candidate 1st International Standard for anti-vaccinia

also had levels of variability in the PRNT that was generally higher than from the ELISA

(Tables, 11 -13).

When samples were calibrated against the 1st British Standard for anti-smallpox serum,

estimates for all samples were greater in the PRNT than in the ELISA (Table 8). However, when calibrated against the candidate 1st International Standard for anti-vaccinia this was not

always the case. Samples A and G produced substantially higher estimates from the ELISA

than from the PRNT, but sample D showed only a relatively a small increase between the PRNT and ELISA (Table 13).

While such a difference for Sample A is not unexpected given the difference seen for candidate 1st International Standard for anti-vaccinia (Samples B and E) in the calibration

relative to 1st British Standard for anti-smallpox serum (close to a 2 fold difference), it is

notable that Sample G is effectively the same as Sample A in calibration relative to the

WHO/BS/10.2134

Page 14

candidate 1st International Standard for anti-vaccinia. What makes this more notable is the fact that Sample G is in fact a 1/10 dilution of the 1st International Standard for anti-variola

and therefore effectively a 1/10 dilution of the 1st British Standard for anti-smallpox serum.

Both samples A and G are therefore derived from the convalescent sera of smallpox patients, whereas the other samples are derived from human serum and plasma collected from donors

whose only poxvirus contact appears to have been with smallpox vaccine i.e. vaccinia virus.

In particular, samples B and E were derived from human plasma collected for the commercial production of vaccinia immunoglobulin (VIG) and sample D from a diluted commercial VIG

preparation. Samples C and F are pooled serum samples collected in the UK from human

donors in the late 1970’s/early 1980’s and therefore from donors that would in all likelihood had been vaccinated against smallpox.

When this is taken into account the contrast between the results between assays types from the calibrations relative to the 1st British Standard for anti-smallpox serum and those relative to

the candidate 1st International Standard for anti-vaccinia is quite obvious. The differences

between the PRNT and ELISA from the calibrations relative to the 1st British Standard for anti-smallpox serum range between 17.9% (Sample G) to 107.3% (Sample C) (Table 14A),

with an overall mean of 68.2% whereas those relative to the candidate 1st International

Standard for anti-vaccinia range between 7.9% (Sample D) and 62.1% (Sample A) with an overall mean of 37.3% (Table 14B). When the values for Samples A and G are excluded from

the latter comparison, the average percentage difference between the two methods amongst

the anti-vaccina sera is even lower at only 29.0%. Moreover, in this instance one of the 4 estimates (Sample D) is higher from the ELISA than from the PRNT – indicating no

consistent bias in the observed differences.

An interesting further insight into why the calibrations against the 1st British Standard for

anti-smallpox serum and the candidate 1st International Standard for anti-vaccinia is so

obviously different may be provided by the ELISA data from laboratories 5A and 5 B (Appendix 2 Tables A3 and A5). In particular in Table A5 and in Figures 2b and 7b, the result

for Sample A is a distant outlier to all of the other values. The ELISA in 5A used purified B5

antigen for coating the plates and the B5 antigen it is only present on the extracellular (EEV) form of vaccinia virus and not on the intracellular mature form (IMV). It has previously been

shown that B5 ELISA results correlate very well with neutralization of EEV (Putz et al, 2005)

and it follows that Sample A contains more neutralizing antibodies to EEV. This 5A ELISA result therefore suggests a quantitative difference in the sera of those infected with smallpox

(Samples A and G) compared to those infected (vaccinated) with vaccinia based on the

relative amounts of anti-EEV antibodies.

Taken together the data generated in the study lead us to conclude that:

1) While it was previously assumed, and indicated by PRNT studies, that the available anti-variola reference sera may be suitable surrogates for anti-vaccinia sera/plasma, this does

not prove to be the case when both PRNT and ELISA are taken into account,

2) The candidate 1st International Standard for anti-vaccinia when used as a calibrant of anti-vaccinia sera alone is a suitable reference material and is fit for the intended purpose in

either PRNT or ELISA assays.

Stability of Candidate 1st International Standard for anti-vaccinia

The candidate 1st International Standard for anti-vaccinia shows a good stability profile in the

lyophilized state (Tables 3 and 4) and after reconstitution and storage at 4ºC. The estimated

WHO/BS/10.2134

Page 15

percentage loss per year at -20°C is 0.002%. Overall the results of the stability studies indicate that the candidate material is suitable to serve as an International Standard.

Assigning a Unitage to the 1st International Standard for anti-vaccinia

One of the possibilities considered for the candidate 1st International Standard for anti-

vaccinia was to assign a separate unitage for the PRNT and for the ELISA. There are

precedents for assigning a separate unitage for a separate test e.g. see WHO (2008) for rabies vaccine. However, in the current case when potencies are expressed against the candidate the

overall spread of data for all samples (i.e. see Figures 2b-7b) do not appear to warrant having

separate unitages for the two assays. It was also suggested that for the PRNT a separate unitage be established based on the challenge virus to be used and/or that the challenge virus

be prescribed. Precedence for this also exists where a unitage for the 1st Ph. Eur. BRP

(European Pharmacopoeia reference) for anti-vaccinia was assigned only on the basis of data obtained for cell-culture derived Lister strains of the virus. In this case the instructions for use

prescribe the challenge virus to be used and that the unitage only applies to PRNT (Fuchs et

al., 2005). In this case however, there was a clear distinction between the cell culture derived and animal flank-derived challenge viruses in the potencies obtained. Again, no such

substantive difference was found in the current study.

Therefore, given the above considerations and our understating of the potential uses for an

anti-vaccinia serum reference we conclude that a single unitage is warranted by the data.

As 05/124 would be the 1st International Standard for anti-vaccinia it would be appropriate to assign it arbitrary unitage. However, from a historical perspective it would be valuable to

maintain the link through the PRNT to the 1st International Standard for anti-smallpox serum

and hence it is proposed that a unitage of 55 IU per ampoule be assigned to the standard. The Instruction for Use for this standard (see Appendix 3) would reflect the historical link

between the two standards and the intended use but should also reflect the possible limitations

of the reference – particularly in the analysis of anti-variola rather than anti-vaccinia sera.

CURRENT STOCKS AND PREDICTED USAGE OF THE PROPSED IS The previously established IS for anti-variola serum (and its parallel reagent the 1st British

Standard) has been used as a surrogate for anti-vaccinia and a small number of ampoules (<30)

have been distributed over the past five years on this basis. Certainly the heavy contamination of the anti-variola standards with Hepatitis B surface antigen mitigated against wider use and

so it may be expected that an uncontaminated IS for anti-vaccinia may find increased usage.

However, we expect this increased usage would be minimal in absolute terms and even with a ten fold increase in use we would anticipate that current stock levels of 05/124 (2700) would

last in excess of 20 years.

RECOMMENDATIONS It is recommended that:

1) that NIBSC 05/124 be established as the 1st International Standard for anti-vaccinia (Plasma).

2) that it be assigned a unitage of 55 International Units per ampoule.

WHO/BS/10.2134

Page 16

REFERENCES Anderson, S.G. and Skegg,J. (1970) The International Standard for anti-smallpox serum. Bull.

Wld. Hlth. Org. 42, 515-22.

Fuchs,F., Poirier,B., Leparc-Goffart,I. and Buchheit,K.H. (2005) Collaborative study for the establishment of the Ph. Eur. BRP batch 1 for anti-vaccinia immunoglobulin.

Pharmeuropa Bio. 2005 (1), 13-18.

Kirkwood, T.B.L. (1979) Geometric Means and Measures of Dispersion. Biometrics 35, 908-09

Pütz, M., Midgeley, C.M., Law, M., and Smith, G.I. (1006). Quantification of antibody

responses against multiple antigens of the two infectious forms of vaccinia virus provides a benchmark for smallpox vaccination. Nature Medicine, 12, 1310-1315.

WHO (2006) Report of a collaborative study to assess the suitability of a replacement for the

2nd international standard for anti-measles serum. WHO/BS/06.2031.

WHO (2008) Report of the WHO collaborative study to calibrate a candidate replacement for

the Fifth International Standard for rabies vaccine. WHO/BS/08.2087.

SUMMARY OF COMMENTS FROM PARTICIPANTS The first draft of the report was sent out for comments on the 15th June 2010 with a deadline

of 25th June for receipt of comments. All laboratories responded to the original draft of the

report.

In the light of comments returned by Laboratory 7 on 25th June, the report was revised and

sent back to participants on 28th June with a specific request for comments on the suggested revisions to the recommendations and on the desirability to include the full copy of the

comments from Laboratory 7 in the final draft. Participants were asked for responses to be

returned to NIBSC by 29th June.

Five laboratories replied to the revised draft, with none of these laboratories disagreeing with

the recommendation to proceed with single unitage for the candidate.

A final draft of this report was generated on 15th July 2010 that incorporated the revised

versions of the tables and text resulting from a reanalysis of the corrected data provided by laboratory 1 (see below).

Laboratory 1 Laboratory 1 made no comments on the recommendations in the first draft of the report and

made no additional comments on the revised draft. They did however note that they had made

two transcriptional errors relating to assays with Samples D and E when originally reporting their ELISA data. This error was detected and notified to NIBSC late in the preparation of the

first draft of this report. A full reanalysis of the data was not possible until after the original

submission to WHO. However, basic analyses of the revised data were performed and were found to have little overall effect on the study results. This version of the report (substantially

the same as the current version in terms of the data and text) was what participants

commented upon.

WHO/BS/10.2134

Page 17

Laboratory 3 A number of minor editorial changes requested. No comments on the recommendations. Final

draft revised in response to comments.

The laboratory made no additional comments on the revised draft.

Laboratory 5 Laboratory 5 made several comments relating to the variability between laboratories and that the nature of the virus used for the neutralization i.e. purified vs unpurified virus can have an

impact on the neutralization titre obtained. Likewise, it was also noted that the purity of the

virus used for producing coating antigen can have a similar effect in ELISA.

Laboratory 7 noted that the above points were in broad agreement with some of the points

made by Laboratory 1. No specific comment was made on the recommendations.

Laboratory 6 A number of minor editorial changes were requested along with some queries in relation to the figures. The draft was corrected in response to the comments. There was agreement with

the recommendations.

Supported the unchanged recommendations in the revised report and the responses to the

comments made by Laboratory 7 and suggested that the full text of the suggestions from

Laboratory 7 and the authors response be included in the final draft.

Laboratory 7 Laboratory 7 made a number of comments relating to some of the analysis and to differences between some of the materials included in the study and differences between the

methodologies used in different laboratories. They did not agree with the assignment of a

single potency for the candidate and suggested multiple potencies be assigned for the reference. The comments from Laboratory 7 are reproduced below in italics. Our responses

and comments are shown in normal text.

1) For the PRNT method: as a whole, the potency is two fold higher where challenge

virus strain is obtained from animals flank: for labs 4 and 6 the titre of sample B is

around 80 VU/vials instead of 45 VU/vial for the others labs where challenge virus

strain is cell-adapted (see both tables 5a and 6). These results confirm the potency

results obtained by the EDQM collaborative study carried out in 2005. Moreover, the

results of labs 4 and 6 are more consistent than those for the other laboratories: it

could be explained by the fact that except for sample G, all samples provide from

human sera vaccinated with the first generation of vaccine produced on animals flank.

2) For the ELISA method: as a whole, potency results for labs 3, 5C and 8 are more

consistent than those for labs 1 and 6 (see both tables 5b and 7). This could be

explained by the fact that the virus used as target antigen is an attenuated vaccinia

virus while inactivated β-propiolactone virus is used by labs 1 and 6. β-propiolactone

is known as a drastic inactivating agent for enveloped virus that can alter/destroy

more or less proteins at the surface of the viral membrane depending of the process

of inactivation. Consequently, the capacity of samples to react with an altered or a

non altered virus is not the same and moreover all sera provide from human

WHO/BS/10.2134

Page 18

immunized with vaccinia (live attenuated vaccine) and not with an inactivated

vaccine.

The above observations are generally correct, although given the overall number of samples in the study it is difficult to extrapolate from these observations too far. The latter is

particularly so in the case of the first statement as this relates primarily to calibration against

the 1st British standard for anti-smallpox. Once the calibration is made against the candidate 1st International Standard for anti-vaccinia, the apparent difference is removed (Sample E

Table 11). In addition, while there may be apparent differences between the estimates in the

collaborative study, when different challenge viruses are used, Table 2 indicates that removal of laboratory to laboratory variation removes the differences between results from different

challenge viruses. Additions to take account of these comments have been made in the report.

In relation to the statement regarding the ELISA results again, while generally correct it

relates primarily to calibration against the 1st British standard for anti-smallpox. Once the

calibration is made against the candidate 1st International Standard for anti-vaccinia, the apparent difference is removed (Table 12 Sample E).

Both of the above observations certainly highlight the difference between calibrations made against the 1st British standard for anti-smallpox and against the candidate 1st International

Standard for anti-vaccinia. Further studies on the effect of challenge virus and the type of

ELISA assay used would certainly be of value in avoiding problems with future reference materials such as that seen with anti-measles serum i.e. where highly significant differences

were found between methods (ELISA and PRNT) in calibrating a new reference (WHO, 2006)

For both methods, please note that results for samples D and G are aspecific compared

to the others maybe because sample D is not a serum but a purified immunoglobulin

against vaccinia virus and sample G is a serum against smallpox virus and not against

vaccinia virus.

It is very important to highlight that both methods are:

- able to discriminate a low anti-vaccinia titre serum (samples C and F) from high

anti-vaccinia titre sera

- reproducible (see results for samples B and E)

Some of the above comments are covered in the text and additions and revisions have been

made to cover the above points.

Taking into account these interpretations of the results and as clearly mentioned in the

report “a significant difference was found between the mean potency estimated by PRNT

and ELISA for the candidate 1st International Standard for anti-vaccinia. This may not

be surprising as the majority of the ELISAs used in the current study measure the

amount of anti-vaccinia IgG and not the amount of neutralizing anti-body", we are not

in favour of the assignment of a unique value (55 IU/vial) for both PNRT and ELISA

methods. We strongly suggest having:

- one assigned value for ELISA with careful recommendation in case of using

inactivated virus with BPL for target antigen

- one assigned value for PRNT. If we were purist we could request to have two different

values for PRNT : one for the method using virus challenge from animals flank and

one for the method using cell-adapted virus challenge.

WHO/BS/10.2134

Page 19

Again the above quote is in relation to the potency determined relative to 1st British Standard

for anti-smallpox serum. Once the calibration is made against the candidate 1st International

Standard for anti-vaccinia, the apparent differences are reduced between the two methods (Tables 14A and 14B) and also between challenge viruses for the PRNT. Additions have been

made to the Draft Instructions form Use to indicate the above possible problems and to draw

attention back to this study report.

In summary, while we agree that there are major issues with calibration of the candidate

against the 1st British Standard for anti-smallpox serum, by treating the candidate as a new product and assigning an arbitrary value, many of the problems with quantification of anti-

vaccinia sera/antibodies are removed. In our opinion a single assigned unitage for the 1st

International Standard for anti-vaccinia is appropriate and warranted.

The laboratory made no additional comments on the revised draft.

Laboratory 8 A few minor editorial changes requested and draft revised in response to the comments. No

comments on the recommendations.

The laboratory made no additional comments on the revised draft.

Laboratory 9. Clarification of the reasoning for some of the data exclusions was requested. No comments on the recommendations were made in either the initial or revised draft.

WHO/BS/10.2134

Page 20

Table 1. Geometric Mean ED50 of a range of candidate plasma/sera and the 1st British Standard

(Standard) shown with Geometric Mean Potency(in VUs) and Geometric Coefficient of Variation (%GCV) for those samples. Challenge virus was a Lister (Sheep skin) strain.

Samples selected for eventual use in the Collaborative Study are highlighted.

Sample

Geomean

Sample

ED50

% GCV

Sample

ED50

Geomean

Standard

ED50

Geomean

Sample

Potency

%GCV

Sample

Potency

65/140 Anti-measles; human serum

96.98 23.24 4807 20.26 37.62

66/161 Anti-rubella; human serum

70.79 27.18 5488 13.28 60.69

67/98 Immunoglobulin Human serum

151.36 24.56 4268 35.48 28.28

69/184 Post-menopausal human plasma

83.82 21.96 4268 19.65 24.46

75/526 Anti-mumps; human serum

6.41 27.64 5348 1.20 15.46

82/528 Anti-D.

pteronyssimus; human serum 192.01 26.09 5188 36.87 22.92

82/585 High cortisone; human serum

96.24 17.75 4786 20.11 17.75

90/662 Serum oestradiol (human)

183.37 49.24 5076 36.59 26.52

Table 2 Geometric Mean ED50 of the Candidate Plasma and the 1

st British Standard (Standard) shown

alongside the Geometric Mean and Geometric Coefficient of Variation (%GCV) of the Sample Potency (in IUs) when assayed using a range of vaccinia virus strains.

Virus Strain

Mean

Candidate

ED50

% GCV

Candidate

ED50

Mean

Standard

ED50

Geometric

Mean

Sample

Potency

%GCV

Sample

Potency

Lister, Sheep skin 855.76 13.68 5425 157.28 23.08

Lister, Chick embryo

fibroblasts 213.75 29.00 1667 123.03 41.04

NYCBH,MRC-5 cells 248.81 16.16 1997 133.86 27.36

NYCBH, Calf Skin 259.33 24.76 2026 129.82 9.75

WHO/BS/10.2134

Page 21

Table 3 Accelerated Degradation Study – all samples exposed at the temperature indicated in the left hand column for the time period shown in the upper row. A) Geometric Mean ED50 and B)

Potencies of samples relative to -20°C sample (expressed as percentage)

A.

26 days 49 days 113 days 243 days 525 days 1187 days

-20O

C 360.08 363.03 345.50 433.32 409.40 410.52

-70O

C - - 322.26 - 393.83 395.06

+4O

C - - 356.58 - 405.55 395.06

+20O

C 391.51 367.88 - - 389.05 251.19

+37O

C 366.78 323.52 - 360.30 - 145.66

+56O

C 256.62 129.80 102.40 86.43 -

B.

26 days 49 days 113 days 243 days 525 days 1187 days

-20O

C 100.00 100.00 100.00 100.00 100.00 100.00

-70O

C 93.27 96.20 96.23

+4O

C 103.21 99.06 96.23

+20O

C 108.73 101.34 95.03 61.19

+37O

C 101.86 89.12 83.15 35.48

+56O

C 71.27 35.75 29.64 19.95

Table 4 Estimated percentage (%) loss per year or per month from Arrhenius Model for accelerated

degradation

Temperature

(°C)

% loss per month % loss per year

-20 - 0.002

4 0.013 0.180

20 0.201 2.552

37 2.665 27.66

WHO/BS/10.2134

Page 22

Table 5a. Summary of cell line, challenge viruses and assay conditions employed in PRNTs reported in

the current study.

Lab

#

Cell

Line

Used

Challenge

Virus Strain

(Substrate)

Target

Number

Plaques

Neutralization

conditions

Serum

inactivation

Assay

conditions

1 Vero Lister (Chick

embryo fibroblasts)

80 60 minutes

37˚C 56ºC, 30’

3 days 37˚C

2A Vero Lister (Primary

rabbit cells) 50

37˚C, 15-18 hours

60ºC, 20’ 5 days 37˚C

2B Vero Lister (Vero) 50 37˚C,

5-18 hours 60ºC, 20’

5 days 37˚C

4 RK13 Lister (Calf

flanks) 75

60 minutes 37˚C

56ºC, 30’ 3 days 37˚C

6 Vero Lister (Sheep

flanks) 35

60 minutes 37˚C

None 4 days 37˚C

7 Vero Lister (Cell

adapted) 35

60 minutes 37˚C

56ºC, 30’ 2 days 37˚C

9 Vero IHD (Vero) 100 90 minutes

37˚C 56ºC, 30’

3 days 37˚C

Table 5b Summary of ELISA assays conditions employed in the current study.

Lab # Assay type Target antigen Detection antibody

1 Indirect in-

house

β-propiolactone inactivated vaccinia virus

(IHD-J strain).

Goat Anti-human IgG: alkaline phosphatase.

3 Indirect in-

house Vaccinia infected-cell

lysate (WR strain). Goat Anti-Human IgG: Horseradish Peroxidase.

5A Indirect in-

house Purified Vaccinia B5

antigen. Anti-human: alkaline

phosphatase.

5B Indirect in-

house Purified Vaccinia A27

antigen. Anti-human: alkaline

phosphatase.

5C Indirect in-

house Vaccinia infected-cell

lysate. Anti-human: alkaline

phosphatase.

6 Indirect in-

house

β-propiolactone inactivated vaccinia virus

(IHD-J strain).

Goat Anti-human IgG: alkaline phosphatase.

8 Indirect in-

house Vaccinia infected-cell

lysate (WR strain). Rabbit anti-human IgG; horse radish peroxidase.

9 Indirect in-

house

β-propiolactone inactivated vaccinia virus

(IHD-J strain).

Goat Anti-human IgG: alkaline phosphatase.

WHO/BS/10.2134

Page 23

Table 6.

The laboratory geometric mean estimates of potency (VU/ampoule) from PRNT relative to

the 1st British Anti-smallpox Standard (assigned potency 1000 IU/ampoule) presented along with the percentage geometric coefficient of variation (%GCV) . The overall results are

shown in the bottom row of the table. The duplicate samples of the candidate 1st International

Standard are shown highlighted i.e samples B and E.

Sample

B C D Lab. #

Pot %GCV Pot %GCV Pot %GCV

1 59.59 2.75 39.33 21.89 183.82 15.20

2A 53.26 38.70 24.72 36.42 128.75 57.92

2B 49.15 3.90 19.88 14.69 150.94 18.91

4 82.72 12.97 45.35 45.74 165.75 20.77

6 78.03 59.21 38.69 56.49 138.32 63.39

7 22.27 33.39 9.20 24.17 78.99 117.17

9 42.69 62.10 16.78 89.74 94.88 20.63

Overall 51.47 55.22 24.46 77.06 129.59 34.46

Sample

E F G Lab #

Pot %GCV Pot %GCV Pot %GCV

1 77.02 24.46 30.09 24.19 207.65 27.62

2A 55.84 32.30 15.10 31.01 80.90 44.34

2B 60.65 34.74 14.30 22.52 203.67 40.31

4 82.38 17.30 31.69 35.52 63.02 6.28

6 77.38 67.50 24.06 64.24 55.80 23.55

7 23.93 7.93 5.21 22.17 120.97 4.79

9 58.05 51.91 10.01 71.91 135.80 37.37

Overall 58.37 52.65 15.91 91.36 110.22 70.33

WHO/BS/10.2134

Page 24

Table 7.

The laboratory geometric mean estimates of potency (VU/ampoule) from ELISA relative to

the 1st British Anti-smallpox Standard (assigned potency 1000 IU/ampoule) presented along

with the percentage geometric coefficient of variation (%GCV). The overall results are shown in the bottom row of the table. The duplicate samples of the candidate 1st International

Standard are shown highlighted i.e samples B and E.

Sample

B C D Lab #

Pot %GCV Pot %GCV Pot %GCV

1 23.66 15.90 5.81 34.63 69.36 19.66

3 22.51 3.93 8.85 2.99 58.69 7.96

5C 19.26 6.48 7.15 5.79 50.84 4.38

6 52.47 11.82 10.93 19.26 154.80 22.26

8 27.09 17.80 5.56 16.90 68.24 3.06

Overall 27.08 47.59 7.41 33.01 73.78 54.19

Sample

E F G Lab #

Pot %GCV Pot Pot %GCV Pot

1 21.46 21.45 6.64 -(1) 95.90 24.97

3 20.59 9.65 4.97 1.22 103.29 8.08

5C 18.52 2.87 4.99 8.33 87.53 2.19

6 59.46 9.35 11.62 13.84 99.49 13.95

8 24.20 10.53 4.20 19.83 76.83 16.68

Overall 25.95 60.52 6.04 49.35 92.10 12.58

(1) ND = Value not determined due to only one value being available for the potency estimate.

WHO/BS/10.2134

Page 25

Table 8.

The overall geometric mean estimates of potency (VU/ampoule) from PRNT and ELISA

relative to the 1st British Anti-smallpox Standard (assigned potency 1000 IU/ampoule)

presented along with the overall percentage geometric coefficient of variation (%GCV). The duplicate samples of the candidate 1st International Standard are shown highlighted i.e.

samples B and E.

PRNT ELISA Sample

Pot %GCV Pot %GCV

B 51.47 55.22 27.08 47.59

C 24.56 77.06 7.41 33.01

D 129.59 34.46 73.78 54.19

E 58.37 52.65 25.95 60.52

F 15.92 91.36 6.04 49.35

G 110.22 70.33 92.10 12.58

Table 9.

Pooled estimates of the within laboratory – between assay %GCV for samples B to G calculated from potency estimates relative to the 1st British Anti-smallpox Standard (Sample

A).

Lab No. Method %GCV

1 ELISA 24.0

3 ELISA 6.4

5A ELISA 4.8

5B ELISA 4.7

5C ELISA 5.4

6 ELISA 15.6

8 ELISA 15.1

9 ELISA 65.7

Overall ELISA 23.4

1 PRNT 20.8

2A PRNT 40.6

2B PRNT 24.8

4 PRNT 25.7

6 PRNT 56.8

7 PRNT 43.4

9 PRNT 57.8

Overall PRNT 40.0

WHO/BS/10.2134

Page 26

Table 10. Within assay – between duplicate variability shown as the percentage Root Mean Square difference (%RMS) in potency estimates of Samples B & E relative to 1st British Anti-

smallpox Standard (Sample A).

Lab No. Method %RMS

1 ELISA 12.8

3 ELISA 10.8

5A ELISA 4.9

5B ELISA 4.2

5C ELISA 5.1

6 ELISA 14.9

8 ELISA 19.5

9 ELISA 10.9

Overall ELISA 11.5

1 PRNT 37.4

2A PRNT 11.4

2B PRNT 34.8

4 PRNT 9.1

6 PRNT 10.1

7 PRNT 25.4

9 PRNT 47.3

Overall PRNT 27.8

WHO/BS/10.2134

Page 27

Table 11.

The laboratory geometric mean estimates of potency (VU/ampoule) from PRNT relative to

the candidate 1st International Standard for anti-vaccinia (Study Sample B: assigned potency 55 VU/ampoule) presented along with the percentage geometric coefficient of variation

(%GCV) . The overall results are shown in the bottom row of the table. The duplicate sample

of the candidate (Sample E) is shown highlighted.

Sample

A C D Lab #

Pot %GCV Pot %GCV Pot %GCV

1 923.05 2.75 36.30 22.92 169.67 16.31

2A 1032.61 38.70 25.55 1.98 132.94 29.25

2B 1119.12 3.90 22.25 17.44 168.93 14.47

4 664.89 12.97 34.17 6.98 110.21 9.27

6 704.75 59.18 27.28 6.30 97.50 2.84

7 2469.46 33.39 22.72 7.69 195.06 66.26

9 1288.59 62.16 21.62 37.75 122.31 36.54

Overall 1068.54 55.22 26.61 23.21 138.49 29.06

Sample

E F G Lab #

Pot %GCV Pot %GCV Pot %GCV

1 75.09 25.81 27.77 23.51 191.67 25.12

2A 57.66 12.58 15.59 18.26 83.54 6.06

2B 67.88 29.72 16.01 21.60 227.96 43.60

4 54.77 11.19 21.07 20.05 41.91 6.86

6 54.53 12.46 16.88 52.01 39.34 43.79

7 2469.46 33.39 22.72 7.69 195.06 66.26

9 1288.59 62.16 21.62 37.75 122.31 36.54

Overall 1068.54 55.22 26.61 23.21 138.49 29.06

WHO/BS/10.2134

Page 28

Table 12.

The laboratory geometric mean estimates of potency (VU/ampoule) from ELISA relative to

the candidate 1st International Standard for anti-vaccinia (Study Sample B: assigned potency 55 VU/ampoule) presented along with the percentage geometric coefficient of variation

(%GCV) . The overall results are shown in the bottom row of the table. The duplicate sample

of the candidate (Sample E) is shown highlighted.

Sample

A C D Lab #

Pot %GCV Pot %GCV Pot %GCV

1 2324.38 23.16 13.50 17.03 161.48 23.22

3 2443.85 3.93 21.62 5.44 143.42 12.11

5C 2855.94 6.48 20.42 0.69 145.20 8.02

6 1048.20 11.82 11.46 6.72 162.27 16.25

8 2030.52 17.80 11.30 32.12 138.55 19.15

Overall 2030.66 47.59 15.05 36.79 149.87 7.51

Sample

E F G Lab #

Pot %GCV Pot %GCV Pot %GCV

1 49.98 8.57 13.39 -- 222.91 6.64

3 50.32 6.42 12.15 4.94 252.41 12.27

5C 52.88 3.81 14.25 2.05 249.99 7.90

6 62.33 7.76 12.18 24.29 104.29 4.69

8 49.13 18.35 8.52 28.00 156.00 11.10

Overall 52.72 10.24 11.92 22.06 187.03 46.29

WHO/BS/10.2134

Page 29

Table 13.

The overall geometric mean estimates of potency (VU/ampoule) from PRNT and ELISA

relative to the 1st International Standard for anti-vaccinia (assigned potency 55 VU/ampoule) presented along with the overall percentage geometric coefficient of variation (%GCV). The

duplicate sample of the candidate (Sample E) is shown highlighted.

PRNT ELISA Sample

Pot %GCV Pot %GCV

A 1068.54 55.22 2030.66 47.59

C 26.61 23.21 15.05 36.79

D 138.49 29.06 149.87 7.51

E 62.86 15.08 52.72 10.24

F 16.99 31.58 11.92 22.06

G 117.77 128.16 187.03 46.29

Table 14A

Differences between estimated potencies with PRNT or ELISA relative to 1st British Anti-smallpox standard (sample A). (Difference expressed as a percentage of average result of

PRNT and ELISA) The duplicate sample of the candidate (Sample E) is shown highlighted.

Sample PRNT ELISA Difference Difference (%)

B 51.5 27.1 24.4 62.1

C 24.6 7.4 17.2 107.3

D 129.6 73.8 55.8 54.9

E 58.4 26.0 32.4 76.9

F 15.9 6.0 9.9 90.0

G 110.2 92.1 18.1 17.9

Mean 68.2

Mean (Excluding G) 78.2

Table 14B Differences between estimated potencies with PRNT or ELISA relative to candidate 1st IS for anti-vaccinia (sample B) (Difference expressed as % of average result of PRNT and ELISA).

The duplicate sample of the candidate (Sample E) is shown highlighted.

Sample PRNT ELISA Difference Difference (%)

A 1068.5 2030.7 -962.1 62.1

C 26.6 15.1 11.6 55.5

D 138.5 149.9 -11.4 7.9

E 62.9 52.7 10.2 17.6

F 17.0 11.9 5.1 35.1

G 117.8 187.0 -69.3 45.5

Mean 37.3

Mean (Excluding A & G) 29.0

WHO/BS/10.2134

Page 30

(b)

(a)

(c) (c)

Figure 1. (a) Ampoules of the 1

st International Standard for anti-smallpox serum shown on

the left and that for the 1st British Standard on the right. The label of the 1st British Standard is held in place by a small rubber band and has been removed and is shown in (b). (c) A composite picture of the label from the 1st International Standard.

FIGURE LEGENDS (Figures 2-7)

Figures 2a-7a. Potencies of samples relative to the 1st British Standard for anti-smallpox

(Sample A). Results from PRNT are shown in the open boxes and those from ELISA in the

shaded boxes.

Figures 2b-7b. Potencies of samples relative to the Candidate 1st International Standard for

anti-vaccinia (Sample B). Results from PRNT are shown in the open boxes and those from ELISA in the shaded boxes.

WHO/BS/10.2134

Page 31

Fig 2a. Potency of Sample B (in VUs) relative to Sample A (1st British Standard for anti-smallpox

0

1

2

3

4

5

6

7

8

9

10

Potencies of B relative to A

0 10 20 30 40 50 60 70 80 90 100

5A 1

3

5C

7

8 9 2A

2B

6

1 5B 4

6

9

Fig 2b. Potency of Sample A (in VUs) relative to Sample B (Candidate 1st International Standard for anti-vaccinia

0

1

2

3

4

5

6

7

8

9

10

Potencies of A relative to B

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500

4

5B

6

9

1

2A

2B

6

9 8 1

3

7

5C 5A

WHO/BS/10.2134

Page 32

Fig 3a. Potency of Sample C (in VUs) relative to Sample A (1st British Standard for anti-smallpox

0

1

2

3

4

5

6

7

8

9

10

Potencies of C relative to A

0 5 10 15 20 25 30 35 40 45 50

1

5A

5C

8

3

6

7

9

9

2B 2A 1

5B

6

4

Fig 3b. Potency of Sample C (in VUs) relative to Sample B (Candidate 1st International Standard for anti-vaccinia

0

1

2

3

4

5

6

7

8

9

10

Potencies of C relative to B

0 5 10 15 20 25 30 35 40 45 50

6

8

9

1 2B

3

5A

5C

9

2A

6

7

5B 1

4

WHO/BS/10.2134

Page 33

Fig 4a. Potency of Sample D (in VUs) relative to Sample A (1st British Standard for anti-smallpox

0

1

2

3

4

5

6

7

8

9

10

Potencies of D relative to A

0 20 40 60 80 100 120 140 160 180 200 220 240 260

5A 1

3

5C

8

7 9 2A 5B

6

2B

4

6

1 9

Fig 4b. Potency of Sample D (in VUs) relative to Sample B (Candidate 1st International Standard for anti-vaccinia

0

1

2

3

4

5

6

7

8

9

10

Potencies of D relative to B

0 20 40 60 80 100 120 140 160 180 200 220 240 260

5B

6

4

9

2A

3

5C

8

1

1

2B

6

9

5A

7

WHO/BS/10.2134

Page 34

Fig 5a. Potency of Sample E (in VUs) relative to Sample A (1st British Standard for anti-smallpox

0

1

2

3

4

5

6

7

8

9

10

Potencies of E relative to A

0 10 20 30 40 50 60 70 80 90 100 110

5A 1

3

5C

7

8

2A

2B

6

9

5B 1

4

6

9

Fig 5b. Potency of Sample E (in VUs) relative to Sample B (Candidate 1st International Standard for anti-vaccinia

0

1

2

3

4

5

6

7

8

9

10

Potencies of E relative to B

0 10 20 30 40 50 60 70 80 90 100

1

3

4

5B

5C

6

8

2A

5A

6

7

9

2B

9

1

WHO/BS/10.2134

Page 35

Fig 6a. Potency of Sample F (in VUs) relative to Sample A (1st British Standard for anti-smallpox

0

1

2

3

4

5

6

7

8

9

10

Potencies of F relative to A

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0

5A 3

5C

7

8

1 9 6 2A

2B

9 6 1 4 5B

Fig 6b. Potency of Sample F (in VUs) relative to Sample B (Candidate 1st International Standard for anti-vaccinia

0

1

2

3

4

5

6

7

8

9

10

Potencies of F relative to B

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0

8 9 1

3

5A

6

7

9

2A

2B

5C

6 4 1

5B

WHO/BS/10.2134

Page 36

Fig 7a. Potency of Sample F (in VUs) relative to Sample A (1st British Standard for anti-smallpox

0

1

2

3

4

5

6

7

8

9

10

Potencies of G relative to A

0 20 40 60 80 100 120 140 160 180 200 220 240 260

4

6

2A

5C

8

1

3

5A

5B

6

7

9

9 1

2B

Fig 7b. Potency of Sample F (in VUs) relative to Sample B (Candidate 1st International Standard for anti-vaccinia

0

1

2

3

4

5

6

7

8

9

10

Potencies of G relative to B

0 50 100 150 200 250 300 350 400 450 500 550 600

4

5B

6

2A

6

9

8 1

1

9

2B

3

5C

7 5A

WHO/BS/10.2134

Page 37

APPENDIX 1

Sylvie Jorajuria, Sylvie Morgeaux, Isabelle Ramon

Agences des Produits Sante (AFSSAPS),

DLC 321, Avenue Jean Jaurès

69007 Lyon

FRANCE sylvie.jorajuria@afssaps.sante.fr

sylvie.morgeaux@afssaps.sante.fr

isabelle.ramon@afssaps.sante.fr

Shlomo Lustig, Tomer Israely, Nir Paran and Sharon Melamed,

Israel Institute for Biological Research

Department of Infectious Diseases, 24 Reuven Street

P.O. Box 19

Ness-Ziona, 74100

ISRAEL

shlomol@iibr.gov.il, tomeri@iibr.gov.il, nirp@iibr.gov.il, sharonm@iibr.gov.il

Hiroyuki Yokote The Chemo-Sero-Therapeutic Research

Institute (Kaketsuken)

1-6-1-Okubo Kumamoto 860-8568

JAPAN

yokote@kaketsuken.or.jp

Joy Bosica Cangene Corporation

155 Innovation Drive

Winnipeg, Manitoba CANADA R3T 5Y3

jbosica@Cangene.com

Ben Johnson, Geoffrey Smith

Section of Virology Division of Infectious Diseases

Imperial College London

St. Mary's Campus London W2 1PG

UNITED KINGDOM

b.johnson@imperial.ac.uk geoffrey.l.smith@imperial.ac.uk

Maureen Bentley, Peter Christian, Dept of Virology

National Institute for Biological Standards and Control (NIBSC)

Blanche Lane, South Mimms Herts EN6 3QG UNITED KINGDOM

maureen.bentley@nibsc.hpa.org.uk

Harry Vennema

National Institute for Public Health &

Environment (RIVM) A.van Leeuwenhoeklaan 9

3721 MA Bilthoven

THE NETHERLANDS harry.vennema@rivm.nl

Nadv Orr, Inbar Gahali-Sass, Orgad Laub

Omrix Biopharmaceuticals,

Weizmann Science Park Bldg 14 Nes-Ziona

ISRAEL

nadav@omrix.co.il, inbar@omrix.co.il orgad@omrix.il

Lasse Vinner Statens Serum Institut (SSI)

Building 85/41

5 Artillerivei DK-2300 Copenhagen S

DENMARK

LVI@ssi.dk

WHO/BS/10.2134

Page 38

APPENDIX 2

Table A1: Individual ED50s from PRNTs conducted as part of the current study. Assays indicated by an asterisk (*) did not neutralize below the 50% level. These data were not used

to calculate potency against the 1st British Standard (Sample A) which were calculated

directly from the probit analysis.

Sample Lab

No. Assay

A B C D E F G

1 2269 137 92 434 185 79 543

2 2225 129 102 457 206 70 416

3 2786 167 87 432 168 65 483 1.

Geomean 2414 143 93 441 186 71 478

1 104194* 3826 1816 8259 4235 1175 5520

2 90709* 6163 2846 12436 5639 1442 9239

3 44026 2667 1219 8646 3033 846 4319 2A.

Geomean ND(1)

2667 1219 8646 3033 846 4319

1 9679 460 224 1269 463 114 1904

2 11153 571 214 2025 945 157 1650

3 8927 435 158 1279 492 157 2593 2B.

Geomean 9877 485 196 1487 599 141 2012

1 1027 98 65 211 99 46 69

2 1465 116 72 210 103 43 88

3 1621 122 49 251 134 40 99 4.

Geomean 1346 112 61 223 111 43 84

1 3842 180 90 309 164 57 174

2 2621 227 120 411 258 105 181

3 1892 221 103 397 208 45 105 6.

Geomean 2671 208 104 369 206 65 149

1 4206 130 49 811 106 28 534

2 5139 91 39 278 127 24 594

3 5630 113 49 266 123 26 680 7.

Geomean 4955 110 45 391 118 26 600

1 2072 81 45 178 85 15 226

2 4291 119 35 363 221 31 505

3 1773 128 48 209 164 33 347 9.

Geomean 2507 107 42 238 146 25 341

All Geomean 3196(2)

258 122 649 292 80 548

(1) No value calculated as data from assays 1 and 2 were not used i.e. the assay did not reach 50% neutralization.

(2) Does not include data from Laboratory 2A.

WHO/BS/10.2134

Page 39

Table A2. Individual estimates from PRNT of potency (in VUs) relative to the 1st British

Standard for anti-smallpox (assigned potency = 1000 VU/ampoule).

Sample Lab # Assay

B C D E F G

1 61.17 42.33 193.14 81.54 36.25 274.44

2 57.94 45.72 205.22 92.64 31.67 186.80

3 59.69 31.43 156.70 60.48 23.72 174.65

Geomean 59.59 39.33 183.82 77.02 30.09 207.65

1

% GCV 2.75 21.89 15.20 24.46 24.19 27.62

1 36.72 17.43 79.26 40.64 11.28 52.98

2 67.94 31.37 137.09 62.17 15.89 101.86

3 60.57 27.69 196.39 68.90 19.22 98.10

Geomean 53.26 24.72 128.75 55.84 15.10 80.90

2A

% GCV 38.70 36.42 57.92 32.30 31.01 44.34

1 47.51 23.12 131.10 47.79 11.76 196.72

2 51.23 19.22 183.15 84.73 14.11 147.94

3 48.77 17.69 143.23 55.10 17.64 290.43

Geomean 49.15 19.88 150.94 60.65 14.30 203.67

2B

% GCV 3.90 14.69 18.91 34.74 22.52 40.31

1 95.02 63.10 205.15 96.41 44.44 67.58

2 78.87 49.11 143.28 70.07 29.05 60.40

3 75.53 30.09 154.94 82.75 24.66 61.33

Geomean 82.72 45.35 165.75 82.38 31.67 63.02

4

% GCV 12.97 45.74 20.77 17.30 35.52 6.28

1 46.92 23.34 80.54 42.78 14.80 45.25

2 86.80 45.70 156.71 98.41 39.92 68.99

3 116.71 54.38 209.83 110.00 23.26 55.73

Geomean 78.03 38.69 138.32 77.38 24.06 55.80

6

% GCV 59.21 56.49 63.39 67.5 64.24 23.55

1 30.82 11.69 192.73 25.28 6.57 126.88

2 17.79 7.67 54.18 24.67 4.63 115.56

3 20.15 8.69 47.20 21.92 4.66 120.74

Geomean 22.27 9.20 78.99 23.93 5.21 120.97

7

% GCV 33.39 24.17 117.17 7.93 22.17 4.79

1 39.07 21.60 85.84 41.14 7.48 108.92

2 27.68 8.11 84.54 51.46 7.15 117.66

3 71.90 26.96 117.84 92.41 18.72 195.44

Geomean 42.69 16.78 94.88 58.05 10.01 135.80

9

% GCV 62.10 89.74 20.63 51.91 71.91 37.37

WHO/BS/10.2134

Page 40

Table A3. Individual estimates from ELISA of potency (in VUs) relative to the 1st British Standard for anti-smallpox (assigned potency = 1000 VU/ampoule.

Sample Lab # Assay

B C D E F G

1 20.32 4.16 60.86 18.63 Excluded 74.14

2 23.90 6.40 85.56 19.78 Excluded 109.15

3 27.28 7.36 64.41 26.79 6.64 109.00

Geomean 23.66 5.81 69.36 21.46 6.64 95.90

1

%GCV 15.90 34.63 19.66 21.45 -- 24.97

1 21.80 9.10 63.35 20.03 4.98 108.77

2 22.26 8.58 58.70 19.10 5.03 107.24

3 23.49 8.87 54.35 22.82 4.91 94.46

Geomean 22.51 8.85 58.69 20.59 4.97 103.29

3

%GCV 3.93 2.99 7.96 9.65 1.22 8.08

1 9.64 3.72 32.74 9.17 2.26 91.57

2 8.88 3.36 32.64 8.91 2.09 95.68

3 9.19 3.77 32.91 9.82 2.35 98.02

Geomean 9.23 3.61 32.76 9.29 2.23 95.05

5A

%GCV 4.21 6.50 0.42 5.12 6.15 3.51

1 74.41 39.18 150.02 69.68 35.87 88.88

2 70.97 35.82 144.96 69.33 33.01 91.71

3 75.80 39.45 140.32 74.89 37.80 93.80

Geomean 73.70 38.11 145.05 71.26 35.51 91.44

5B

%GCV 3.45 5.53 3.40 4.41 7.07 2.74

1 20.40 7.54 52.33 18.85 5.41 87.82

2 18.01 6.74 51.89 17.92 4.61 89.29

3 19.44 7.19 48.40 18.79 4.98 85.53

Geomean 19.26 7.15 50.84 18.52 4.99 87.53

5C

%GCV 6.48 5.79 4.38 2.87 8.33 2.19

1 59.59 13.32 175.51 63.42 10.40 113.46

2 48.34 9.50 122.78 53.69 11.26 87.38

3 50.15 10.32 172.15 61.73 13.40 99.33

Geomean 52.47 10.93 154.80 59.46 11.62 99.49

6

%GCV 11.82 19.26 22.26 9.35 13.84 13.95

1 22.51 5.90 69.41 22.96 4.04 65.51

2 28.72 6.26 65.90 27.16 5.11 89.13

3 30.74 4.66 69.46 22.72 3.58 77.66

Geomean 27.09 5.56 68.24 24.20 4.20 76.83

8

%GCV 17.80 16.90 3.06 10.53 19.83 16.68

1 68.15 13.49 204.68 80.17 14.76 124.76

2 58.51 11.27 177.51 62.71 12.33 77.47

3 165.79 28.31 400.78 170.91 30.45 223.80

Geomean 87.11 16.27 244.19 95.07 17.70 129.33

9

%GCV 75.50 62.94 54.49 68.64 61.37 70.12

WHO/BS/10.2134

Page 41

Table A4. Individual estimates from PRNT of potency relative to the candidate 1st International Standard for anti-vaccinia (Study Sample B: assigned potency = 55

VU/ampoule).

Sample Lab # Assay

A C D E F G

1 899.13 38.06 173.66 73.32 32.59 246.76

2 949.26 43.40 194.81 87.94 30.06 177.32

3 921.43 28.96 144.39 55.73 21.86 160.93

Geomean 923.05 36.30 169.67 71.09 27.77 191.67

1

% GCV 2.75 22.92 16.31 25.81 23.51 25.12

1 1497.82 26.11 118.72 60.87 16.90 79.36

2 809.54 25.40 110.98 50.33 12.86 82.46

3 908.04 25.14 178.33 62.56 17.45 89.08

Geomean 1032.61 25.55 132.94 57.66 15.59 83.54

2A

% GCV 38.70 1.98 29.25 12.58 18.26 6.06

1 1157.65 26.76 151.77 55.32 13.61 227.73

2 1073.59 20.63 196.63 90.97 15.15 158.83

3 1127.74 19.95 161.53 62.14 19.89 327.53

Geomean 1119.12 22.25 168.93 67.88 16.01 227.96

2B

% GCV 3.90 17.44 14.47 29.72 21.60 43.60

1 578.83 36.52 118.75 55.81 25.72 39.12

2 697.35 34.25 99.92 48.86 20.26 42.12

3 728.19 31.91 112.83 60.26 17.96 44.66

Geomean 664.89 34.17 110.21 54.77 21.07 41.91

4

% GCV 12.97 6.98 9.27 11.19 20.05 6.86

1 1172.21 27.36 94.41 50.15 17.35 53.04

2 633.64 28.96 99.30 62.36 25.29 43.72

3 471.25 25.63 98.88 51.84 10.96 26.26

Geomean 704.75 27.28 97.50 54.53 16.88 39.34

6

% GCV 59.18 6.30 2.84 12.46 52.01 43.79

1 1784.56 20.86 343.94 45.11 11.72 226.42

2 3091.62 23.71 167.50 76.27 14.31 357.27

3 2729.53 23.72 128.83 59.83 12.72 328.56

Geomean 2469.46 22.72 195.06 59.04 12.87 298.43

7

% GCV 33.39 7.69 66.26 30.06 10.56 28.55

1 1407.73 30.41 120.84 57.91 10.53 153.33

2 1986.99 16.11 167.98 102.25 14.21 233.79

3 764.95 20.62 90.14 70.69 14.32 149.50

Geomean 1288.59 21.62 122.31 74.80 12.89 175.00

9

% GCV 62.16 37.75 36.54 33.44 19.16 28.55

WHO/BS/10.2134

Page 42

Table A5. Individual estimates from ELISA of potency relative to the candidate 1st International Standard for anti-vaccinia (Study Sample B: assigned potency = 55

VU/ampoule).

Sample Lab # Assay

A C D E F G

1 2706.69 11.26 164.67 50.43 Excluded 200.67

2 2301.26 14.73 196.90 45.85 Excluded 251.18

3 2016.13 14.84 129.86 54.01 13.39 219.76

Geomean 2324.38 13.50 161.48 49.98 13.39 222.91

1

%GCV 23.16 17.03 23.22 8.57 - 6.64

1 2522.94 22.96 159.83 50.53 12.56 274.42

2 2470.80 21.20 145.04 47.19 12.43 264.97

3 2341.42 20.77 127.26 53.43 11.50 221.17

Geomean 2443.85 21.62 143.42 50.32 12.15 252.41

3

%GCV 3.93 5.44 12.11 6.42 4.94 12.27

1 5705.39 21.22 186.80 52.32 12.89 522.44

2 6193.69 20.81 202.16 55.19 12.94 592.61

3 5984.77 22.56 196.96 58.77 14.06 586.63

Geomean 5957.91 21.52 195.20 55.36 13.29 566.31

5A

%GCV 4.21 4.30 4.11 5.99 5.03 7.25

1 739.15 28.96 110.89 51.50 26.51 65.70

2 774.98 27.76 112.34 53.73 25.58 71.07

3 725.59 28.62 101.82 54.34 27.43 68.06

Geomean 746.29 28.44 108.25 53.18 26.50 68.24

5B

%GCV 3.45 2.21 5.49 2.87 3.55 4.01

1 2696.08 20.33 141.09 50.82 14.59 236.77

2 3053.86 20.58 158.47 54.73 14.08 272.68

3 2829.22 20.34 136.93 53.16 14.09 241.98

Geomean 2855.94 20.42 145.20 52.88 14.25 249.99

5C

%GCV 6.48 0.69 8.02 3.81 2.05 7.90

1 922.97 12.29 161.99 58.54 9.60 104.72

2 1137.77 10.81 139.70 61.09 12.81 99.42

3 1096.71 11.32 188.80 67.70 14.70 108.94

Geomean 1048.20 11.46 162.27 62.33 12.18 104.29

6

%GCV 11.82 6.72 16.25 7.76 24.29 4.69

1 2443.36 14.42 169.59 56.10 9.87 160.06

2 1915.04 11.99 126.20 52.01 9.79 170.69

3 1789.20 8.34 124.28 40.65 6.41 138.95

Geomean 2030.52 11.30 138.55 49.13 8.52 156.00

8

%GCV 17.80 32.12 19.15 18.35 28.00 11.10

1 807.04 10.89 165.19 64.70 11.91 100.69

2 940.01 10.59 166.86 58.95 11.59 72.83

3 331.71 9.39 132.96 56.70 10.10 74.25

Geomean 631.34 10.27 154.18 60.02 11.17 81.66

9

%GCV 75.51 8.19 13.69 7.02 9.24 19.93

WHO/BS/10.2134

Page 43

APPENDIX 3

PROPOSED INSTRUCTIONS FOR USE

1st INTERNATIONAL STANDARD FOR ANTI-VACCINIA (PLASMA)

NIBSC CODE: 05/124

1. CAUTION

This preparation contains material of human origin which has been tested and found

negative for HBsAg, HCV antibody and HIV antibody.

As with all materials of biological origin, the preparation should be regarded as

potentially hazardous to health. The container and its contents should be used and

discarded according to your own laboratory procedures. Such procedures probably will

include the wearing of protective gloves and avoiding the generation of aerosols. Care

should be exercised in opening the containers to avoid cuts.

THIS MATERIAL IS NOT FOR ADMINISTRATION TO HUMANS

2. BACKGROUND

Prior to this resurgence in interest, standardization of anti-poxvirus titres had centered around

the First International Standard for anti-smallpox serum which was established in 1966. This Standard was produced from a serum pool collected from smallpox-convalescent patients

during the early 1960’s (WHO Committee on Biological Standardization; Eighteenth Report.

WHO Tech. Rep. Series No. 329 p18). As implied in the report of the Collaborative Study used to establish this Standard (Anderson and Skegg, 1970) the International Standard was

only generated from part of this pool. The remainder of the pool was used to make the 1st

British Standard for anti-smallpox serum (established in 1965) – which was assigned the same unitage as the International Standard i.e. 1000 IU/ampoule.

This is the first International Standard for anti-vaccinia (plasma) and is intended for use in the characterization of anti-vaccinia plasma and sera and was established through an International

Collaborative study (WHO, 2010) which included the 1st British Standard for anti-smallpox

serum. While the collaborative study to establish the current standard had involved (primarily) the neutralization of the vaccinia virus, the collaborative study for the current standard

demonstrated that it is not suitable for the calibration of anti-smallpox sera/plasma. For

further information on the relationship between the available anti-poxvirus references users are referred to the full collaborative study report (WHO, 2010).

3.UNITAGE

The reconstituted material will contain 55 IU anti-vaccinia activity per ampoule.

If you have any further questions concerning the unitage or use of this material then please contact NIBSC. A full copy of the collaborative study report is also available upon request

(WHO, 2010).

4. CONTENTS AND USE

4.1 Contents Each ampoule contains a freeze-dried residue comprising (under an atmosphere of nitrogen) human plasma containing antibodies against vaccinia virus. Each ampoule should be

WHO/BS/10.2134

Page 44

reconstituted in 1ml of distilled water.

4.2 Preparation of Standard

The candidate standard, NIBSC Code 05/124 was produced from a pool of defibrinated plasma supplied a commercial manufacturer of vaccinia immunoglobulins. The plasma was

filled, lyophilized and sealed into ampoules at NIBSC in June 2005.

The mean weight of the fill was 1.0063gms (mean of 42 ampoules) with a coefficient of

variation of 0.97%. The mean dry weight of the fill measured by coulometric Karl Fischer

was 0.0722g (taken from a mean of 6) and the residual moisture content 0.56%.

The preparation has been tested and found negative for HBsAg, HCV antibody, HIV antibody

and HCV RNA by PCR.

The ampoules have been stored since production at -20oC at NIBSC. A summary of the

product characteristics is shown below.

Product Summary for the Candidate 1st International Standard for Anti-Vaccinia

(Plasma) (05/124)

Presentation Ampoule

Excipients/additives None

Coefficient of variation of the liquid fill 0.97%

Residual Moisture 0.56%

4.3 Storage and Use

Unopened ampoules should be stored at -20°C or below until use. It is recommended that

samples be used as soon after receipt as possible.

After re-constitution samples may be aliquotted and stored at 2-8°C for further use. Studies have

shown that reconstituted samples are stable for up to 28 days at this temperature. For longer periods of storage recipients should use their own in-house criteria to determine the length of

time for which reconstituted samples can be retained.

The sample may be inactivated after reconstitution (56ºC for 30 minutes) without substantive

loss of activity. However, it is recommended that if the reference is treated in this way that all

samples to be calibrated/tested against the reference also be treated in a similar manner.

Please note that the 1st IS is provided as a reagent for calibrating your own in-house reference

material(s). With this in mind recipients should remember that the supply of this reagent will be limited to 3 ampoules per organization per year.

IT IS NOT INTENDED THAT THIS PRODUCT BE USED AS A WORKING

REFERENCE AND SHOULD ONLY BE USED TO CALIBATE YOUR OWN

REFERENCE.

In addition, this Standard is intended for use in the characterization of anti-vaccinia plasma and sera and was established through an International Collaborative study (WHO, 2010) that involved the use of Plaque Reduction Neutralization Test (PRNT) and Enzyme-linked Immunosorbent assays (ELISA) to characterize the candidate reference. When using this

WHO/BS/10.2134

Page 45

reference in either of these methods for calibration of samples care should be taken in the choice of factors such as challenge viruses, coating antigens and other assay conditions and the user is referred to the Collaborative Study Report (WHO, 2010) for further details. Samples of some of the other materials used in the original collaborative study may also be available from NIBSC.

5. DIRECTIONS FOR OPENING THE DIN AMPOULE

DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow ampoule stem

joins the wider ampoule body.

Tap the ampoule gently to collect the material at the bottom (labelled) end. Ensure that the

disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the

disposable ampoule breaker covering the ampoule stem between the thumb and first finger of

the other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily using the hand holding the plastic collar.

Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for example, by the use of suitable gloves and an eye shield. Take care that no material is lost

from the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas

at slightly less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN ampoule.

6. CITATION

In all publications in which this preparation is used as an assay calibrant, it is important that

name and address of NIBSC are cited correctly.

7. PRODUCT LIABILITY

7.1 Information emanating from NIBSC is given after the exercise of all reasonable care

and skill in it compilation, preparation and issue, but is provided without liability in its application and use.

7.2 This product is intended for use as a standard or reference material in laboratory work in relation to biological research, manufacturing or quality control testing of biological

products or in the field of in vitro diagnostics. It is the responsibility of the user to

ensure that he/she has the necessary technical skills to determine the appropriateness of this product for the proposed application. Results obtained from this product are likely

to be dependent on conditions of use and the variability of materials beyond the control

of NIBSC.

NIBSC accepts no liability whatsoever for any loss or damage arising from the use of

this product, whether loss of profits, or indirect or consequential loss or other wise, including, but not limited to, personal injury other than as caused by the negligence of

NIBSC. In particular, NIBSC accepts no liability whatsoever for:

a) results obtained from this product; and or

b) non delivery of goods or for damages in transit.

WHO/BS/10.2134

Page 46

7.3 In the event of any replacement of goods following loss or damage, a customer accepts as a condition of receipt of a replacement product, acceptance of the fact that the

replacement is not to be construed as an admission of liability on NIBSC’s behalf.

REFERENCES WHO (2010). Report on a Collaborative Study to Assess the Suitability of a First

International Standard for Anti-Vaccinia Plasma, Human

Anderson, S.G. and Skegg,J., 1970. The International Standard for anti-smallpox serum. Bull. Wld. Hlth. Org. 42, 515-522.

WHO/BS/10.2134

Page 47

1st INTERNATIONAL STANDARD FOR ANTI-VACCINIA (PLASMA)

NIBSC CODE: 05/124

MATERIAL SAFETY SHEET

Physical properties (at room temperature)

Physical appearance White/yellowish freeze-dried cake.

Fire hazard None

Chemical properties

Stable Yes Corrosive: No

Hygroscopic No Oxidising: No

Flammable No Irritant: No

Other (specify) None

Handling: For in vitro use only, not for administration to humans.

Toxicological properties

Effects of inhalation: Not established

Effects of ingestion: Not established

Effects of skin absorption: Not established

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 a

virucidal agent. Rinse area with a virucidal agent followed by water.

Absorbent materials used to treat spillage should be treated as biologically hazardous waste.