From bedside to bench and back : future options for antiretroviral drugs in non-B HIV-1 subtypes

HIV-1 drug resistance remains a burden in lowand middle-income countries (LMIC). Regardless of the advances in antiretroviral (ARV) therapy, there is an increase in the trend of acquired and pre-treatment drug resistance mutations (DRM) in LMIC affected by diverse HIV-1 subtypes. In the work presented in this thesis, I investigated the prevalence of HIV-1 acquired drug resistance (ADR) in South Africa from the period 2006-2014 (Paper I). Additionally, the in vitro proteaseinhibitor (PI) drug sensitivity assay (DSA) response of HIV-1 subtype C (HIV-1C) derived recombinant viruses was compared to non-C viruses to understand their susceptibility profile to PI drugs. Results for the prevalence of ADR, specifically for patient sequences treated with a PI-based regimen (n=1043), was found to be <9%. No significant subtype-specific differences were seen for viruses tested in the DSA for Darunavir (DRV), Lopinavir (LPV) and Atazanavir (ATV) susceptibility. In Paper II, it was hypothesized that the gag gene could play a role in the response of PIs in the absence of known PI primary mutations. Hence to understand the role of Gag in impacting PI susceptibility and viral fitness, the gag-p6 region was specifically investigated. The study showed an increase in viral fitness for HIV-1C viruses carrying the PYxE insertion in gag-p6 when compared to the wild-type (WT) HIV-1C viruses. Furthermore, some PYxE-carrying viruses had low sensitivity to LPV and Tenofovir alafenamide (TAF) when tested in DSAs. Clinical data analysis, showed a higher pre-therapy viral load and a decrease in CD4 T-cell counts for patients harboring PYxE-carrying viruses when compared to WT. It was also essential to understand the inhibitory potential of most clinical (new and old) drugs used mainly to treat non-B HIV-1 subtypes. Hence, in Paper III, the newer antiretroviral drug 4’-Ethynyl-2’-Fluoro-2’ deoxyadenosine (EFdA), was compared to TAF, first and second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs). It was demonstrated that EFdA has a high inhibitory potential independent of HIV-1 subtype and high antiviral activity against resistant viruses. However, HIV-1C viruses had a significantly reduced susceptibility to NNRTIs, specifically Rilpivirine and Etravirine. Finally in Paper IV, the drug susceptibility of Integrase strand transfer inhibitors (INSTIs) against diverse HIV-1 subtypes was investigated. Results indicated that INSTIs such as Dolutegravir (DRV), Bictegravir (BIC) and Cabotegravir (CAB) inhibited non-B subtypes significantly as compared to HIV-1B subtypes. Finally, inferences suggest that subtype-specific differences play an essential role in influencing the ARV susceptibility which could further impact the treatment efficacy in sub-optimal adherence. To reduce the trend of increasing DRMs in non-B HIV-1 subtypes which are mainly dominating in LMICs, adherence support and viral load monitoring should be prioritized. A rapid adaptation of INSTIs and newer drugs that have long-acting potential is encouraged. However, pre-clinical studies and clinical trials that are mainly restricted to HIV-1B enrolled patients, should be inclusive of non-HIV-1B infected patients before the massive roll-out of INSTIs and newer drugs continues in non-HIV-1B dominated settings. LIST OF SCIENTIFIC PAPERS I. Njenda T. Duncan, Mikasi S. Given, Ambikan T. Anoop, Adetayo E. Obasa, Sarafianos G. Stefan, Sönnerborg Anders, Neogi Ujjwal, Engelbrecht Susan and Jacobs B. Graeme – HIV-1 acquired drug resistance mutations in South Africa and phenotypic susceptibility in HIV-1 subtype C against protease inhibitors (Manuscript) II. van Domselaar Robert, Njenda T. Duncan, Rao Rohit, Sönnerborg Ander, Singh Kamalendra, Neogi Ujjwal, (2019) HIV-1 subtype 1 C with PYxE insertion has enhanced binding of Gag-p6 to host cell protein ALIX and increased replication fitness J. Virol. 93:e00077-19. DOI:10.1128/JVI.00077-19 [PMID: 3076057] III. Njenda T. Duncan, Aralaggupe Shambhu, Singh, Kamalendra; Rao Rohit; Sönnerborg Anders, Sarafianos Stefan; Neogi Ujjwal, 2018. Antiretroviral potency of 4’-Ethnyl-2’-Fluoro-2’-deoxyadenosine, Tenofovir alafenamide and second-generation non-nucleoside reverse-transcriptase inhibitors across diverse HIV-1 subtypes J Antimicrob Chemother (2018); 73: 2721–2728 DOI: 10.1093/jac/dky256 [PMID: 30053052] IV. Neogi Ujjwal, Singh Kamalendra, Aralaguppe G. Shambhu, Rogers C. Leonard, Njenda T. Duncan, Sarafianos G. Stefan, Hejdeman Bo and Sönnerborg Anders, (2018). Ex-vivo antiretroviral potency of newer integrase strand transfer inhibitors cabotegravir and bictegravir in HIV type 1 non-B subtypes. AIDS. 2018 Feb 20;32(4). DOI: 10.1097/QAD.0000000000001726 [PMID: 29239896] LIST OF RELATED SCIENTIFIC PAPERS NOT INCLUDED IN THESIS I. Mikasi SG, Gichana JO, Van der Walt C, Brado D, Obasa AE, Njenda DT, Messembe M, Lyonga E, Assoumou O, Cloete R, Ikomey GM, Jacobs GB. HIV-1 Integrase Diversity and Resistance-Associated Mutations and Polymorphisms Among Integrase Strand Transfer Inhibitor-Naive HIV-1 Patients from Cameroon. AIDS Res Hum Retroviruses. 2020 Jan 13. doi: 10.1089/AID.2019. 0264 PubMed [PMID: 31830799] II. Hill KJ, Rogers LC, Njenda DT, Burke DH, Sarafianos SG, Sönnerborg A, Neogi U,Singh K. Strain-specific effect on biphasic DNA binding by HIV-1 integrase.AIDS. 2019 Mar 1;33(3):588-592. doi: 10.1097/QAD.0000000000002078. PubMed [PMID:30475264]; III. Ikomey GM, Assoumou MCO, Gichana JO, Njenda DT, Mikasi SG, Mesembe M, Lyonga E, Jacobs GB. Observed HIV drug resistance associated mutations amongst naïve immunocompetent children in Yaoundé, Cameroon. Germs. 2017 Dec 5;7(4):178-185. doi: 10.18683/germs.2017. 1124. eCollection 2017 Dec. PubMed [PMID: 29264355] LIST OF ABBREVIATIONS 3TC Lamivudine ADR Acquired drug resistance AIDS Acquired Immunodeficiency Syndrome ARV Antiretroviral BIC Bictegravir CA Capsid CAB Cabotegravir cART Combination antiretroviral therapy DNA Deoxyribonucleic Acid DRM Drug Resistance Mutation DSA Drug sensitivity assay DTG Dolutegravir EfdA 4’-Ethynyl 2-fluro deoxyadenosine ENV Envelope EVG Elvitegravir FDA Food and Drug Administration FTC Emtricitabine GRT Genotypic resistance testing HIC High income countries HIV Human Immunodeficiency Virus HIV-1B Human Immunodeficiency virus type 1 subtype B HIV-1C Human Immunodeficiency virus type 1 subtype C HTS High throughput sequencing IN Integrase INSTI Integrase strand transfer inhibitor LMIC Low and middle-income countries LTR Long terminal repeats MA Matrix NC Nucleocapsid NOP Naturally occurring polymorphism NNRTI Non-Nucleoside Reverse Transcriptase Inhibitor NRTI Nucleoside analogue Reverse Transcriptase Inhibitor PDR Pre-treatment drug resistance PI Protease inhibitors RAL Raltegravir RNA Ribose nucleic Acid RT Reverse transcriptase SS Sanger sequencing TAF Tenofovir alafenamide TMC125/ETR Etravirine WHO World Health Organization