Chief, Laboratory of Molecular Virology, CBER, FDA Surveillance of HIV variants in Cameroon and evaluation of their impact on HIV diagnostics Indira K. Hewlett, Ph.D Chief, Laboratory of Molecular Virology, CBER, FDA

Genetic diversity of HIV Two major types of HIV: HIV-1 and HIV-2 3 distinct HIV-1 groups identified to date: M (major), O (outlier) and N (non-M, non-O) Group M consists of many subtypes of viruses (A-H) and group O (3 prototype classes) 5 major HIV-2 subtypes Increasing numbers of circulating recombinant forms (CRFs) of HIV-1

Mosaic structure of HIV circulating recombinant forms

Worldwide distribution of predominant HIV-1 group M subtypes and CRFs CRF14_BG CRF01_AE Cameroon B Adapted from Thomson et al. Lancet Infect Dis 2002.

HIV genetic diversity: diagnostic implications 1994: Schable et al. from CDC tested 9 sera of HIV group O from Cameroon, Africa with 10 different FDA licensed tests 2/9 sera were not detected by 3 assays used for blood screening; 1/9 was not detected by 5/10 tests Tests that detected all 9 sera were based on whole virus lysate (WVL) antigens Some tests based on synthetic peptides or recombinant antigens did not detect all sera

Diagnostic implications- con’t CDC study caused heightened awareness of impact of HIV genetic diversity on sensitivity of diagnostic tests FDA requested manufacturers to modify tests for detection of HIV-1 group O by including group O antigens FDA considered need for in-house research in HIV diversity

Methods of detection Target EIA antibodies Western Blot antibodies IFA antibodies P24 ELISA HIV antigen NAT HIV RNA

Study goals Evaluate ability of existing and new blood screening, rapid and other diagnostic tests to detect and quantitate HIV variants Determine appropriate strategies to modify tests for sensitive detection of HIV variants Investigate co-infection of HIV with HHV-8 Study HIV and HHV-8 epidemiology in Cameroon blood bank setting Characterize and genotype emerging HIV variants Develop reagents (panels) for standardizing new tests for variant HIV

CBER HIV surveillance efforts CBER initiated dialogue with Cameroonian investigators in 1998/99 and CDC to study HIV diversity in-country Cameroon has all known HIV genetic variants Interagency agreement (3-5 yr.) with CDC for cooperative surveillance developed last year Additional collaboration with Johns Hopkins and DoD

Study Plan and Methods Blood samples were collected from sites around Yaounde, tested by a rapid HIV assay used to screen blood donors in Cameroon. Plasma were shipped to FDA At LMV samples were tested by FDA licensed HIV antibody, antigen and nucleic acid tests (NAT) Confirming HIV infection of Discordant samples using in-house test Genotyping HIV positive samples Samples were also tested for HHV-8 antibodies and its co-infection with HIV

Results A total 239 blood samples were evaluated. There are 149 HIV positive samples and 90 HIV negative samples. Six different FDA approved tests were used: EIA (2) IFA (1) Western Blot (1) NAT (2)  

Results Original report FDA tests result HIV+ samples: 149 Confirmed HIV+:131 Confirmed HIV-: 4 Indetermined: 14 HIV- samples: 91 Confirmed HIV-: 80 Conformed HIV+: 2 Indetermined: 9

Discordant result Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1   HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 CE01 R R ++ Pos R <LDL CE17 NR NR neg NR <LDL CE29 NR R + Ind CE33 NR NR LT11 - LT13 R R nt <LDL LT21 R <LDL CE62 NR R NR <LDL CE64 CE84 LT61 R R +/- R 5.1x103 CE164 R Ind CE182 LT130 Ind <LDL

Discordant result Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1   HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 CE11 NR R - Ind NR <LDL CE18 NR NR neg CE39 nd CE76 CE87 NR <LDL LT01 LT36 R NR LT16 NR NR R <LDL LT05 CE107 R R Pos R 7.5X104 CE223 R 2.4X103

Viral load in HIV positive samples #of samples 101 2 102 11 103 47 104 41 105 29 106 1

No HIV group O was detected by using an in-house ELISA. None of the samples were detected by the p24 antigen test In-house PCR is performed to aid the determination of HIV infection

239 plasma samples from urban areas around Yaounde Investigate discordant samples to identify new HIV variant. Searched for new HIV variant through genetic characterization (Subtype determination) 239 plasma samples from urban areas around Yaounde % RT-PCR + Set #1: n= 41 80.5 Set #2: pos. n=59 78 neg. n=40 0 Set #3: pos. n=48 48 neg. n=51 1.9

Methods PCR amplification: env (gp41) and gag (p17) Sequencing: PCR product forward and reverse PCR primers ABI prism 310 Genetic Analyzer Editing: Sequence Navigator software Alignment: CLUSTAL W software Fhylogenetic Analysis: Matrix distance (2 Kimura parameters) Neiborg-joining (tree) Bootstrap (fidelity of the tree) Maximum likelihood

O N M SET #1 DNA Matrix Distances GP41 (n=33) 1000 976 1000 652 O4 O1 CPZ-2 CPZ-3 N2 M N1 976 1000 A3 A5 A4 652 LT051 LT02 LT34 CPZ-1 A1 A6 LT04 LT28 LT22 A2 LT32 CRF02 AG3 CRF02 AG4 LT17 CE38 LT26 LT07 LT03 CE42 CE01 CRF02 AG2 LT25 CE41 CE32 LT33 G3 J2 J1 LT19 LT09 G2 LT27 G4 CRF02 AG1 CE46 LT24 LT10 B2 B3 LT31 G1 B1 LT29 CRF11 AGJ3 CE09 B4 CRF11 AGJ4 CE35 CPZ-5 CRF11-AGJ1 CE34 D1 D4 CRF11 AGJ2 D2 D3 C1 CE03 LT06-gp4 C4 C2 K2 F1-3 C3 CRF01 AE3 F1-1 CRF01 AE-U CRF01 AE1 H1 CRF01 AE2 LT141 K1 H2 F1-2 F2-1 H3 0.1 CE36 F2-2

SET #1 A CRF02_AG J G B CRF11_AGJ D C CRF01_AE K H F DNA Matrix Distances SET #1 A3 A (5, 15.2%) GP41 (n=33) A4 LT05 A5 LT02 CRF02_AG (18, 54.5%) LT34 A1 A6 LT28 CRF02 AG4 A2 LT32 CRF02 AG3 LT22 LT04 LT17 LT25 CE38 LT26 LT07 CE41 J CRF02 AG2 CE42 G (3, 9.1%) LT03 LT19 CE32 CE01 LT09 G3 J2 LT10 J1 B G2 LT24 LT33 G4 CRF02 AG1 LT27 CE46 B2 B3 974 LT31 B1 G1 LT29 208 922 B4 CRF11 AGJ3 CRF11 AGJ4 817 498 737 CRF11_AGJ (4, 12.1%) CE09 CE35 CRF11-AGJ1 CE34 D (1, 3%) D1 D4 D3 CRF11 AGJ2 D2 995 C1 LT06 C2 CE03 C4 F1-3 K2 C3 CRF01 AE3 F1-1 C CRF01 AE1 H1 CRF01 AE4 K1 H2 CRF01 AE2 LT14 F1-2 CRF01_AE K H3 F2-1 CE36 H F (2, 6.1%) F2-2 0.1

SET #1 C G H F K CRF11_AGJ J D CRF01_AE B A CRF02_AG Maximum Likelihood SET #1 C G (3, 9.1%) H C1 GP41 (n=33) C2 H3 F (2, 6.1%) LT06 AGJ2 C4 K CE09 C3 CRF11_AGJ (4, 12.1%) H2 AGJ3 CE34 H1 F2-2 CE03 K1 CE36 G3 F1-2 AGJ4 G1 K2 J AGJ1 G2 LT27 F2-1 LT14 J2 F1-1 J1 F1-3 LT31 G4 D (1, 3%) LT29 D4 CE35 D2 D1 D3 CRF01_AE AE2 B2 LT22 AE3 AE-4 AE1 A6 B3 B1 A5 B LT33 A1 A2 LT19 B4 LT25 AG1 LT34 AG2 A4 LT10 CE01 CE32 CE42 LT07 A3 A (4, 12.1%) AG3 LT24 LT02 LT05 LT17 LT26 LT03 CE41 CRF02_AG (19, 57.6%) AG4 LT04 CE38 CE46 LT09 LT32 LT28 0.1

N O M SET #1 DNA Matrix Distances P17 (n=23) 961 1000 503 719 1000 CPZ2 O2 CPZ1 N N2 N1 O 961 O4 1000 O3 503 719 O1 1000 CPZ3 CE03 LT05 LT26 LT31 LT04 LT28 CRF01 AE4 CRF01 AE2 LT24 CRF02 AG4 CRF01 AE3 CRF01 AE1 LT02 CFR02 AG1 CRF02 AG2 CRF02 AG3 CRF11 AGJ2 CE09 CRF11 AGJ1 CE34 LT33 LT25 CE32 CRF11 AGJ3 CRF11 AGJ4 CE41 LT03 LT29 LT06 CE38 A1-2-AF069 LT32 LT27 G3 A1-4 A1-1-AF004 A1-3 G4 A2-1-AF286 M G1 G2 H1 H2 H3 B1 B3 B2 J2 D3 B4 D2 J1 D4 LT22 C2 CE35 D1 C3 F1-1 C4 K2 LT14 F2-1 F1-3 F1-2 F2-2 C1 K1 0.1

O SET #1 N CRF01_AE CRF02_AG CRF11_AGJ G A H B J D C F K DNA Matrix Distances O N SET #1 p17 (n=23) CRF01_AE (5, 21.7%) CE03 CRF02 AG2 LT28 LT05 CRF02 AG4 CRF01 AE4 LT26 CFR02 AG1 LT04 LT24 CRF02_AG (10, 43.5%) CRF01 AE3 CRF01 AE2 LT31 CRF02 AG3 CRF01 AE1 LT02 CRF11_AGJ (2, 8.7%) CE09 CRF11 AGJ2 CE34 CE32 LT33 CRF11 AGJ1 LT25 CE41 CRF11 AGJ4 LT29 LT03 CRF11 AGJ3 CE38 980 G (1, 4.3%) LT06 540 LT32 175 A1-2 LT27 A A1-4 493 860 G3 A1-1 535 A1-3 956 G4 650 889 720 A2-1 G1 778 533 585 G2 H1 955 512 B2 H H2 H3 B1 B3 1000 B J2 B4 D3 D2 J D4 J1 LT22 CE35 D1 D (1, 4.3%) C2 F1-1 C3 C4 K2 LT14 F1-3 C (1, 4.3%) F2-1 F1-2 F2-2 F (1, 4.3%) 0.1 K C1 K1

SET #1 O N B J D C G K F H CRF02_AG CRF11_AGJ A CRF01_AE Maximum Likelihood SET #1 B p17 (n=23) J D (1, 4.3%) B4 C (1, 4.3%) C3 H2 J1 B2 H3 B3 C2 D4 D1 J2 H1 B1 C4 CE35 D2 F1-3 D3 K1 F1-1 C1 LT22 G (1, 4.3%) F1-2 K G2 A2-1 G1 K2 G3 LT27 G4 F2-1 LT14 A1-1 A1-4 F (1, 4.3%) AG3 A1-2 AG2 LT26 LT06 F2-2 LT28 AG4 LT29 CE38 LT32 AGJ4 H LT03 CE32 CE34 CRF02_AG (11, 47.8%) LT33 AG1 AGJ2 AE2 A1-3 AGJ1 CE41 AGJ3 LT25 AE1 LT02 CE09 O N AE3 LT24 CRF11_AGJ (2, 8.7%) LT05 A AE4 LT04 LT31 CE03 CRF01_AE (5, 21.7%) 0.1

70% with VL>1000 RNA cop/ml SUBTYPE DETERMINATION: SET DATA #1 10 samples amplified gp41 but not p17: 8 CRF_02-AG 1 A 1 F 70% with VL>1000 RNA cop/ml 23 samples amplified both gp41 and p17: Gp41 A AG AGJ D F G P17 AE C AG AGJ D F G n 2 1 10 gp41 (AGJ) p17 (?)

SUBTYPE DETERMINATION: SET DATA #2 7 samples amplified gp41 but not p17: 6 CRF02_AG 1 A 85% with VL>1000 RNA cop/ml 39 samples amplified both gp41 and p17: Gp41 A AG AGJ G F2 P17 A AE AG G F2 AGJ n 3 1 25

SET #3 DNA Matrix Distances GP41 (n=24) 0.1 O1 O3 O2 CE208-gp41 LT110-gp41 CE133-gp41 LT117-gp41 CE173-gp41 LT165-gp41 LT89-gp41 AG4 CE217-gp41 CE145-gp41 LT115-gp41 CE198-gp41 CE202-gp41 LT160-gp41 CE180-gp41 CE129-gp41 A2-2 AG3 LT107-gp41 AG1 CE207-gp41 CE177-gp41 CE175-gp41 CE115-gp41 A2-1 CE187-gp41 LT88-gp41 CE174-gp41 B1 AG2 A1-1 A1-3 B2 A1-2 B3 CE107-gp41 D3 N2 D1 AE3 D2 AE1 AE2 C1 G1 G2 LT129-gp41 CE189-gp41 AGJ1 C2 AGJ2 H1 N1 AGJ3 J1 J2 F1-2 F1-1 K2 H2 K1 0.1 F2-1 F2-2

SET #3 DNA Matrix Distances GP41 (n=24) CRF02_AG (20, 83.3%) A LT110 LT165 CE217 LT89 GP41 (n=24) CE208 CE133 LT117 CRF02_AG (20, 83.3%) CE145 CE173 CE202 AG4 LT160 CE180 LT115 CE198 A (1, 4.2%) CE177 AG3 CE207 CE175 A2-2 LT107 AG1 CE187 A2-1 B1 CE174 LT88 B AG2 B2 A1-3 B3 A1-1 A1-2 CE107 D (1, 4.2%) D3 D1 D2 AE3 CRF01_AE AE1 C1 AE2 G1 G2 C LT129 C2 AGJ1 CE189 G (1, 4.2%) AGJ2 J1 H1 H2 AGJ3 J2 F1-2 K2 H F1-1 K1 K F2-1 0.1 F F2-2

SET #3 Maximum Likelihood GP41 (n=24) G (2, 8.3%) A (1, 4.2%) D CE173 LT89 AG1 CE208 AG4 CE217 AG2 CE180 AG3 LT88 CE177 CE207 CE187 CE198 CE175 CE174 LT115 CE145 CE133 LT110 LT117 LT160 CE202 LT165 A1-3 A1-1 LT107 A2-2 A2-1 J2 J1 CE189 G2 LT129 G1 AGJ3 AGJ2 AGJ1 AE1 AE3 AE2 H2 H1 C2 C1 F1-1 F1-2 K2 K1 F2-2 F2-1 D3 D1 CE107 D2 B2 B1 B3 CRF02_AG (20, 83.3%) A (1, 4.2%) D G (2, 8.3%) 0.1

SUMMARY Subtype % 5.8 1.9 2.9 70 4.8 3 CRF02_AG A B C D F G CRF01_AE 1.9 2.9 70 4.8 3 CRF02_AG B C D F G CRF01_AE CRF02_AG CRF011_AGJ AC AF GF

Discordant result AG Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1   HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 CE01 R R ++ Pos R <LDL CE17 NR NR neg NR <LDL CE29 NR R + Ind CE33 NR NR LT11 - LT13 R R nt <LDL LT21 R <LDL CE62 NR R NR <LDL CE64 CE84 LT61 R R +/- R 5.1x103 CE164 R Ind CE182 LT130 Ind <LDL AG

Discordant result D Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1   HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 CE11 NR R - Ind NR <LDL CE18 NR NR neg CE39 nd CE76 CE87 NR <LDL LT01 LT36 R NR LT16 NR NR R <LDL LT05 CE107 R R Pos R 7.5X104 CE223 R 2.4X103 D

SUMMARY AND CONCLUSIONS Six different FDA licensed tests detected additional HIV positive specimens. However, discordant results are not rare among all six tests when testing African samples. EIA results from different manufactures do not always agree with each other. WB may not resolve some discordant results. Current licensed HIV NAT can detect most of the subtypes and recombinant HIV variants. The sensitivity may be different between manufacturer’s tests. HIV viral load varies in Cameroon blood donors, the majority viral load around 103-105 RNA/ml.

SUMMARY AND CONCLUSIONS – con’t CRF02_AG was the most prevalent viral strain found in Cameroon samples (70%) New CRF AC, AF and GF were found All the subtypes and CRF identified and sequenced to date in Cameroonian plasma samples are detected by FDA licensed HIV NAT assays.

FUTURE DIRECTIONS Establish primary isolates from these plasma samples Genotyping of the virus will be continue performed Analyze discordant and negative samples with degenerate primers and PCR Enhance RT assay (PERT) to identify emerging retroviruses Optimize new technologies including Microarray for identification of HIV groups and subtypes Samples collected from blood banks outside Yaounde and in rural areas will be analyzed in future studies

Acknowledgements Cameroon Ministry of Health Leopold Zekeng Bih Awazi LMV/DETTD/OBRR Indira Hewlett Owen Wood Sherwin Lee Stephen Kerby Sylvester Daniel Ana Machuca Jinjie Hu Dr. Hsia CDC Thomas Folks Marcia Kalish