Dynamics of drug-resistant minor viral populations and phenotypic drug susceptibility in diverse HIV-1 subtypes

Antiretroviral therapy (ART) has significantly reduced mortality in human immunodeficiency virus type 1 (HIV-1) infection, both in highand low/middle-income countries (LMIC). However, the development of drug resistance following exposure to subtherapeutic concentrations of antiretrovirals (ARV) and transmitted drug resistance mutations (DRM) adversely affects the outcome of ART. In my thesis, drug resistance patterns of diverse HIV-1 subtypes were described, at both genotypic and phenotypic level. A high throughput sequencing (HTS) method was utilized to amplify and sequence the gag-pol of different HIV-1 subtypes for identification and quantification of DRM present in <20% of the viral population. A novel bioinformatics pipeline was developed, MiDRMpol, which integrates genomic variations and mapping of minor populations with DRM. Phenotypic drug sensitivity assays were performed to study in vitro potency of newer ARV. Also, polymerase independent increase of virulence and replication competence of HIV-1, which may influence the outcome of ART, was studied. Paper I describes the pipeline, MiDRMpol, which can be used without any prior knowledge and does not require on-site bioinformatics support. The raw data from HTS in the fastq format are uploaded to get an easily readable format. One of the unique features when compared to other available pipelines is the FastUniq tool, which removes the duplicate reads generated by PCR, thus reducing the pseudo-bias of few variants. Another feature is the subtype-specific adaptation during the analysis. In Paper II and Paper III, I studied the potency of three newer ARV. 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), a novel translocation defective reverse transcriptase inhibitor (RTI), was found to inhibit both wildtype and RTI resistant viruses efficiently in a subtype-independent manner. Similarly, the second-generation integrase strand inhibitors (INSTI) cabotegravir (CAB) and bictegravir (BIC) were shown to have equal or higher potency against non-B subtypes as compared to HIV-1B. This confirms the suitability of these drugs for use in countries dominated by non-B subtypes. In Paper IV, I observed that a PYxE insertion in the gag plays a role in increased virulence and replication capacity in HIV-1C viruses and seems to be associated with suboptimal CD4 + T-cell gain following ART initiation. Even though there was no effect of PYxE or PTAPP on the susceptibility to 20 ARVs, the enhanced replication capacity might increase the time to reach viral suppression during ART and thereby increased risk for the virus to develop DRM. In conclusion, identification and quantification of DRM at frequencies <20% is a major hurdle in current ART monitoring, and our MiDRMpol facilitates the analysis of such HTS data. However, we found that also polymerase independent mutations which increase the replication capacity/virulence of HIV-1 may influence the outcome of ART. The new ARVs EFdA, CAB, and BIC suppress the viral load in vitro in a subtype independent manner. This is important since if not all subtypes are suppressed efficiently the risk of a global increase of acquired DRM would be further increased. LIST OF SCIENTIFIC PAPERS I. Aralaguppe SG, Ambikan AT, Njenda DT, Manickam A, Kumar MM, Hanna LE, Amogne W, Sönnerborg A, Neogi U. MiDRMpol: A high-throughput multiplexed amplicon sequencing workflow to identify protease, reverse transcriptase and integrase inhibitors minor drug resistance mutations. Manuscript submitted. II. Njenda DT, Aralaguppe SG, Singh K, Rao R, Sonnerborg A, Sarafianos SG, Neogi U. Antiretroviral potency of 4'-ethnyl-2'-fluoro-2'-deoxyadenosine, tenofovir alafenamide and second-generation NNRTIs across diverse HIV-1 subtypes. J Antimicrob Chemother 2018,73:2721-2728. III. Neogi U, Singh K, Aralaguppe SG, Rogers LC, Njenda DT, Sarafianos SG, Hejdeman B, Sönnerborg A. Ex-vivo antiretroviral potency of newer integrase strand transfer inhibitors cabotegravir and bictegravir in HIV type 1 non-B subtypes. AIDS 2018,32:469-476. IV. Aralaguppe SG, Winner D, Singh K, Sarafianos SG, Quinones-Mateu ME, Sönnerborg A, Neogi U. Increased replication capacity following evolution of PYxE insertion in Gag-p6 is associated with enhanced virulence in HIV-1 subtype C from East Africa. J Med Virol 2017,89:106-111. LIST OF PUBLICATIONS NOT INCLUDED IN THESIS I. Aralaguppe SG, Siddik AB, Manickam A, Ambikan AT, Kumar MM, Fernandes SJ, Amogne W, Bangaruswamy DK, Hanna LE, Sonnerborg A, Neogi U. Multiplexed next-generation sequencing and de novo assembly to obtain near fulllength HIV-1 genome from plasma virus. Journal of Virological methods 2016; 236:98-104. II. Siddik AB, Haas A, Rahman MS, Aralaguppe SG, Amogne W, Bader J, Bader J, klimkait T, Neogi U. Phenotypic co-receptor tropism and Maraviroc sensitivity in HIV-1 subtype C from East Africa. Sci Rep 2018; 8(1):2363. III. Kalu AW, Telele NF, Aralaguppe SG, Gebre-Selassie S, Fekade D, Marrone G, Sonnerborg A. Coreceptor Tropism and Maraviroc Sensitivity of Clonally Derived Ethiopian HIV-1C Strains Using an in-house Phenotypic Assay and Commonly Used Genotypic Methods. Curr HIV Res 2018; 16(2):113-120. IV. Ashokkumar M, Aralaguppe SG, Tripathy SP, Hanna LE, Neogi U. Unique Phenotypic Characteristics of Recently Transmitted HIV-1 Subtype C Envelope Glycoprotein gp120: Use of CXCR6 Coreceptor by Transmitted Founder Viruses. J Virol 2018,92 Apr 13;92(9). pii: e00063-18. doi: 10.1128/JVI.00063-18. Print 2018 May 1.