Update on CBER HIV NAT panelsand International panels

Indira Hewlett, Ph.DIndira Hewlett, Ph.DChief, Lab. of Molecular Virology Chief, Lab. of Molecular Virology

DETTD/OBRR/FDADETTD/OBRR/FDAMay 28, 2009May 28, 2009

XXI SoGAT meetingXXI SoGAT meeting

Phillipe

Nyambi

NYU

HIV-1 Molecular Epidemiology: Historical Timeline and Key Milestones

1983

HIV

1998

Nomenclature

2001CRF

Variation – HIV-2

1988A

URF

AD

URF

C

A

URF

Kenya

Uganda

Tanzania

2002URF

Group O1993

Co-infection, dual- & super-infections

2003

1995

Recombination

2004SGRs

2nd gen.

recombinant

1992

Subtypes

14 new CRFs ha14 new CRFs haveve been been assignassigned in year 2007ed in year 2007

Worldwide distribution of predominant HIV-1 group M subtypes and CRFs

North and Central America

B

South America

B, F1, CRF12_BF

Western Europe

B, A, C, G CRF14_BG

Eestern Europe

A CRF03_AB

China

B CRF07_BC, CRF08_BC

Southeast Asia

BCRF01_AE

B

South AsiaC

CRF01_AE

AustraliaAustraliaBB

South Africa

C

Central AfricaMost CRFs, A, C, D, G, H, J, K, O. N

Western Africa

A, GCRF02_AGEast Africa

A, D, C

Impact of HIV genetic diversity on diagnosis

Schable, C., et al Lancet. (1994) Sensitivity of US licensed antibody tests for detection of HIV-1 group O infection. 344,

Amendola, et al., J AIDS, (2002) Under-evaluation of HIV -1 Plasma Viral Load by a Commercially Available Assay in a Cluster of Patients Infected With HIV -1 A/G Circulating Recombinant Form (CRF02).

Colson, P., et al J. Clin. Virol., 2007. Impaired quantification of plasma HIV-1 RNA with a commercialized real-time PCR assay in a couple of HIV-1 infected individuals

Zouhair, S., et al JCM, (2006), Group O HIV-1 infection that escaped detection in two immunoassays

Lee, S., et al. AIDS Res and Hum Retr.( 2007) Detection of Emerging HIV variants in blood donors from urban areas of Cameroon

Yao JD, et al . J. Clin. Micro., (2008). Plasma Load Discrepancies between the Roche Cobas Amplicor Human Immunodeficiency Virus Type 1 (HIV-1) Monitor Version 1.5 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Assays

CBER Efforts in panel development

CBER HIV-1 RNA subtype panel

CBER developed an HIV-1 RNA subtype panel currently in use for lot release since 2004

Panel consists of one primary isolate each of group M subtypes A-G and groups O and N cultured in PBMC and characterized by sequencing

Virus isolates are inactivated by heat treatment at 600C for 60 mins)

Panel composed of 3 members of each subtype at 103, 104 and 105 copies/ml spiked in negative plasma

CBER HIV-2 RNA panel

Seven isolates of HIV-2 subtype A were cultured in PBMC cultures and characterized by partial sequencing

Viruses were inactivated by heat treatment (600C for 60 mins) and spiked into negative plasma

Testing was performed in 3 laboratories Results from labs were in good agreement HIV-2 panel was formulated at 5, 10, 50,100 copies/ml

and is currently in use for lot release testing of HIV-2 NAT assays

CBER HIV-2 Panel Isolate Testing Summary (Log 10) Isolate ID Manufacturer

A

Manufacturer

B

Manufacturer

C

Mean Standard

Deviation

B2 8.7782 8.4116 9.3424 8.8441 0.4689

B3

8.5798 8.6702 9.3424 8.8642 0.4167

B4

8.6128 8.6263 9.4133 8.8841 0.4583

B5

8.6812 8.5988 9.4425 8.9075 0.4651

B7 8.8451 8.3522 9.1732 8.7902 0.4133

B8 8.6721 8.1847 9.1732 8.6767 0.4943

B9 8.2788 7.7924 9.0969 8.3894 0.6593

CBER panel for CRF02_AG and CRF01_AE Emerging new HIV strains are circulating recombinant forms

(CRFs)

CRF02_AG and CRF01_AE are currently the most prevalent CRF strains; need for standards

Five primary virus isolates each of CRF02_AG and CRF01_AE cultured in PBMCs were characterized by full genome sequencing

Heat inactivated virus isolates spiked into negative plasma were tested in five laboratories

Results were in good agreement between labs; data analyzed statistically and values assigned

Panel will consist of 3 members for each isolate spiked in negative plasma at 103, 104 and 105 copies/ml (log)

Isolate ID Lab A Lab B Lab C Lab D Lab E Mean SD

AE-1 8.71 8.65 8.19 8.06 8.47 8.42 0.25AE-2 8.87 8.84 8.51 8.31 8.68 8.64 0.21AE-3 8.70 8.69 8.34 8.12 8.32 8.43 0.23AE-9 8.92 8.85 8.52 8.34 8.52 8.63 0.22AE-10 8.69 8.61 8.24 8.15 8.47 8.43 0.21NYU 360 8.57 8.79 8.08 8.71 9.27 8.68 0.38NYU2395 9.86 9.53 9.33 n/a 9.15 9.47 0.26NYU4730 9.36 9.43 9.03 n/a 9.19 9.25 0.16NYU5203 9.30 8.84 9.35 8.81 9.23 9.11 0.23NYU5466 8.58 9.59 8.90 8.73 9.21 9.0 0.36

CRF01_AE and CRF02_AG Isolate Testing Summary

Ongoing CBER HIV panel development

Project initiated in Cameroon in 2001 to study HIV evolution and have access to diverse HIV strains known to emerge in this region for panel development needs

Study Goals: Collect specimens, viruses; genotyping and virus tropism Evaluate sensitivity of blood screening and diagnostic tests

for ability to detect diverse strains Identify new strains for future use as reference reagents Develop reference panels for emerging HIV variants for lot

release and assay standardization Future panels will target B/C, B/F and A/B recombinants

Summary of Genotyping Findings

CRF02_AG is most prevalent strain in Cameroon (60-70%) in both study groups

Virus is continuing to evolve - pure subtypes and new CRFs,

URFs, cpx strains identified in 2002

Viruses from 2006 –present are mostly recombinants of CRFs,

esp. CRF02_AG with other CRFs

Only 2.6% were pure subtype in 2006 compared with 15.8% in

2002

Globally CRF02_AG prevalence is currently 6.7% compared

with subtype B at 10%

CRF02_AG (68.4%)

URF(23.7%)

Unknown (2.6%)

CRF06_cpx (2.6%)F2 (2.6%)

HIV subtypes in Cameroon blood donors – 2006 to present

number %

CRF02 26 68.42URF 9 23.7CRF06 1 2.6F2 1 2.6Unknown 1 2.6Total samples 38

URF %

CRF02 + F2 22CRF02 + CRF35 11CRF02 + CRF37 11CRF02 + B 22CRF11 + A1 11CRF11 + CRF13 11

CRF04 + U 12

International Collaboration for HIV diversity studies and panel development

Inter-agency PHS working group (IWG) formed in 2006 to monitor reports of HIV diversity and impact on diagnostics

Need for reference panels for emerging variants was identified

In 2008 NIAID formed an International Collaborative Group to develop studies to assure that screening, diagnostic and confirmatory assays detect circulating strains

— Assays presently are based on prototype HIV strains— Numerous studies showed failure of assays to detect and

accurately quantify divergent subtypes— Evidence of viral divergence in the donor pool could accelerate

development of robust serological and NAT assays for donor, diagnostic and clinical management

Blood donor studies

Blood donors are a “convenience sample” likely to represent the larger population

— Studies in donors permit population based monitoring of recently transmitted viruses, including drug resistant phenotypes.

— Knowledge of virus variation is critical to public health strategies for AIDS prevention

Detection of variants in blood donors allows access to large volume plasma components for test development, evaluation and Quality Control

HIV Viral Panels Project: Purpose

In cooperation with other HIV surveillance efforts, to establish a set of fully characterized viruses from early acute HIV infections that are consistent with the degree of viral evolution present globally, for

-Developing new assays

-Validating assay platforms

-Assisting regulators to evaluate test kits

-Monitoring HIV drug resistance

-Informing vaccine development

Acknowledgments

DETTD/CBER

Owen Wood Sherwin LeeJiangqin ZhaoRagupathy ViswanathShixing TangStephen KerbyNYU Phillipe NyambiSherri Burda

ManufacturersCollaborative Study Group

Supported by NHLBI IAG- Y1-HB-5026-01CBER Critical Path Grant

Intl. Collab study groupM. Busch, BSRIM. Schito,NIAIDS. Peel, WRAIRM. Manak, SeracareS.Stovanabutraand others