HIV Genetic Diversity Consequences for diagnosis viral load

HIV Genetic Diversity Consequences for diagnosis, viral load and treatment monitoring Prof François SIMON & Jean - Christophe PLANTIER University Paris Diderot - Hospital Saint Louis – Paris 10

HIV GENETIC DIVERSITY : FROM SIV TO HIV § Different cross–species transmission events in the recent past - Two types : HIV 1 & HIV 2 - 4 HIV-1 groups (M, N, O & P) and 9 HIV 2 groups (A to I) Ø Different HIV-1 group M subtypes (A to K) q High rate of HIV super/dual infections and HIV recombinant strains Increasing number of HIV-1 group M Circulating Recombinant Forms (CRF) and M- O inter group recombinant and HIV-2 inter-group recombinant strains v High replication - Volatile genome in host, high rate of mutations - Low genetic barrier against anti-retroviral and immune-responses

THE ORIGINS OF SIV/HIV : RECOMBINATION & INTER-SPECIES TRANSMISSIONS HIV-2 groups A to I HIV-1 groups M, N mnd 1 mnd 2 HIV-1 groups O, P Adapted from P Sharp, B Hahn, Cold Spring Harb Perspect Med 2011; 1: a 006841

REPLICATIVE FITNESS AND HIV-1 GENETIC DIVERSITY v High replication = high rate of mutations Low genetic barrier against anti-retroviral and immune- responses Ariën et al. Nature Reviews Microbiology 5 (February 2007) |

HIV-1 GENETIC DIVERSITY IN 2016 Group N Non M : N O P Group M Subtypes A-K 80 CRF Group O

HIV-1 NON- GROUP M GENETIC DIVERSITY IN 2016 Mourez et al CMR 2013

ESTIMATION OF MRCA S : THE EXEMPLE OF HIV-O DIFFERENT VIROLOGICAL AND EPIDEMIOLOGICAL WAVES 1970 1950 1930 Marginal posterior density courves obtained using 4 different population growth models Marie Leos, Plos Pathogen, 2015.

HIV-O MAIN CHARACTERISTICS • 1% of HIV-infected patients in Cameroon 143 cases diagnosed in France mainly in Cameroonian Pts • Pathogenic, sexual and vertical transmission documented • Lower replicativity than HIV-1 group M • 70% of the HIV-O are naturally resistant (Y 181 C) to NNRTI according to the phylogenetic position • Sensitive to enfuvirtide in vitro and in vivo despite N 42 D in 100%

HIV-N MAIN CHARACTERISTICS • Group N is close from the SIV cpz : cross species transmission occurring in the last decades • Pathogenic, sexual and vertical transmission documented • Low prevalence but strain trafficking outside of Cameroon, in Europe • Sensitive to all the ARV like group M strains • Low epidemic (to date) • a lower transmission rate ?

HIV-2 INFECTION : A COMPLEX MONITORING A • HIV-2 is less pathogenic than HIV-1 with a slower decline in CD 4 count • Lower replication : 50 % have “undetectable” plasma RNA (<100 copies/ml) • Not specific commercial HIV-2 Viral Load assays • Naturally resistant to NNRTI (including etravirine et rilpivirine) and fusion inhibitor enfuvirtide • Less sensitive to amprenavir, atazanavir in vitro • Sensitive to maraviroc, raltegravir, elvitegravir, dolutegravir • Poorest CD 4 response = HAART must be initiated as early as 500 CD 4 cells/m. L B

HIV-1 GROUP MAJOR M : GENETIC DIVERSITY IN 2016 http: //clinicalgate. com/human-immunodeficiency-virus

HIV-1 GROUP M DIVERSITY IN AFRICA CRF 02_AG Mali Ivory Coast Cameroon Congo Gabon Senegal D G English speaking countries : Subtype C FC Javaugue

HIV-1 group M diversity Main consequences in 2016 • Detection of HIV-1 non B during primary infection • Viral Load miss-identification • Drug sensitivity

VIROLOGICAL LIMITATIONS IN HIV SCREENING IN 2016 HIV-1 ANTIGENIC DIVERSITY • Highly divergent strains are circulating in Central Africa and elsewhere • Ab not always fairly detected by all the screening assays • Different non-B p 24 antigen epitopes • Up to 6 months following the seroconversion, antibodies have a low binding avidity to the antigen • RDTs lack of sensitivity during AHI • Follow-up of children born to HIV seropositive mothers mainly after HAART initiation : false negativity in RTD

Mc. Cord Hospital outpatient department Acute HIV screening study, Durban, South Africa 994 RAPID TESTS NEGATIVE OR DISCORDANT, OUT PATIENTS AHI = 1% percent of outpatients with negative or discordant detectable through pooled serum HIV RNA screening. Bassett IV, HIV Med. 2011 AHI in adult population presenting fever ; Southern Mozambique 37. 8% ( 32. 7– 43. 2) of adults had undiagnosed established HIV infection. AHI = 3. 3% ( 1. 3– 6. 7 ) Serna-Bolea Celia , AIDS 2010 Acute HIV-1 infection is as common as malaria in coastal Kenya 3602 adults , HIV-1 prevalence 3. 9% AHI in 5 of 506 HIV-1 -negative or discordant patients Eduard J. Sanders, AIDS 2014

LACK OF SCREENING TEST SENSITIVITY DURING HIV-1 NON-SUBTYPE B SEROCONVERSIONS B B Non-B 0. 94± 0. 35 0. 84± 0. 31 EIA INDIRECT Optical densitiy 12 subtype B vs 9 ‘non-B’ During early primary infection 4. 52± 4. 3 0. 91± 1. 21 EIA SANDWICH New EIA Sandwich : Increasing sensitivty for anti-subtype B antibodies only C Apetrei , AIDS 1996

HIV RAPID DIAGNOSTIC TESTS LACK OF SENSITIVITY DURING PRIMARY INFECTION Saint Louis Hospital , Paris : evaluation of 5 RDT EC-approved on the whole blood Prospective study, 200 HIV– 1 B & non-B infected-patients - 3 acute primary infections (subtype B-2 and F-1) : 5/5 RTD negative -1 HIV-1 group O-infected patient : 3/5 RDT negative Pavie, Plos. One 2010

p 24 ANTIGENIC DIVERSITY AND THE 1 st COMBO FOURTH-GENERATION Ag-Ab RAPID TEST Combo Rapid Test : Lack of Antigen p 24 Detection Supernatants of Various Subtype N % Ag p 24 positivity A B C D 9 13 12 10 22. 2 61. 5 41. 7 60 F, G , H , J CRF 01_AE CRF 02_AG CRF 06_cpx CRF 11_cpx HIV-1 g. O HIV-1 g. P HIV-2 22 7 8 2 2 12 2 20 0 28. 5 25 0 0 0 S Laperche , JID 2012

VIRAL LOAD ASSAYS AND HIV DIVERSITY IN 2016 • • • 20 years for viral load Previously based only onto subtype B quantification High sensitivity performances (LOD 20 copies/m. L) Overpassing (now) the genomic diversity Random, Automation high throughput or unitary

IN 2001 b. DNA QUANTIPLEX 3. 0 IN HIV-1 NON-B INFECTED PATIENTS WITH UNEXPECTEDLY LOW VIRAL LOAD ON MONITOR ROCHE Patient Birthplace Subtype CD 4 cell count, cells/mm 3 3 Nigeria Non-B 167 4 Liberia Non-B 167 6 Nigeria Non-B 18 7 New York Non-B 153 8 New York Non-B 360 9 New York Non-B 39 11 Benin Non-B 398 12 Ghana ? 7 14 Ivory coast Non-B 309 15 Ghana Non-B 507 Virus load, copies /m. L RT-PCR MONITOR ROCHE b. DNA test 1314 543 < 400 232 613 1900 < 50 78, 168 < 400 < 50 92, 112 51, 088 88, 000 10, 295 37, 985 555, 900 3408 7, 202, 040 76, 142 5099 Elizabeth R. Jenny-Avital, and Sara T. Beatrice Clin Infect Dis. 2001; 32: 1227 -1230

IN 2016 RNA VIRAL LOAD ASSAYS ARE OVERPASSING THE HIV-1 GENOME DIVERSITY Subtypes ANRS 2013 HIV-1 viral Load : quality control , ANRS

COMPARISON OF 4 VIRAL LOAD ASSAYS , PLASMA PANEL samples P A N E L JC Plantier 2014 A B C D F G H K CRF 01 CRF 02 CRF 06 CRF 09 CRF 12 CRF 14 Non B 8 33 1 3 4 2 1 1 6 27 1 1 2 3 1 samples

VIRAL LOAD AND HIV DIVERSITY : FROM 1996 TO 2016 • To survey the emergence of resistance • Rare but real discrepancies between the different assays whatever the subtype • Improved sensitivity for the large HIV-1 group M diversity (B and non B) • Often, adapted to DBS and to DNA detection Discrepancies between a low viral load /failure of genotyping and low CD 4 number suggestive of HIV variant

HIV- 1 GROUP M NON-B SUBTYPES ACQUIRED RESISTANCE TO ARV • Active sites for polymerase and integrase are highly – conserved • Subtype C propensity for K 65 R TDF-associated resistance mutation : limited consequences ( E White JID 2016) • Etravirine & Rilpivirine : 10 % non-B strains harbour a mutation present in the HIV-1 subtype B score for resistance • Natural polymorphism overlapping the acquired mutation againts PI • Anti-integrase resistance profiles similar whatever the subtypes

HIV DIVERGENT STRAINS Groups N-O- P, inter-Group M/O and HIV-2 CONSEQUENCES IN DIAGNOSIS & MONITORING - Different cross-species transmissions (HIV-1 group M non B, non M) - trafficking in human population with a - Potential lack of sensitivity of the low prevalence screening assays during AHI with RDT - differences in pathogenicity - WB atypical profile (non B, non M) - Children born to HIV mothers HIV-1 group B & non B, non M Limitation in monitoring by molecular assays - Need of viral load assay adapted - Genotyping : failure of sequencing HIV-2 and HIV-O natural resistance to non-nucleoside inhibitors HAART against HIV-2 must be initiated as early as 500 CD 4 cells/m. L

HIV diversity is underlining the need for a rigorous epidemiological monitoring around the world François SIMON CHU saint Louis- Paris- France Jean-Christophe PLANTIER Marie LEOZ CHU Charles Nicolle, Rouen France Cristian APETREI University of Pittsburgh - USA