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NYU. Phillipe Nyambi. Update on CBER HIV NAT panels and International panels. Indira Hewlett, Ph.D Chief, Lab. of Molecular Virology DETTD/OBRR/FDA May 28, 2009 XXI SoGAT meeting. Group O 1993. Subtypes. HIV. Nomenclature. Co-infection, dual- & super-infections. CRF. 1998. 1983.

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Update on CBER HIV NAT panels and International panels

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Update on cber hiv nat panels and international panels

NYU

Phillipe Nyambi

Update on CBER HIV NAT panels

and International panels

Indira Hewlett, Ph.D

Chief, Lab. of Molecular Virology

DETTD/OBRR/FDA

May 28, 2009

XXI SoGAT meeting


Update on cber hiv nat panels and international panels

Group O

1993

Subtypes

HIV

Nomenclature

Co-infection, dual- & super-infections

CRF

1998

1983

1992

2003

2001

1988

1995

2002

2004

URF

SGRs

2nd gen. recombinant

URF

A

Recombination

URF

Variation – HIV-2

Kenya

D

A

Uganda

C

URF

A

Tanzania

HIV-1 Molecular Epidemiology: Historical Timeline and Key Milestones

14 new CRFs have been assigned in year 2007


Update on cber hiv nat panels and international panels

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

Eestern Europe

CRF03_AB

A

Western Europe

CRF14_BG

B, A, C, G

North and Central America

China

B

CRF07_BC, CRF08_BC

B

Western Africa

CRF01_AE

Southeast Asia

A, G

CRF02_AG

CRF01_AE

B

East Africa

A, D, C

Central Africa

South Asia

B

Most CRFs, A, C,

D, G, H, J, K, O. N

C

South America

CRF12_BF

B, F1,

South Africa

C

Australia

B


Impact of hiv genetic diversity on diagnosis

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 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

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

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


Cber panel for crf02 ag and crf01 ae

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)


Update on cber hiv nat panels and international panels

Isolate IDLab ALab BLab CLab DLab EMean SD

AE-18.718.65 8.19 8.068.478.420.25

AE-28.878.84 8.51 8.318.688.640.21

AE-38.708.69 8.34 8.128.328.430.23

AE-98.928.85 8.52 8.348.528.630.22

AE-108.698.61 8.24 8.158.478.430.21

NYU 3608.578.79 8.08 8.719.278.680.38

NYU23959.86 9.53 9.33 n/a9.159.470.26

NYU47309.369.43 9.03 n/a9.199.250.16

NYU52039.308.84 9.35 8.819.239.110.23

NYU54668.589.59 8.90 8.739.219.00.36

CRF01_AE and CRF02_AG Isolate Testing Summary


Ongoing cber hiv panel development

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

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%


Update on cber hiv nat panels and international panels

number%

CRF022668.42

URF 923.7

CRF06 12.6

F2 12.6

Unknown 12.6

Total samples 38

CRF06_cpx (2.6%)

F2 (2.6%)

Unknown (2.6%)

URF

(23.7%)

URF %

CRF02 + F222

CRF02 + CRF3511

CRF02 + CRF3711

CRF02 + B22

CRF11 + A111

CRF11 + CRF1311

CRF04 + U12

CRF02_AG

(68.4%)

HIV subtypes in Cameroon blood donors – 2006 to present


International collaboration for hiv diversity studies and panel development

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 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

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


Update on cber hiv nat panels and international panels

Acknowledgments

Manufacturers

Collaborative Study Group

DETTD/CBER

Owen Wood

Sherwin Lee

Jiangqin Zhao

Ragupathy Viswanath

Shixing Tang

Stephen Kerby

NYU

Phillipe Nyambi

Sherri Burda

Intl. Collab study group

M. Busch, BSRI

M. Schito,NIAID

S. Peel, WRAIR

M. Manak, Seracare

S.Stovanabutra

and others

Supported by NHLBI IAG- Y1-HB-5026-01

CBER Critical Path Grant


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