Results of Pilot NIH Study of Global HIV Variants

Transcription

1 Results of Pilot NIH Study of Global HIV Variants SoGAT Blood Virology Meeting Vilnius, Lithuania April 2012 Mark Manak, Ph.D. MHRP, USA The opinions expressed herein are those of the authors and should not be construed as representing the official views of the U.S. Department of Health and Human Services, Department of the Army, Department of Defense, or the Food and Drug Administration.

2 Challenges of HIV-1 Global Diversity HIV-1 Subtype Classification Group M: 9 Major Subtypes (A-D, F-H, J, K) >50 CRF Numerous URF Group N, Group O, Group P Persons living with HIV Persons newly infected AIDS-related deaths Sub-Saharan Africa 22,900,000 1,900,000 1,200,000 South and South-East Asia 4,000, , ,000 Eastern Europe & Central Asia 1,500, ,000 90,000 Latin America 1,500, ,000 67,000 North America 1,300,000 58,000 20,000 Western & Central Europe 840,000 30,000 9,900 East Asia 790,000 88,000 56,000 North Africa & Middle East 470,000 59,000 35,000 Global Total 34,000,000 2,700,000 1,800,000 Ref:

3 Major HIV-1 Subtypes A1 B B, C and BC A, B and A3 G, CRF02_AG, CRFs F,G,H, J, K, CRF01 C B and BF Insufficient Data D B and CRF01_AE Worldwide Distribution Rapid Global Dissemination Emergence of Circulating Recombinants

4 Challenges of Extensive Diversity of HIV Diagnostic and Virus Monitoring Tests Blood screening Epidemiological surveillance Therapeutic monitoring Nucleic Acid Assays Target different HIV genes Different amplification approaches Limitations of assays on non-b subtypes Screening tests failure Under-quantification by Viral Load assays Failures with CRFs, Group N or Group O

5 Viral Panels Working Group National Institute of Allergy and Infectious Diseases (NIAID) National Heart Lung and Blood Institute (NHLBI) Food and Drug Administration (FDA) Centers for Disease Control and Prevention (CDC) Walter Reed Army Institute (WRAIR) National Institute for Biological Standards & Control (NIBSC) In Partnerships with International Blood Banks Industry Academia Goals Establish an updated Subtype Panel of fully characterized viruses representative of the current global epidemic. Panel Uses Assay development Performance evaluations of post market assays (i.e. viral load evaluation) Monitoring HIV drug resistance Informing vaccine design/development

6 Panel Criteria and Approach Acquire acute or early infections from plasma sources Closer to transmitted/founder virus Target viruses representative of all major subtypes, Groups, Circulating Recombinant Forms in circulation Blood and plasma donor populations Partner with others in the field Full characterize Full Length Sequence Viral Load Co-receptor usage Serological reactivity Infectivity Prepare HIV subtype panels Plasma panels Cultured virus panels

7 HIV Markers During Early Infection ECLIPSE Fiebig Stage I II III IV V VI HIV RNA HIV Antibody HIV p24 Ag Fiebig Stage Markers Duration (days) Eclipse None 10 I RNA Pos 7 II p24 Pos 5 III EIA Pos 3 IV WB IND 6 V WB Pos, no p31 band 70 VI WB Pos, w/ p31 Open Estimated days since infection Early stages more reflective of initial founder virus Before accumulation of mutated forms

8 Comparisons of HIV Quantitative Assays: Subtype B and C Roche Amplicor Monitor v1.5 Roche TaqMan v1.0 Roche TaqMan v2.0 Siemens bdna v3.0 Subtype B Subtype C

9 Characterization of Candidate Plasma Samples Country of Origin Specimen ID Viral Load (copies/ml) p24 EIA WB Fiebig Stage Country of Origin Specimen ID Viral Load (copies/ml) p24 EIA WB South Africa BP00022 < Neg Neg South Africa BP Pos Ind South Africa BP00024 < Neg Neg South Africa BP , Pos Ind South Africa BP00017 < Neg Neg South Africa BP , Pos Ind South Africa BP00026 < Neg Neg South Africa BP , Pos Ind South Africa BP Neg Neg USA BP , Pos Ind South Africa BP Neg Neg South Africa BP , Pos Ind South Africa BP Neg Neg USA BP , Pos Ind South Africa BP Neg Neg USA BP Neg Neg USA BP Pos Pos South Africa BP Neg Neg USA BP , Pos Pos South Africa BP Neg Neg USA BP , Pos Pos South Africa BP Neg Neg USA BP , Pos Pos South Africa BP Neg Neg USA BP , Pos Pos Poland BP Neg Neg I USA BP , Pos Pos USA BP , Neg Neg USA BP , Pos Pos USA BP , Neg Neg Brazil BP , Pos Pos South Africa BP , Neg Ind South Africa BP , Pos Pos South Africa BP , Neg Neg South Africa BP , Pos Pos South Africa BP , Neg Neg USA BP , Pos Pos Poland BP , Neg Neg Brazil BP , Pos Pos Poland BP , Neg Neg South Africa BP , Pos Pos South Africa BP , Neg Neg USA BP , Pos Pos USA BP , Neg Neg South Africa BP ,510, Pos Pos South Africa BP , Neg Neg South Africa BP , Neg Neg Cameroon BP Pos Pos+p31 USA BP , Neg Neg Brazil BP Pos Pos+p31 USA BP , Neg Neg USA BP Pos Pos+p31 Brazil BP Pos Pos+p31 South Africa BP , Neg Ind Greece BP , Pos Pos+p31 Poland BP , Neg Neg Brazil BP , Pos Pos+p31 South Africa BP , Neg Ind Cameroon BP , Pos Pos+p31 South Africa BP , Neg Neg USA BP , Pos Pos+p31 South Africa BP , Neg Neg Brazil BP , Pos Pos+p31 II South Africa BP , Neg Neg Greece BP , Pos Pos+p31 South Africa BP , Neg Neg Brazil BP , Pos Pos+p31 South Africa BP ,190, Neg Neg South Africa BP , Pos Pos+p31 South Africa BP ,480, Neg Neg Cameroon BP , Pos Pos+p31 USA BP ,200, Neg Neg Greece BP , Pos Pos+p31 Greece BP , Pos Pos+p31 South Africa BP ,360, Pos Neg III Cameroon BP , Pos Pos+p31 Brazil BP , Pos Pos+p31 Greece BP , Pos Pos+p31 Brazil BP , Pos Pos+p31 Brazil BP , Pos Pos+p31 Fiebig Stage IV V VI

10 Characterization of Candidate Plasma Samples Country of Origin Specimen ID Viral Load (copies/ml) p24 EIA WB Fiebig Stage Infectvity Country of Origin Specimen ID Viral Load (copies/ml) p24 EIA WB Fiebig Stage Infectvity South Africa BP00022 < Neg Neg - South Africa BP Pos Ind - South Africa BP00024 < Neg Neg - South Africa BP , Pos Ind - South Africa BP00017 < Neg Neg - South Africa BP , Pos Ind - South Africa BP00026 < Neg Neg - South Africa BP , Pos Ind IV - South Africa BP Neg Neg - USA BP , Pos Ind + South Africa BP Neg Neg - South Africa BP , Pos Ind - South Africa BP Neg Neg - USA BP , Pos Ind + South Africa BP Neg Neg - USA BP Neg Neg - USA BP Pos Pos - South Africa BP Neg Neg - USA BP , Pos Pos - South Africa BP Neg Neg - USA BP , Pos Pos - South Africa BP Neg Neg - USA BP , Pos Pos - South Africa BP Neg Neg - USA BP , Pos Pos - Poland BP Neg Neg I - USA BP , Pos Pos - USA BP , Neg Neg - USA BP , Pos Pos + USA BP , Neg Neg - Brazil BP , Pos Pos V - South Africa BP , Neg Ind - South Africa BP , Pos Pos + South Africa BP , Neg Neg - South Africa BP , Pos Pos - South Africa BP , Neg Neg - USA BP , Pos Pos + Poland BP , Neg Neg + Brazil BP , Pos Pos + Poland BP , Neg Neg - South Africa BP , Pos Pos + South Africa BP , Neg Neg - USA BP , Pos Pos + USA BP , Neg Neg - South Africa BP ,510, Pos Pos + South Africa BP , Neg Neg - South Africa BP , Neg Neg - Cameroon BP Pos Pos+p31 USA BP , Neg Neg - Brazil BP Pos Pos+p31 USA BP , Neg Neg - USA BP Pos Pos+p31 - Brazil BP Pos Pos+p31 South Africa BP , Neg Ind - Greece BP , Pos Pos+p31 + Poland BP , Neg Neg + Brazil BP , Pos Pos+p31 South Africa BP , Neg Ind - Cameroon BP , Pos Pos+p31 South Africa BP , Neg Neg - USA BP , Pos Pos+p31 - South Africa BP , Neg Neg + Brazil BP , Pos Pos+p31 II South Africa BP , Neg Neg + Greece BP , Pos Pos+p31 VI South Africa BP , Neg Neg + Brazil BP , Pos Pos+p31 - South Africa BP ,190, Neg Neg - South Africa BP , Pos Pos+p31 - South Africa BP ,480, Neg Neg + Cameroon BP , Pos Pos+p31 + USA BP ,200, Neg Neg + Greece BP , Pos Pos+p31 + Greece BP , Pos Pos+p31 + South Africa BP ,360, Pos Neg III - Cameroon BP , Pos Pos+p31 + Brazil BP , Pos Pos+p31 Greece BP , Pos Pos+p31 + Brazil BP , Pos Pos+p31 + Brazil BP , Pos Pos+p31 +

11 Isolation and Expansion of Virus in Culture Virus Isolation Clarify plasma by low speed spin Concentrate virus by high speed spin Incubate with CD8 depleted PBMC Monitor virus for 21 days by p24 Virus Expansion Incubate with PHA stimulated PBMC Original isolate supernatant Original isolate PBMC 2 Rounds of Expansion 25 cm 2 75 cm 2 Flask Clarify Supernatant Aliquot and characterize Quantitative RNA p24 Ag

12 Success of Virus Isolation as function of Plasma Viral Load or Fiebig Stage No. Samples 10 No. Culture Pos 5 100% 80% 60% % Culture Pos 40% 20% No. Samples 15 No. Culture Pos 10 5 Culture Pos Samples % Culture Pos 100% 80% 60% 40% 20% % Culture Pos 0 0% 0 I II III IV V VI 0% Viral Load (copies/ml) Fiebig Stage Fiebig Stage Pos/ Attempted % Pos I 2/17 7.4% II 8/ % III 0/1 0.0% IV 2/7 28.6% V 9/ % VI 9/ %

13 The full length genome amplification strategy vpr vpu tat gag vif rev nef 5 LTR pol env 3 LTR 3 Tat primer HIV-1 RNA LH 1.5 kb RH 2 half genomes Plasma RNA SGA cdna cdna dilutions First round PCR (RH or LH) Nested PCR Sequence if less than 30% of the wells are positive

14 BP Determination of diversity of HIV-1 quasi species in 3 cultured samples 5 half genome 3 half genome

15 No significant sequence drift following virus culture Translation Sample ID Plasma (Mean + SD) Pairwise Diversity (%) Groups Comparison Poisson Model PBMC (Mean + SD) SUP (Mean + SD) Plasma vs PBMC (Mean + SD) Plasma vs SUP (Mean + SD) PBMC vs SUP (Mean + SD) Star Phylogeny Days (CI) BP YES 38 (27,48) BP YES 33 (22,43) BP YES 43 (30,55)

16 Phylogenetic Analysis Phylogenetic Tree of Viral Isolates from Pilot Study Manak et al. AIDS Res Hum Retroviruses. 2012, Feb 24

17 HIV-1 Isolates from EQAPOL Isolates from Pilot Study Sample ID Country of Origin HIV-1 Subtype RNA copies/ml BP00047 Brazil B 2.10E+09 BP00075 Poland B 1.50E+09 BP00074 Poland B 2.80E+09 BP00055 USA B 2.40E+09 BP00058 USA B 2.70E+08 BP00063 USA B 4.40E+07 BP00064 USA B 1.10E+08 BP00067 USA B 3.40E+07 BP00070 USA B 7.20E+07 BP00069 USA B 1.30E+08 BP00044 Brazil BF 1.70E+08 BP00040 Brazil C 1.60E+09 BP00002 South Africa C 2.20E+09 BP00012 South Africa C 1.30E+08 BP00014 South Africa C 2.10E+09 BP00016 South Africa C 9.10E+08 BP00018 South Africa C 7.90E+06 BP00025 South Africa C 6.20E+08 BP00028 South Africa C 1.10E+09 BP00010 South Africa C 1.60E+07 BP00005 South Africa C 5.10E+08 BP00008 South Africa C 4.40E+08 BP00029 South Africa C 4.70E+08 BP00009 South Africa C 1.20E+09 BP00078 Cameroon CRF_AG 1.40E+08 BP00079 Cameroon CRF_AG 1.60E+08 BP00048 Greece CRF04_CPX 3.60E+07 BP00049 Greece CRF04_CPX 4.80E+09 BP00052 Greece CRF04_CPX 1.20E+08 BP00053 Greece CRF04_CPX 2.60E+09 Ratio of ng p24 to Viral Load ng/ml p RNA Copy number/ml Coefficient of correlation: 0.686

18 HIV-1 Isolates from EQAPOL Isolates from Pilot Study Additional Isolates from Follow up Study Sample ID Country of Origin HIV-1 Subtype RNA copies/ml Sample ID Country of Origin HIV-1 Subtype RNA million copies/ml Sample ID Country of Origin HIV-1 Subtype BP00047 Brazil B 2.10E+09 DE05ES001 Spain A1 2.54E+09 DE08NG001 Bolivia B BP00075 Poland B 1.50E+09 DE09ES005 Tanzania A1 1.81E China B BP00074 Poland B 2.80E+09 DE09ES002 Spain A1B (URF) 1.16E+10 BP France B BP00055 USA B 2.40E+09 DE05FR001 France B 1.15E+10 DE99DE001 Germany B BP00058 USA B 2.70E+08 DE09US003 US B 2.57E+09 DE08CM003 Poland B BP00063 USA B 4.40E+07 DE09US002 US B 4.44E+09 DE08UY001, Spain B BP00064 USA B 1.10E+08 DE09ES006 Spain BF 3.07E+09 DE09BO001 Uruguay B BP00067 USA B 3.40E+07 DE09ES003 Spain BF 3.55E+09 ARP1183 Venezuala B BP00070 USA B 7.20E+07 DE00GR002 Brazil C 6.26E+09 DE07BR003 Angola C BP00069 USA B 1.30E+08 DE02FR001 France C 5.19E+09 DE07AO001 Nigeria C BP00044 Brazil BF 1.70E+08 DE09ZA009 South Africa C 1.59E+09 DE07ZA011 S Africa C BP00040 Brazil C 1.60E+09 DE99PL001 South Africa C 1.50E+10 DE10ZA001 South Africa C BP00002 South Africa C 2.20E+09 DE09ZA008 South Africa C 2.93E+09 DE06ES003 Spain C BP00012 South Africa C 1.30E+08 DE10DZ001 Angola CRF02_AG 1.58E+10 DE09CN003 China CRF_001 BP00014 South Africa C 2.10E+09 DE08CM004 Cameroon CRF02_AG 8.18E+09 DE09CN004 China CRF_001 BP00016 South Africa C 9.10E+08 DE10CM009 Cameroon CRF02_AG 1.24E China CRF01_AE BP00018 South Africa C 7.90E+06 DE06ES002 Spain CRF02_AG/A3 5.86E+09 DE08ES001 Algeria CRF02_AG/CRF06_cpx BP00025 South Africa C 6.20E+08 DE08ZA011 Greece CRF04_CPX 1.00E+10 DE08CM001 Cameroon CRF02_AG BP00028 South Africa C 1.10E+09 DE10CM008 Cameroon D 6.89E+09 DE08CM002 Cameroon CRF02_AG BP00010 South Africa C 1.60E+07 DE10CM001 Cameroon F2 1.81E China CRF07_BC BP00005 South Africa C 5.10E+08 DE05TZ001 Cameroon F2 1.96E+10 ARP1083 Brazil CRF14_BG BP00008 South Africa C 4.40E+08 DE10CM007 Cameroon G 5.35E+09 ARP1176 SPAIN F1 BP00029 South Africa C 4.70E+08 DE06AO001 Spain G 2.09E+10 BP00009 South Africa C 1.20E+09 DE07ES002 Spain G 1.54E+10 BP00078 Cameroon CRF_AG 1.40E+08 BP00079 Cameroon CRF_AG 1.60E+08 BP00048 Greece CRF04_CPX 3.60E+07 BP00049 Greece CRF04_CPX 4.80E+09 BP00052 Greece CRF04_CPX 1.20E+08 BP00053 Greece CRF04_CPX 2.60E+09 Sequence data is deposited in Los Alamos Database (

19 Summary Plasma from Acute/Early Infection was obtained from collaborating blood banks and HIV test centers around the world Virus was characterized, isolated in culture, and expanded Virus cultured from plasma at early Fiebig Stages was very homogenous and essentially identical to that in the source plasma. Full genome sequencing of 33 cultured isolates identified 2 subtype CRF02_AG (Cameroon), 4 subtype CRF04 (Greece), 16 subtype C (South Africa and Brazil), 10 subtype B (USA, Poland and Brazil), and 1 BF URF (Brazil). Additional subtypes continue to be added through EQAPOL. The EQAPOL Viral Panel will provide critical resource of viral isolates representative of the current global HIV epidemic Virus Cultures are available from EQAPOL-Duke University (

20 Acknowledgements DAIDS, NIH Marco Schito, HJF Contractor Jim Lane BSRI Mike Busch Leslie Tobler FDA Indira Hewlett Viswanath Ragupathy Jiangqin Zhao American Red Cross Susan Stramer SPAIN, ISC-III Lucía Pérez-Álvarez NIBSC Harvey Holmes Meghna Ramaswamy SeraCare Pat Garrett Bharathi Anekella USMHRP Sheila Peel Nelson Michael Jerome Kim Sodsai Tovanabutra Silvana Sina Eric Sanders-Buell South Africa, NBS Marion Vermeulen Lynn Morris Brazil, U Sao Paulo Ester Sabino Ricardo Sophie Diaz Poland, IHT Ewa Brojer Piotr Grabarczyk EQAPOL-Duke Tom Denny Ana Sanchez This project was funded in part NIH NIAID Contract No. HHSN C and by a cooperative agreement W81XWH between the HJF and U.S. Department of Defense (DOD).