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