national institute for biological standards and control assuring the quality of biological medicines...
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National Institute for Biological Standards and ControlAssuring the quality of biological medicines
Development of an HIV-2 RNA International Standard
Harvey Holmes, Clare Morris, Neil Berry, Alan Heath and Collaborative Study Group
HIV-2: Background
• HIV-2 is a diverse group of viruses closely related to and thought to be derived from the SIV of sooty mangabey monkeys
• First isolated in 1986 from AIDS patient in West Africa
• Generally confined to W Africa and in countries with close links such as Portugal.
• Different subtypes exist; A and B main subtypes infecting humans
• Different disease progression to HIV-1
– Lower virulence – slower progression to AIDS
– Lower viral load – lower transmission rates
• Poorly or not detected by most HIV-1 assays.
• International Standard for HIV-2 RNA would be valuable for assays that detect HIV-2
HIV-2 Virus Isolates
• Genotype A most common HIV-2 subtype affecting humans
• Although most subtype A strains have been grown in culture, subtype B strains less easily cultured
• In consultation with WHO and CBER/FDA, two subtype A strains were identified that were available and for which full length sequences had been published:
– HIV-2 ROD – isolated 1985 in Cape Verde Islands, Senegal
– HIV-2 CAM2 – isolated 1987 in Guinea Bissau
• Low passage isolates acquired that grew well in T-cell lines
NIB
SC H
IV-2
RO
D
HIV2 RODHIV2 MCN13
HIV2 MCR35
HIV1 ISY
NIBSC CAM-2
HIV2 CAM
HIV2 M
D2 HI
V2 A
LI
HIV2
BEN
HIV2 GH1
HIV2 D194
HIV2 UC2
SIV MMH4
SIV 32H
SIV 239
SIV 251 1A11
HIV-2 7312A
HIV2 KR020
HIV2 EH
O
HIV2 UC1
HIV2 D205
0.02
HIV-2 gag sequences
HIV-2 subtype A
HIV-2 subtype B
SIVmac/smm
27-28 May 2009 4SoGAT XXI
Preparation of candidate standards
• Virus cultured in CEM cells and stocks stored down at ≤ -80oC
• RNA concentration determined using in house real time PCR assay (LTR)
• Virus heat inactivated at 600C for 60 minutes
• Inactivation confirmed using tissue culture– No growth with inactivated samples
• 2500 vials of each virus freeze dried
RNA concentration
Reagent Status HIV-2 CAM-2 HIV-2 ROD
Pre heat inactivation 9.62 9.51
Post heat inactivation 9.25 9.05
Pre freeze dried 3.66 4.26
Post freeze dried 3.47 4.06
• Virus stocks tested pre and post heat inactivation and pre and post freeze drying
NIBSC real-time PCR assay - values shown as copy number (log10)
International Collaborative Study
• 29 laboratories took part in the collaborative study.– Including Europe, USA, Canada, Japan, Australia, South Africa
• Each Lab was sent 4 vials of each of candidate labelled S1-S4
– S1 and S2 were HIV-2 CAM2
– S3 and S4 were HIV-2 ROD
• Requested to test in at least 3 assays, with the first assay containing 10 fold dilutions, then 0.5 log dilution around the end point
• Majority of results were from qualitative assays – from which end-point dilutions were determined
• 9 labs provided quantitative data from in-house assays
– Quantitative estimates used where all results positive or limited range of dilutions used
– Both quantitative and qualitative estimates used where full set of set of dilutions across end-point provided
• Results analysed by NIBSC statistician (Alan Heath)
27-28 May 2009 8SoGAT XXI
S1 Qualitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
26 23A 20 11 14
05
01
06
09
28
10
23B
27
02
06
18
22
25
24
13
04
08
32
07
29 15IH IH IH IH IH
MPX
IH
IH
IH
IH
MPX
MPX
PRO
MPX
MPX
MPX
MPX
MPX
IH
MPX
IH
IH
IH
MPX
MPX IH
In-House Roche MPX Procilex Nuclisens EasyQ
S2 Qualitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
26 23A 20 11
05
06
23B
14
02
18
27
06
25
01
08
09
28
32
10
13
22
04 24
07
29 15IH IH IH IH
MPX
MPX
MPX
IH
MPX
MPX
PRO
IH
MPX
IH
IH
IH
IH
IH
MPX
MPX
MPX
IH IH
MPX
MPX IH
In-House Roche MPX Procilex Nuclisens EasyQ
HIV-2 CAM2HIV-2 CAM2
27-28 May 2009 9SoGAT XXI
S3 Qualitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
23A
26
30 23B 20 09
11
24
02
05
06
13
22
27
19
06
08
14
28
32
25
10
18
01 04
07
29 15IH
IH
IH MPX IH IH
IH
IH
MPX
MPX
MPX
MPX
MPX
PRO
EZQ
IH
IH
IH
IH
IH
MPX
MPX
MPX
IH IH
MPX
MPX IH
In-House Roche MPX Procilex Nuclisens EasyQ
S4 Qualitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
23A 30 20 01
11
02
06
13
22
23B
25
27
19
09
05
18
06
08
14
24
28
32
07
10
29 04 15IH IH IH IH
IH
MPX
MPX
MPX
MPX
MPX
MPX
PRO
EZQ
IH
MPX
MPX
IH
IH
IH
IH
IH
IH
MPX
MPX
MPX IH IH
In-House Roche MPX Procilex Nuclisens EasyQ
HIV-2 RODHIV-2 ROD
S1 Quantitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
31 15
26
03 08 04 16
28
32IH IH
IH
IH IH IH IH
IH
IH
In-House Roche MPX Procilex Nuclisens EasyQ
27-28 May 2009 10SoGAT XXI
S2 Quantitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
31 15
26
03 08 04 16 28 32IH IH
IH
IH IH IH IH IH IH
In-House Roche MPX Procilex Nuclisens EasyQ
HIV-2 CAM2HIV-2 CAM2
27-28 May 2009 11SoGAT XXI
S3 Quantitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
31 19 15 03
26
08 04
16
28 32IH EZQ IH IH
IH
IH IH
IH
IH IH
In-House Roche MPX Procilex Nuclisens EasyQ
S4 Quantitative Assays
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
31 19 03
15
26
04
08
16 28 32IH EZQ IH
IH
IH
IH
IH
IH IH IH
In-House Roche MPX Procilex Nuclisens EasyQ
HIV-2 RODHIV-2 ROD
Potency of S2 relative to S1 (=4.0 log10 units/ml)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
15 08
32
26
31
06
23B
04
23A
03
04
15
16
32
02
05
13
18
25
27
01
06
11
14
20
26
28
08
07
10
22
29
09
24
28
IH IH
IH
IHQ
IHQ
MPX
MPX
IH
IH
IHQ
IHQ
IHQ
IHQ
IHQ
MPX
MPX
MPX
MPX
MPX
PRO
IH
IH
IH
IH
IH
IH
IH
IHQ
MPX
MPX
MPX
MPX
IH
IH
IHQ
In-House Qualitative Roche MPX Procilex In-House Quantitative
27-28 May 2009 12SoGAT XXI
(CAM2 relative to CAM2)
Potency of S3 relative to S1 (=4.0 log10 units/ml)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
08
15
31
13
23B
23A
24
32
32
02
06
27
06
03
04
15
16
28
22
25
04
09
28
08
26
05
07
29
11
14
20
26
10
18
01IH
IH
IHQ
MPX
MPX
IH
IH
IH
IHQ
MPX
MPX
PRO
IH
IHQ
IHQ
IHQ
IHQ
IHQ
MPX
MPX
IH
IH
IH
IHQ
IHQ
MPX
MPX
MPX
IH
IH
IH
IH
MPX
MPX
IH
In-House Qualitative Roche MPX Procilex In-House Quantitative
27-28 May 2009 13SoGAT XXI
(ROD relative to CAM2)
Potency of S4 relative to S1 (=4.0 log10 units/ml)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Estimated Units (log10/ml)
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
23A 13 01
08
15
32
04
31
06
07
27
03
08
16
26
32
02
22
23B
25
29
06
20
24
28
15
28
18
04
09
11
05
10
14IH MPX IH
IH
IH
IH
IHQ
IHQ
MPX
MPX
PRO
IHQ
IHQ
IHQ
IHQ
IHQ
MPX
MPX
MPX
MPX
MPX
IH
IH
IH
IH
IHQ
IHQ
MPX
IH
IH
IH
MPX
MPX
IH
In-House Qualitative Roche MPX Procilex In-House Quantitative
27-28 May 2009 14SoGAT XXI
(ROD relative to CAM2)
Stability - Accelerated Degradation Studies
•Vials of freeze dried CAM-2 and ROD stored at range of elevated temperatures•Tested at intervals of 4, 8, 12 months, 2,3,4,5 years •Can predict stability using Arrhenius equation
Conclusions and proposal
• Considerable variation between the results from different labs and different assays
• Use of relative potency improves agreement between labs and assay methods
• Good agreement between S1 and S2 (HIV-2 CAM2) and between S3 and S4 (HIV-2 ROD)
• Statistically, results were similar and there was no preference for either S1/S2 or S3/S4
• We suggest that HIV-2 CAM2 (S1/S2) be proposed to WHO ECBS as 1st International Standard for HIV-2 RNA with a unitage of 10,000 IU/vial/ml
• We welcome feedback and comments from the SoGAT Working Group
Acknowledgements
• NIBSC Project Team
• Dr. Indira Hewlett, CBER/FDA, USA
• Dr Ana Padilla, WHO, Switzerland
• Collaborative Study Group– 29 international participants – Thank You !!!!!
Other HIV-1 Standards
• Current HIV-1 2nd IS has stocks that will last 3-4 years• Need to start planning replacement now• Virus HIV-1 genotype B stock used for previous IS still available
– shall we use this??• Should we heat-inactivate the virus? This makes processing
and shipping more straight-forward
• 2nd genotype panel – work is underway• Collaborative study: 2010