WHOfBSi05.2009 ENGLISH ONLY
EXPERT GQMhllXTTEE ON BIOLOCIGAL STANDAWLZATION Geneva, 24 to 28 October 2005
m P O R T OF A GOLLABOWTNE: STUDY TO ASSESS THE SUITABILITY OF G;INDIDATE WFEENGIE: MATERIAL TO SERVE AS THE FIRST WHO mFERENGE =AGENT DENGUE WRLIS MTIBODU AN13 TO S T M D A m I Z E THE
PU'EUTMLIZATION TEST FOR DEIVGUE VTRtjSES Morag ~ e r ~ o n " S u z a e ~ohnes' and AIan ~ e a t h '
'~ivision of Virolog?l and 2fnfomatics Laboratov N E S G
A collaborative study was undertdken to assess the suitability of a polyvalent mti-dengue virus 1 +2+3+4 s e w and 4 monovalent prepaations, mti-dengue virus types l, 2,3 and 4, to serve as International Standads. Thirteen participants in 6 countries took part in the study which was divided into two phase: In the first phase participants were sent coded samples which incIuded a duplicate sample of the candidate standad and a negative control. They also received Vero cells and 4 dengue virus serotypes. In the second phase, participants assayed uncoded smples using the method, cells and viruses in routine use in their laboratory. Data from a total of 32 assays on each of the 4 dengue virus serotypes were analysed. 50% Plaque reduction neutralisation titres were calculated for each smple when assayed against each virus and potencies relative to the candidate standard for results which were expected to be reactive.
O World Health Organization 2005
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WHOIBSI05.2009 Page 2
The expression of neutralising antibody titre relative to the candidate standard only marginally improved the percentage geometric coefficient of variation, a measure of the variation between laboratories and assays. The pofpafent anti-dense rims 1121-3~4 serum (NIBSC code 921 185) had reasonable titres against all four dengue virus serotypes tested, as did the anti-dengue 3 and 4
preparations. However, the mti-dengue f preparation is likely to be of too lols. titre to be usek1 b.
as it was scored be negative in some assays f ntroduction
At present there are no internationall)~ available reference materials i'or antibodies to d e n e e virus. The WHO Steering ComrniMee on Fla\~ivimses identified the need for reference materials in this Geld as it was enliisaged that the use of standards calibrated in lntemationaf Units would facilitate the comparison of data between laboratories and the responses to different vaccines.
The availabiliQ of such materials, along with Vero cells and 4 serotypes of d e n p e virus for use in dengue neutralization tests would facilitate the standardization of antibody assajrs for the detection of specific antibodies to dengue virus. These materials will be of use in the assessment of antibodies induced in vaccine trials as well as naturally infected individuals in diapostic
laboratories.
a
Naturally immune individuals with antibodies to either a single serotpe or all serotypes have been identified by Dr Sutee at Mahidof University, Thailand. Collaborative studies were undertaken to assess the suitability of a freeze-dried polyvalent plasma (designate NIBSC code 0211 86) to serve as the International Standard for dengue virus antibodies with an assigned unitage in International Units per ampoule and to assess the variatiorr in neutralisation assays for antibodies to dengue virus. There is no international conventional reference measurement procedure and this measurand is not traceable to SI. Although enzyme immunoassays are now being developed for diagostic use, no such assays were included in the studies undertaken so fw.
Materials
Candidate standard
The Candidate Standard for antibodies to dengue 1+2+3+4 is a batch of ampoules coded 02il86 containing freeze-dried pooled plasma obtained fiom two normal blood donors in Thailand. The
donations have been tested and found negative for HBsAg, antibodies to HIV 1+2, antibodies to
a
HCV and WCV RNA- This material was filled in ampoules and freeze dried at NIBSC following documented procedures. This material is stored at -20'6 at NIBSC. Details of this preparation are given in Table 1 .
Monovalent rnmarations
4 freeze dried monovalent plasma preparations have been prepared as a candidate reference panel. The materials were included in the study as coded smples. All constituent donations of these materials have been tested and found negative for WBsAg, antibodies to HTV 11-2, antibodies to HCV and HCV RXA. This material was filled in ampoules and freeze dried at YlBSC foliowing documented procedures. These matefials me stord at -205G. Details of the monovalent prepasations are listed in Table 1. In the studies in which coded samples were distn'buted, smples C, D, E and F contained antibodies to DENI, 2,3 and 4 respectively Additional sa-tadl: reparations
A freeze dried negative conh-ol s m p l e prepared from plasma taken from six US individuals prior to imunization with JE vaccine was included as study sample A. This material was filled in ampoules md tkeeze dried at NIBSC following docmented procedures and is stored at -20°C.
WH01BSf05.2009 Page 3 A coded duplicate of the candidate standard was also dist~buted to some participants as coded sample E3 to facilitate intra-assay variability to be assessed.
Participmts were asked to store all freeze-dried ampoules at -20° and to reconstitute in 0.5rnl distil led water.
A master cell bank comprising 20 mpoules was derived from Vero cells (Biowhittaker reference 76-1 08A, lot 8CB0657, NIBSG accession number NIfi3SG-011038.) sent to NIBSC in October 2001 for rnycoplasrna testing and ceIl banking. This bank was tested for mycoplasrna (culture and PCR), st&liV, kwologq. and iso e anatysis. 3 working cell b&s totally 500 mpoules have been prepared and characterised.
h p o u l e s of Vero cells from one of the working cell b d s were supplied to pwicipants in Phase 1 of the collaborative studies. They were asked to prepare a working eel1 bank and use these cells in their assays.
Viruses
Freeze dried stocks of each of the serotypes of dengue virus have been prepared by Dr Putnak, WRAIR. These are stored at -86 'C at NUBSC. A vial of each of the 4 dengue virus serotypes was supplied to each participant in phase 1 of the collaborative sstudies. Participants were asked to reconstitute each vial in lml and to produce a working virus stock. The virus serotypes and titres are listed in Table 2.
Accelerated degradation studies have been undertaken on the fi-eeze-dried anti-DEN 1+2+3+4 preparation 02i186. Samples of each preparation were stored at elevated temperatues (+4"C, +20°C and 137°C) for 27 months. These samples were assayed by Dr R Putnak, WRAFR.
Ampoules of 02/186 stored at -20°C and elevated tmperatures were also assayed for neutrdizing
a
antibodies to measles virus in a plaque reduction assay by Mrs M Bentley at NIBSC as we considered that there should be no diRerence in stability of neutralizing antibodies to the two enveloped viruses.Data were available fi-om a series of 3 independent assays of measles neukalising mtibody for 0211 86, and single assays (with triplicate dilution series) for neutralising antibody to dengue types l - 4 inclusive. The potencies of the samples stored at elevated tempaames were expressed relative to the sample stored at -20°G, along with approximate 95% confidence internals. The results are summarised in Table 3.
Tbe results are very v ~ a b f e , reflecting the poor precision of the neutralisation assay. The
congdence limits for the estimated potencies relative to the -20°C smple are wide, md &thou&
there are obsmed drops in potency at higher temperawres, as expected, only the +37"6 smples are simificmtly Iower than the baseline smple.
For the dengue assax the sample stored at 120°C for over 2 years still has an estimated activity of 70% or better than the - 2 0 T sample. For the measles assayy this figure is 6 1 %,
WHOISS/05.2009 Page 4
m e data was fitted to the usual Anhenius model. It was not possible to obtain any estimates for the measles or the dengue-1 or dengue-4 assays. For the dengue 2 and 3, a %loss per year of 0.46% and 2.90% respectively was predicted. However, the standard error of the estimates of the depradation rates were hi& relative to the actual estimate. Consernative upper confidence limits (95'6) for the predicted loss were 33% and 50% per year respectively. This does not indicate that the materials are unstable, but that the assay results are too variable for the Arrhenius model to provide useful infomatian.
Combining the data from the measles and the dengue 1-3 assays (the dengue-4 had very low MEP's and was not felt reliable) the predicted loss of potency when stored at -20° obtained from the Anhenius mode1 was 1.6% per year. If this material is accepted, continuing stability studies will be necessary.
No studies have been m d e ~ e n to assess the stability of the reconstituted candidate standard.
Horvever, previous experience with antibody standad vvhich are sawn or plasma, indicate that aliquots could be stored frozen for use at a later date
Design of study and assav methods
Collaborative studies were carried out in two phases. In the first phase samples were distributed coded to participants 1-8 in the tables and figures. In order to standardise the neutralisation assay method, participants were asked to perform assays using the method used in Dr R Putnak's laboratory and the Vero cells and serotypes of virus supplied. However, most participants used the method in routine use in their laboratory. Differences in methods included use of CMC or agarose as overlay and temperature/time of incubation of the virusiantibody mixture.
In the second phase, samples were distributed uncoded to additional laboratories with a specific interest in vaccine development. These laboratories used the assay methods in routine use which involved the use of the cell line and viruses in routine use..
Participants in both phases of the study were requested
>
to perform 3 independent assays for antibodies against each of the study viruseson different days, preferably 1 week apart.
P to prepare and test a series of dilutions from the candidate standard and each of
a
the coded samples.
P to include all study samples in each assay so that the concentration of antibodies relative to the candidate standard could be calculated
Participants
13 participants from 6 c o m ~ e s are partici-oating in the studies. All participants are listed in
Appendix 1. Pmicipants are identified by a code nmber, randomly assigned and not necessarily in
the order listed in the Appendix. Two of the participants s u b ~ t t d data &om assays undertalcen at c djfferent times and the se data are distinguished in the analysis i.e. l a b o r a t ~ ~ e s 6
and
10 are thes m e pmicipmt as are laborato~es 2 and 13,
\YHOiSS/05.2009 Page 5
Statistical Methods
Raw assay data in the form of replicate plaque comts over a series of dilutions were proirided by pasticipating laboratories. For each assay the mean plaque count for the virus conml was
calculated. The plaque counts for the test smples at each dilution were expressed as a proportion of the mean for the trims conml. Any counts greater than the virus control mean were set equal to the virus conml mean (so no propoftion &.as greater than l .01, If the mean of replicate proportions did not fall below 50% at any of the dilutions tested. the sample was considered negative for dengue mtibody for the particular serotype, and the plaque counts were not analysed fkrther. For positirre samples a 50% Plaque Reduction NeuCralisation Titre (PRNTS0) was
detemined using the probit metbod (Fimey, 1978). This represents the dilution at which tbe plaque count is expected to be 50% of that of the virus control mean. The probit calculations were perfomed independently for each sample, and no validity criterion were applied. W e r e the PRNTjo was calculated as <l 0, the sample was considered negative.
Geomehic mean PRNTsos were calculated across assays for each laboratory, virus serotype and sample. %'here a sample u7as negative or < 10 in an individual assay, the PRNTjO was set to 10 for calculating the laboratory mean. The variability between PRNTSOs was expressed as
geometric coefficients of variation f% GCVs) (Kirkwood: 1979). These were calculated across all individual assays, and reflect both between assay and between laboratory variation.
The potencies of the individual study samples relative to the candidate standard were calculated and expressed as a percentage (equivalently, taking the candidate standard as being l00 units of dengue antibody for the particular serotype). The potencies were calculated by taking the PRNTSo for the sample divided by the PRNTjo for the candidate standard, and multiplied by 100. The potencies were only calculated if the sample was positive. Laboratory mean estimates of potency were based on the ratio of the appropriate laboratory mean PRNTjOs.
An overall mean estimate was obtained from as the geometric mean of the laboratory mean estimates
.
Variability between relative potencies was expressed as % GCVs.Results Data Received
Data were received from eight laboratories in the first phase and seven in the second. Not all laboratories were able to undertake 3 assays on all dengue virus types. The number of assays against each virus serotype performed by each participant is given in Tables 4a-d.
Data from multiple assays submitted by laboratories 9 and 12 were inadvertently analysed as replicates from a single assay. In addition, data from an in-house control s a m was aMributed to being from sample B, the coded duplicate of the cmdidate standard sent to the participants in the first phase of the assay but not sent to Laboratory 12. Any reported results for sample B from this laboratory should be ignored
Samples were scored positive or negative, as described in the statistical methods. The negative results are indicated for each type in Tables 3a-3d. PRNTjO's were calculated for samples scored as positive as described in the statistical methods and the calculated PRNTSOs for the individual assays are shown in Tables 4a-d. The samples which are considered monovalent (see Table 1)
WL3(01BS/05.2000 Page 6
gave titres >l 0 for other viruses in some instances, In addition, the negative control semm (smpf e A) was scored positive in 5 assays g e ~ o m e d by laboratories 4 or 12 against types Den*e 3 or 4 (data not shown). These data were not considered k&her in the statistical analysis.
The calculated PRNTjos for the candidate standard md duplicate s m p l e B are ploned in Figures l a-d and for the monovalene smples C-F in F i m e s 2a-d. m e r e smples were fomd negati~~e, they are plotted at "4 0". The laboratory mean PMTTsos for the candidate standard, the coded duplicate smple B and the smple homologous to the \.ins serotype used are s b o ~ q in Tables 5a-d.
From the tables and fipres it c m be seen that sample C (monovalent smple for Dengue 1) is _found negative or of low titre by the majofity of laboratories. In addition sample D (monovalent sample for Dengue 2) is also of low titre, Both of these are considerabley lower titre than the titre of 0211 85 against the same virus. The range of PMTsos from individual assays md of the
laboratory mems obtained by the different pafticipants is very wide, orrer tenfold in most instances.
Relative Potencies
Potencies of the duplicate candidate standard, and the monovalent smples, were calculated relative to the candidate standard for each virus, as described in the statistical methods are listed in Tables 6a-d. Histogms of the potency for Sample B calculated from individual assays are shown in Figure 3a-d and for the monovalent samples in Figure 4a-d.
Overall geome-tric mean potencies are listed in Table 7. As the analysis of individual assays was undertaken at MBSG on two occasions, the overall mean potencies obtained in the two phases of the study were compared. These were in good agreement as can be seen in Table 6 (brackets).
Variabilip of assays witfun and between laboratories
%GCVs for PRNTSOs were calculated for each sample and dengue virus serotype with which it should be positive, The %GCV of individual PRNT50% are shown in Table 8. This is an
indication of overall assay variation, both within and between laboratories. Although as several laboratories only perfomed single assays, it includes a cctmponent of assay variability. The same calculations were performed for the potencies relative to the candidate standard
and
these values are shown in Tables 9.The PWTsos vs potency
The PRNTSns and potencies were reviewed to see whethef expression of potency improved the comparabiliq behtleen laboratories. The mean PRNTSo for the candidate standad and duplicate
smple B are shown in F i w e s la-d and relative potencies for sample B in Figure 3a-d. The i mem PRHTSo and relative potaeies the monovalent sarnples are shorn in Fipres 2a-d
and
4a-drespectively, Close inspection of the rmges of PRNTsos and potencies in these figures sumests that the expression of potency does not improve t~ariabi1it-y beween laboratories. However, the % GCVs of the relative potencies were margnally less than those fur the PR-?VlTSOs.
WH01BS105.2009 Page 7 Discussion and conclusions
Tihe candidate standard has reasonable titres against each d e n p e virus. The type
DEN
3 and 4 monovalent preparations appear to have antibody content approximately equal to or seater than the cmdidate standard. Wowe~~er theDEN
1 and 2 prepasations are of lower titre and theDEX
1 preparation gave negative results in some assays. These prqaralions were produced from the only plasma available and al&ou& they bad keen assayed in two laboratories, the results were divergent. Nevertbefess they were processed and included in the studyThe expression of potency relative to candidate standard DEN 1-t-2131-4 marginally reduced behTfeen laboratorfl vasiation for dengue 2 and 4 as indicated in Tables 8 and 9. Howeva, the interpretation of the GCV's is complicatd by the n m b e r of apparent negatives obtained.
S m p l e C, DEN 1, is of too low a titre to be able to detect any improvement. For s m p l e D,
DEN2,
smpleE, DEN
3 and s m p l e FDEN
4 there is margnal improvement thou& the use of relative potency. The majorie of laboratories were in reasonable ageement with the absolute PRNTsa3. The use of a relative potency is of greatest benefit for comparing results between laboratories that have big differences in assay sensiti\rity.The numbers of mpoules of the
DEN
1+2+3+4 prqaration 021186 is limited due to the amomt of serum available for processing at the time this project was initiated. Nevertheless, thispreparation will be of use in facilitating the standardization of neutralization assays for antibodies to dengue virus. The dengue type 3 and 4 sera may of use as reference reagents for specificity or monitoring assay sensitivity. However, the a s s i m e n t of a unitage based on the results of this study to these preparations is questionable due to the variation in assays between laboratories.
Efforts are currently undemay to identify alternative sources of plasma containing antibodies to
DEN
1+2+3+4.Proposal
As there are iasufficient ampoules of the anti-DEN 1+2+3+4 preparation 021186 available to allow long term usage, we propose that it be made available as a reference reagent with an assigned unitage of 100 units so that laboratories assaying sera from recipients of vaccines currently in development can compare results.
In addition, we propose that the monovalent anti-DEN-l, 2,s and 4 preparations be issued as a reference panel, but with no unitage assigned, for use in assay vatida~on studies.
References
Fimey, DJ. Statislical me&ods in biological assay. 3rd edn London: Cbasles Griffin 1978 G r h o o d , TBL. Geometric means and measures of dispmion, Biome~cs 1979; 35: 908-909 Figure legends
Figure 1 a-d PRNTsos of individual assays for the candidate standard and sample B, the coded duplicate of the candidate standard, for a
-
DEN I ; b -DEN 2
c - DEN3 md d-
DEN4Figure 2 a-d PWTSOS of individual assays for the conesponding monovalent smple for each dengue virus serotype for a
- DEN
I ; b -DEN
2; e -DEN3
and d -DEN4
WHOIBSIO5.2009 Page 8
Figure 3 a-d Ceomet~c mean potency of individual assay for the smple B, the coded duplicate of the candidate stasldard, eqressed as a percentage of the
c d i d a t e stmdard for a
-
DEN 1; b - DEN 2; c-
DEN3 and d - DEN4 F i p e 4 a-d Geornelsic mean potency of individual assays for thecoxesponding monovalent sample for each denme virus seratpe expressed as a percentage of the cmdidate standad for a
-
DEN l ; b - DEN 2; c - DEN3 md d - DEN4E
WHOISS/O5.2009 Page 9
Appendix
Dr Alan Barrett
Depafiment of Patholoa Room 4.130, Keiller Building 301 University Blvd
University of Texas Medical Branch Galveston TX 77555-0609
USA
Dr J Gardosa
hstitute of Health and Cornunity Medicine Unitlersity Malaysia Sarau7ak-i
Sarawak Kola Samrahan 94300 Malaysia
Dr Beth-Ann Coller Hawaii Biotech Inc., 99-1 93 Aiea Heights Drive Suite 200, Aiea,
Hawaii 9670 1, USA
Dr D Crevat
Global Clinical b u n o l o g y Aventis Pasteur
Discovery Drive Swifturater PA 18370 USA
Dr K Eckels (W) Department of Virus Diseases
Walter Reed h yhstitute of Research 503 Robert Grant Avenue, Suite 3A12 Silver Spring
MD 209 10-7500 USA
Dr Richard Kimey
hbovirus Diseases Branch Division Vector-borne diseases CDC
PO Box 2087
Fort Gollins, Colorado 80522
National hstitute of hfectious Diseases Department of Virolog?i
l -23- 1 Toyama Shinjuh-ku T O ~ O 162-8640
Japan
Dr M Mammen
Department of 'rliroioa 3 1513 16 R;?javithi Road Ban&ok 10400
Thailand Dr K Porter
Naval Medical Research Center W I R C ) , Department of Viral and Rickettsia1 Diseases 503 Robert Grant Avenue, Suite 3A12 Silver Spring
MD 2091 0-7500 USA
Dr R Putnak
Department of Virus Diseases
Walter Reed Army Institute of Research 503 Robert Grant Avenue, Suite 3A12 Silver Spring
MD 209 10-7500 USA
Dr Vance Vorndam
(current contact Elizabeth Hunsperger) Dengue Branch, CDC, Puerto Rico Centers for Disease Control (CDC) 2 Calle Canada
San Juan, Puerto Rico 00920 Puerto Rico
Dr Steve %%itehead
Laboratory of Infectious Diseases Bldg. TBIII, Room 3W-13E 12735 Twinbrook Parkway Betbesda, MD 20852 USA
Dr S Uoksan
LLlahidol University at Sataya Centre for Vaccine Developme&
251 15 Phutthamonthon 4 N&onpathom 73 170 nailand
WHOfBS105.2009 Page 10
Table 1 Details of candidate stand& for dengue 1+2+3+4 antibodies and monovalent preparations
*
pool of pre-bleed samples from people imunized with JE vaccine for preparation of candidate JE antibody standardTable 2 Dengue virus preparations supplied for use in the studies
1
virus/
TitreTable 3 PRNTSOs Erom assays on samples of the candidate DEN l+2+3+4 standard 0211 86 stored at different temperatures for 27 months
Measles 37°C I
* -
too low to measure in assay dilution rangeOlfQS105.2009 Page f 1
-ve = <l 0;
HT
= not testedOiBSf05.2009 Page 12
Table 4b
WEOiffS/05.2009 Page 13 Table 4c
WHOn&SfO5.20@9 Page 14
Table 4d
Table 4 Geometric Mean PRliiTSO's across
WHOIBSi05.2009 Page 15 Table 5c
assays for each laboratory f-br
a - D E N I: b - D E N 3 ; c-DEN3 a n d d - DEN4
Table 5a
I
;
Dengue Virus STD Sample Sample 11 Strain 1 i l
1
B C - \l L I 1
/
DEN l1
Lab l , 1 581
1
NT 15j
Table 5b
Lab 14 Lab 15
Dengue Virus Strain 2
Table 5d
NT = Kot tested
263
f
NT 1831
272Dengue Virus Strain 4
Lab 1 STD
103 223
STD
11 1 Sarnple
B
Sample D -
Sarnple B NT
Sample F - DEN 4
40
WHOIBS105.2009 Page 16
Table 5 Geometric mean potency across assays were for laboratory for samples B and the conesponding monovalent sample for each d e n s e virus serot).pe expressed as a
percentage of the candidate standard for a
-
Table 6c L)ENl;b-DEN?;c-DEN3mdd-DEN4 Table 6a
Table 6b
Table 6d
WHOISS/05,2009 Page 17
Table 7 Overall geometric mean potency of duplicate sample B and monovalent smples across labs & assays with the mean potencies from the two phases of the st-udy in brackets
Dengue Virus Straia
*Sample B was only included in 2 assays analysed in the Phase 2 study so only overall means reported
Table 8 Between laboratory %GCV for PRNTSOs calculated for each sarnple and dengue virus serotype that it should be positive with.
Table 9 Between laboratory %CGV for potency estimates cdculated for each sample and dmgue v i m serotype that it should be positive with.
WWO/BS/05.2009 Page 18
' 1
Figure l a
-
DEN 1Figure I c
-
DEN3"1
*%
i Figure 1 b - DEN 2
l
a
*l Figure 1 d
-
DEN 4Figure 2a
-
DEN lWHOISS/05.2009 Page 19
Figure 2b
-
DEN 24
l"!
*i
Figure 2c
-
DEN 3"1 Figure 2d
-
DEN 4%hiks
WH01SS105.2009 Page 20
Figure 3a
-
DEN 2"; Figure 3b
-
DEN 21
n, Figure 3c
-
DEN 3:j
li
16 5
1 2
b . l l S l S C R I M I I
wb%-(%i W a x y e T o w p ,
WH01IZSf05.2009 Page 21
Figure 4a
-
DEN 1":
Figure 4c
-
DEN 3* * Figure 4b
-
DEN 2Figure 4d
