PhD Students Visiting Researcher Emmanuel Obasa Duncan T Njenda - - PowerPoint PPT Presentation

Ex-vivo vivo and in vi vitr tro antire retroviral ral p potency o y of newer integras rase stra rand tran ansfer i r inhibi bitors i in diverse H HIV-1 s 1 sub ubtype

Ujjwal Neogi, M.Sc., PhD

Assistant Professor/Group Leader Division of Clinical Microbiology Department of Laboratory Medicine Karolinska Institutet, Stockholm, Sweden AREVIR, 04 May 2018

Viral DNA Target DNA Outer IN Outer IN Inner IN Inner IN

Duncan T Njenda Maike Sperk Anoop Ambikan

PhD Students

Wang Zhang

Robert van Domsealer

Sara Svensson

Shambhu Aralaguppe

Associated Members

Ashokkumar M

Visiting Researcher

Emmanuel Obasa Naveen Muppani

PostDoc

Hemalatha Babu

Study Designing: Drugs: RAL, EVG, DTG, CAB and BIC Biochemical Assays: The integrase genes (HXB2: 4230-5096) of four randomly selected INSTI-naïve patients infected by HIV-1B, C, CRF01_AE, and CRF02_AG, respectively, were cloned into the pRSFDuet-integrase vector with N-terminal H6-tag, purified and used for inhibition of 3'-end processing (IC50-3P) and strand transfer (IC50-ST) Virological assay: Drug sensitivity (DSA) for five INSTIs were done using patient-derived integrase or gag-pol genes from subtypes (HIV-1B: n=6; HIV-1C: n=14; HIV-1A1: n=1; HIV- 1CRF_01AE: n=2; HIV-1CRF_AG: n=1) . Clinical Data: Integrase from INSTI-naïve (n=270) and experienced (n=96) patients were sequenced.

In In vi vitro tro inhib ibit ition ion of

• f stra

trand tra ransfer (IC IC50

50-ST ST) and

nd 3’- end nd proc

• cessin

ing activ ivit itie ies (IC IC50-3P)

• No potential subtype specific difference
• DTG, CAB and BIC has slightly better IC50-ST in non-B subtypes

F.

CAB DTG BIC EVG RAL

Ex Ex vi vivo vo drug rug sensitiv ivit ity assa ssay in in TZM ZM-bl bl cell ll li lines

Primary integrase DRM 270 INSTI-naïve individuals, infected with HIV-1B (n=92), C (n=82), 01_AE (n=25), 02_AG (n=15), A1/A2 (n=22), other pure subtypes (n=13), and recombinant forms (CRFs or URFs) (n=21).

• One HIV-1B patient had a major

primary INSTI-DRM, T66I.

• 6.3% (17 of 270) patients had one

major accessory DRM: T97A (n=3), E157Q (n=12), or A128T (n=2). Acquired Drug Resistance Mutations

Genot

• typ

ypic Re Resi sist stance Testing ng: Inf InfCare-Coh

• hor
• rt

• Absence of major RAMs in the cohort collected before the broad availability of

combination antiretroviral therapy (cART) and INSTI in South Africa.

• One of 87 samples carried the accessory mutation G140E.

10 20 30 40 50 60 70 80 90 100 K14R D25E V31I M50I I72V L74M/I F100Y L101I T124A K136Q D167E V201I T218I L234I A265V R269K S283G

Frequency (%) A1/A2 (n=483) B (n=4379) C (n=1155) 01_AE (n=1581) 02_AG (n=522)

K14R D25E V31I M50I I72V L74M/I F100Y L101I T124A K136Q D167E V201I T218I L234I A265V R269K S283G D64 D116 E152

N-terminal domain (NTD) the catalytic core domain (CCD) C-terminal domain (CTD) Rogers et al Manuscript under communication

Naturally ally Occur urri ring Pol

• lymor

ymorphis isms ms

M50/I50 vDNA

Tsiang et al Antimicrob Agents Chemother. 2016

Muta tati tion t to watch ch: M M50I

Rogers et al Manuscript under communication

• DTG, CAB, and BIC had higher antiretroviral potency not only in HIV-1B but also in

non-B subtypes compared to RAL and EVG,

Con

• nclusion
• ns

• DTG, CAB, and BIC had higher antiretroviral potency not only in HIV-1B but also in

non-B subtypes compared to RAL and EVG,

• No significant difference between the newer INSTIs was seen with regard to efficacy on

various subtypes.

Con

• nclusion
• ns

• DTG, CAB, and BIC had higher antiretroviral potency not only in HIV-1B but also in

non-B subtypes compared to RAL and EVG,

• No significant difference between the newer INSTIs was seen with regard to efficacy on

various subtypes.

• Patient derived sequences from INSTI-naïve individuals both in Swedish cohort and

South African cohort have very low prevalence of primary INSTI DRM by Sanger Sequencing.

Con

• nclusion
• ns

• DTG, CAB, and BIC had higher antiretroviral potency not only in HIV-1B but also in

non-B subtypes compared to RAL and EVG,

• No significant difference between the newer INSTIs was seen with regard to efficacy on

various subtypes.

• Patient derived sequences from INSTI-naïve individuals both in Swedish cohort and

South African cohort have very low prevalence of primary INSTI DRM by Sanger Sequencing.

• There are five patients in Sweden who has high level of resistance to all INSTIs.

Con

• nclusion
• ns

• DTG, CAB, and BIC had higher antiretroviral potency not only in HIV-1B but also in

non-B subtypes compared to RAL and EVG,

• No significant difference between the newer INSTIs was seen with regard to efficacy on

various subtypes.

• Patient derived sequences from INSTI-naïve individuals both in Swedish cohort and

South African cohort have very low prevalence of primary INSTI DRM by Sanger Sequencing.

• There are five patients in Sweden who has high level of resistance to all INSTIs.
• The relatively high rate of accessory INSTI mutation (6.3% in Swedish Cohort) but not

in South African cohort.

Con

• nclusion
• ns

Accessory mutations: 15.1% 23.1% in HIV-1A, 8.7% HIV-1C, 11.6% HIV-1D, 25% HIV-1G and 23.8% in recombinants.

• DTG, CAB, and BIC had higher antiretroviral potency not only in HIV-1B but also in

non-B subtypes compared to RAL and EVG,

• No significant difference between the newer INSTIs was seen with regard to efficacy on

various subtypes.

• Patient derived sequences from INSTI-naïve individuals both in Swedish cohort and

South African cohort have very low prevalence of primary INSTI DRM.

• There are five patients in Sweden who has high level of resistance to all INSTIs.
• The relatively high rate of major accessory INSTI mutation (6.3% in Swedish Cohort)

but not in South African cohort.

• Naturally occurring polymorphisms in the catalytic core domains mainly near the active

sites (D64, D116 and E152) and M50I may affect the stability of the enzyme subsequently the DNA binding affinity followed by drug binding.

Con

• nclusion
• ns

Country Treatment Naive First line Failure (NRTIs+NNRTI) Second line failure (NRTIs+ PI/r) Third Line (NRTI+RAL) Status South Africa 30 30 30 15* PCR Completed Kenya 30 30 30 ? Sample collection going

• n

Tanzania 30 30 30 ? Samples are already stored Cameroon 30 30 30 ? Sample collection going

• n

India 30 ? ? ? Samples are already stored Ethiopia 30 30 30 ? To be started Sweden 200 ? ? ? Sample collection started

Study sites and sample collection

*All the samples will have clinical information, mostly from private clinics

Understanding minor pol mutation in HICs and LMICs

19% 16% 21% 4% 16% 8% 6% 10% A1 (/A1 like) B C D AE G AG Other-CRF

Success: Swedish Cohort : 91% (49/54) South African Cohort: 80% (84/106)

Methods and sensitivity

High t h thro hroug ughp hput ut s sequenci cing

MiDRMpol

Jonas Söderquist’s Stipendium for Experimental Virology and Immunology Research-2016

Anders Sönnerborg Piotr Nowak

• Asso. Prof. Kamlendra Singh, University of Missouri, US
• Dr. Luke Elisabeth Hanna, NIRT, Chennai
• Prof. Thomas Klimkait, University of Basel, Switzerland
• Dr. Graeme Jacobs, Stellenbosch University

Dr Christa Kasang, Mwanza, Tanzania

• Dr. George Mondinde Ikomey, University of Yaoundé, Cameron
• Dr. Marc Noguera Julian, Institut de Recerca de la SIDA - IrsiCaixa
• Prof. Stefan Sarafianos, Emory University, US

Establishment Grant Sweden-China