HIV-1 subtype C in Ethiopia and India HIV-1 subtype C Isolated - PowerPoint PPT Presentation

HIV-1 subtype C in Ethiopia and India

HIV-1 subtype C • Isolated 1988, Ethiopia (Sönnerborg et al, 1990) • Dominating subtype globally: 50% • Biological characteristics – 3 NF- κ B sites (Sönnerborg, 1995) – 4 NF- κ B strains in India (Bachu, 2012) – Higher set-point after acute infection (Novitisky 2011) (?) – HIV drug resistance pattern, K65R – Insertions p6 during ART failure – Co-receptor usage

HIV in Ethiopia • First HIV seropositive identified in 1984 • 2001 : 2 million adults + 200,000 children; 2011 : 800,000 persons • HIV prevalence – 1% 1989, 5.2% 1996; 5.6% 2005, 3.5% 2007, 2.6% 2009/11 – Urban centers : 11.5% 2003, 5.5% 2009 – Rural areas : 4% 2003, 1.4% 2009 • In 2005, a national ART program was rolled out • In 2011, 336 000 treated • Ethiopia is one of the six sub-Saharan African countries showing a decline of >25% in new HIV infections

Delatorre. PLoS One, July 2012 1,981 HIV-1C pol sequences: ”South African clade” ”Heterogeneous clades” ”East African clade” ” Subtype C` or Ethiopian 2 clade

Two HIV-1C clades in East Africa Based on 1,981 HIV-1C pol sequences: • >70% belongs to a single regional-specific monophyletic group, originating from Burundi in 1960 • A second major Ethiopian subtype C lineage Delatorre. PLoS One, July 2012

Ethiopian samples 170 plasma from treatment naive Ethiopian AIDS-patients, 2007-2008 • Advanced immunosuppression (average CD4 cells 104 , range 4-199) • The RNA was reverse transcribed and then PCR-amplified using specific • primers for the pol-gene (ref HXB2 inner primers: forward 2135-2158, reverse 3315-3338). Sequencing of the env-gene was attempted on the samples for which pol- • sequencing had been succesful (ref HXB2 inner primers: forward 7003-7021, reverse 7648-7668). env-seq pol-seq

HIV-1 subtypes in Ethiopia Rega RIP pol (n= 127) C 127 126 C/A1/F2 - 1 env (= 102*) C 73 82 A1 1 1 ”close to C” 9 - *19 of these had good sequence only for V3 and could not be subtyped or used for phylogenetic analysis 1. http://dbpartners.stanford.edu/RegaSubtyping/ 2. http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html

Phylogenetic analysis (prel), methods 83 Ethiopian samples with good sequence for both pol (average seq length • 939 bp) and env (average seq length ≈480 bp) References from LANL pre-made alignments [6], 45 ref-seq for different • subtypes + 28 subtyp C seq from different geographic regions Phylogenetic analysis was performed in BEAST [7], evolutionary model • GTR+I+ Γ , run for 10 million generations, sampling every 10 000 gen. The posterior probability value (corresponding to bootstrap in non-Bayesian • phylogenetics) was 1 (100%) for the differentiation between the C and C’ clusters for both pol and env in this analysis. 6. http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html 7. http://beast.bio.ed.ac.uk/

Tree, pol-seq H A, Blue= ref subtype C-seq 01_AE G, Cyan=C-seq from different regions B 02_AG Green=Ethiopian seq from this study which are either C or C’ for both pol and env F D Other colours – potentially recombinant viruses C K J C’

D B J Tree, env-seq G 01_AE Blue= ref subtype C-seq H Cyan=C-seq from different regions A, Green=Ethiopian seq from this study which 02_AG are either C or C’ for both pol and env Other colours – potentially recombinant viruses F C J K 0 C’

Prel phylogenetic analysis, results Out of 83 Ethiopian samples sequenced for pol and env , the following distribution was found in the C and C’ clades: 34 viruses (41%): C-clade for both pol and env (C - C) • 28 viruses (34%): C’-clade for both pol and env (C’- C’) • one C-C and one C’-C’ virus moved substantially within the clades and • associated with strains from Europe/India and South America rather than with other Ethiopian strains for the env-region 10 viruses (12%): C’ for pol and C for env (C’- C) • 8 viruses (9.6%): C for pol and C’ for env (C - C’) • One strain was found to be a C-A1 recombinant •

Conclusion The two Ethiopian lineages have resulted from independent founder • strains, originated in eastern and southern African regions In total, 18/83 (23%) Ethiopian strains analyzed fall into distinctly • different clades for pol and env -> recombinants between C and C’ appear to be common in Ethiopia In contrast, only 1/28 (3.6%) of the randomly selected subtype C strains • from other geographic regions outside Ethiopia appeared to be a recombinant, this was a C’- C strain from Israel These results indicate that circulating recombinant forms C-C’ and C’-C • are spreading in Ethiopia alongside the non-recombinant clades C-C and C’-C’

Co-receptor tropism, methods 102 V3-sequences were tested by: • ₋ geno2pheno [4] ₋ WebPSSM (three algorithms) [5] ₋ CoRSeqV3-C [3] ₋ (the 11/25+net charge rule, 2011) No test repeat • In the cases of ambiguous base pairs (double peaks) all possible translations • were analyzed. ₋ most sequences (73/102) had only one translation while 13 had 2, and 16 had between 3 and 32 possible translations (3: N=1; 4: N=7; 8: N=2; 16: N=3; 28: N=1; 32: N=2). 3. http://www.burnet.edu.au/facilities/8; Gorry et al. 4. http://coreceptor.bioinf.mpi-inf.mpg.de/ 5. http://indra.mullins.microbiol.washington.edu/webpssm/

Co-receptor tropism, results Total: 102 V3 All three G2P WebPSSM CoRSeqV3-C algorithms FPR 20 (sinsi C) R5 70 (68.6%) 82 (80.4%) 83 (81.4%) 89 (87.3%) X4 6 (5.9%) 20 (19.6%) 19 (18.6%) 13 (12.7%) Discordant 26 (25.5%) NA NA NA X4 prevalence : WebPSSM sinsi B: 4/102 X4R5: 5/102 11/25: 2/102

Co-receptor tropism, results (70 samples consequently designated as R5 not shown) Number of alternative Geno2Pheno, WebPSSM Sample # V3-LOOP translations coreceptor CoRSeqV3-C sinsi-C >Et111 CTRPNNNTRRGIGIGPGHTFYTTGQIIGDIRKAYC 1 5 CXCR4 CXCR4 >Et86 CTRPNNNTRKSIGIGPGRAFYATGDIIGNIRQAHC 1 5,4 CXCR4 CXCR4 >Et136 CTRPSNNTRRNIGIGPGQTFFAPGTIIGDIRRAYC 1 7,8 CXCR4 CXCR4 X4 >Et191 CTRPNNNTRKGIRIGPGQMFYAAGEIIGNVRQAFC 1 13,2 CXCR4 CXCR4 N= 6 >Et124 CTRPNNNTRKSIRIGPGQAF[HY]ATG[KNRSEDG]IIGDIRQA[HY]C 28 14,4 CXCR4 (9) CXCR4 (3) >Et73 CTRPSNNTRKSVEIGPGRAIYATGDIIGDIRQAHC 1 15,4 CXCR4 CXCR4 >Et107 CTRPNNNTSKSIRIGPGQAFYATERIIGNIRQAYC 1 4,6 CCR5 CXCR4 >Et62 CTRPNNNTRKGIGIGPGQMFYATDAIIGDMRDNC 1 4,8 CCR5 CXCR4 >Et21 CTRPNNNTIESIRIGPGQAFYAT[TR]RIIGDIRQAYC 2 5 CCR5 CXCR4 (2) >Et150 CTRPNNNTRKSMRIGPGQTFYATGKIIGDIRKAYC 1 5 CXCR4 CCR5 X4? >Et49 CTRPGNNTRRSVRIGPGQAFYTTGEIIGDIRRAHC 1 5,3 CCR5 CXCR4 N= 8 >Et78 CTRPNNNTRKGIGIGPGQTFYAAEEIIGNIRNAYC 1 6,9 CCR5 CXCR4 >Et68 CVRPGNNTRKSIRIGPGQAFYAEGGIIGDVRQAHC 1 10,5 CCR5 CXCR4 >Et35 CARPGNNTRKSMRIGPGQTFYATGDIIGNIRKAHC 1 11,7 CXCR4 CCR5 >Et20 CMRPGNNRRKSIRIGPGQTFYATGEIIGDIRQAYC 1 3,7 CCR5 CCR5 >Et82 CARPGNNTRKSTRIGPGQTFYATGDIIGDIRQAHC 1 6,7 CCR5 CCR5 >Et105 CTRP[NS]NNTRKSMRIGPGQTFYAMGVIGDIRQAYC 2 8,7 CCR5 CCR5 >Et31 CTRPNNNTRKSIRIGPGQAFYTTNIIGDIRQAHC 1 20,9 CCR5 CXCR4 ? >Et88 CERPNNNTRKSIRIGPGQAFYTTGQIIGDIRQAHC 1 23,6 CCR5 CXCR4 N= 8 >Et96 CTRPNNNTRKSIRIGPGQAFYATGDIIGDIRHAFC 1 51,9 CXCR4 CCR5 >Et142 C[TM]RPNNNTRESIGIGPGQTLFAIGAIIGDIRQAHC 2 84,5 CCR5 CXCR4 (2) >Et128 CTRLNNNTRKSIRIGPGQTFYATGGIIGNIRLAHC 1 95,5 CXCR4 CCR5 >Et92 C[IM]RPNNNTRKSMRIGPGQTFYATGEIIGDIRQAHC 2 15,4 CCR5 CCR5 >Et166 C[TM]RPNNNTRKS[IV]RIGPGQAFYATG[ED]I[IV]G[ND]IRQAHC 32 16,4 CCR5 CCR5 >Et149 CTRPNNNTRKSVRIGPGQTYYATGDIIGDIRRAHC 1 19,4 CCR5 CCR5 >Et81 CTRPNNNTRKSI[RG]IGPGQ[TA]FYATGEIIG[ND]IRKA[HY]C 16 21 CXCR4 (2) CCR5 >Et63 CTRPTNNTRKSVRIGPGQ[TA]FYATGEVIGDIR[KN]AHC 4 21,8 CCR5 CXCR4 (2) R5? >Et135 CTRPSNNTRTSMRIGPG[QP*S]TFYATGDIIGDIRKAYC 3 25,3 CXCR4 (2) CCR5 N= 10 >Et152 C[TI]RPGNNTR[KR]SVRIGPGQ[AV]FY[TA]TGEIIGDIREAHC 16 25,6 CCR5 CXCR4 (2) >Et47 CTRPN[NS]NTRKSMRIGPGQTFYATGDI[IV]G[KNED]IRQA[HY]C 32 63,1 CXCR4 (4) CCR5 >Et84 CIRPSNNTR[KE]S[IM]RIGPGQTFYATG[ND]I[TI]GDLRQAHC 16 79,5 CCR5 CXCR4 (4) >Et97 CTRP[NS]NNTRRGIRIGPGQTFFAIGEIIGDIRQAHC 2 90,3 CCR5 CXCR4 (1) Numbers within parantheses show the number of possible translations that were categorized as X4-tropic

Conclusion, Co-receptor tropism CCR5-inhibitors are likely to be effective in around 75%-85% of ART-naïve • Ethiopian subtype C infected AIDS-patients The classification of the co-tropism was disconcordant in 25.5% of the • sequences between G2P, WebPSSM sinsi C and CoRSeqV3-C The patients with X4- strains had lower CD4-counts (average±stdev: • 86.2±65.1), compared with the undetermined (91.8±48.0) and R5-using strains (108.0±49.7), however, these differences were not statistically significant.

HIV in India • First HIV seropositive identified in 1986, Tamil Nadu • Estimate of 2.4 million infections as of 2009 • A prevalence of 0.31% • Despite the low prevalence, India is the third largest country • Currently 334 000 on ART Figure 2. State wise adult HIV-1 prevalence in India (2009 estimates). Data adapted from National AIDS Control Programme Phase III, State Fact Sheets, March 2012

Indian HIV-1 infected subjects, 2007-2011 168 HIV-1 seropositive subjects from 7 different provinces Punjab Haryana Manipur Madhya Pradesh Andra Pradesh Karnataka Tamil Nadu