Author:

Abstract:

Since the mid-nineties, the repertoire of antiv iral drugs for the treatment of human immunodefici ency virus type 1 (HIV-1) has diversified substant ially enabling the prescription of highly active a ntiviral therapy, even in patients who have failed multiple antiretroviral regimens. Despite th e potency of current antiviral drugs, therapy fail ures do occur. Viral replication in the prese nce of ART results into the generation and se lection of resistance mutations. If prolonged viro logical failure occurs, multiple resistance m utations can accumulate affecting more drugs withi n the prescribed regimen, as well as other drugs d ue to cross-resistance within the same drug class. Timely therapeutic interventions are ne eded in order to safeguard the options f or subsequent drug regimens and the long-term surv ival chances of the patient. HIV-1 drug resis tance tests are valuable tools for composing potent regimens but these tests are challenged by the high viral genetic variability. In Europe , genotypic drug resistance testing is the preferr ed and recommended method above phenotypic testing . However, the interpretation of genotypic dr ug resistance tests can be so complex that special ized expertise is needed. In order to refine¨ and improve genotypic drug resistance interpretati on systems, they need to be continuously updated w ith information on (interactions between) new ¨mutations. Before newly detected mutations are in cluded, their impact on in vitro dru g susceptibility and replication needs to be¨ investigated, as well as their impact on ¨the in vivo virological response. Up unt il ten years ago, basic and clinical HIV-1 researc h was mainly performed on HIV-1 subtype B, wh ich predominated in resource-rich settings. Insigh ts into the potency of antiviral drugs and regimen s as well as into the emergence of drug resistance ¨in non-B subtypes, was lacking. This triggered re search in this field, which was reviewed in chapte r 1. In the subsequent research sections, we contributed to a better identification and characterization of HIV-1 drug resistance by investigatin g whether specific antiviral drugs retained their activity against yet uncharacterized genotype s and assessing the requirement of updating g enotypic interpretation systems. In chapter¨ 2, we focused on the amino acid position 70 in rev erse transcriptase (RT) that plays an important ro le in NRTI resistance. K70R is a thymidine an alogue mutation, but also other amino acid changes ¨at that position have already been associate d with nucleoside RT inhibitor (NRTI) re sistance, such as K70E and K70G. We investigated t he impact of the mutations K70S and K70T on t he in vitro phenotype, two mutations ¨observed in a treated patient and in a subse quent in vitro cultivation experiment. Th e mutations had only a minor impact on in vitro drug susceptibility and replication ca pacity. The selection of K70S/T and their phenotyp es are influenced by the presence of other mutatio ns in RT. However, the low impact on the in vi tro phenotype, the low in vivo preva lence, the exclusive direct association with known major RTI mutations and the yet unknown correlation with in vivo response d o not warrant the inclusion of K70S and K70T¨ into drug resistance interpretation syst ems. In chapter 3, we described the impact t hat a unique insertion of 30 nucleotides at positi on 67 in HIV-1 RT had on the in vitro phenotype and we characterized the origin of thi s insertion. The insertion did not result fro m duplications of neighboring HIV-1 sequences, but ¨had very likely a human origin. The presence of a ¨similar insertion in an epidemiologically unlinke d Japanese patient suggests that a particular sequ ence within chromosome 17 is prone to horizontal g ene transfer into the HIV-1 RT finger subdomain. T he codon use of subtype A and CRF01_AE at position ¨68 in RT favors the insertion of the human s equence in these subtypes. The insertion in the st udy patient conferred high-level in vitro ¨NRTI resistance. The in vivo retention of the insertion for more than 6 years with hi gh viral load, together with the high levels of in vitro NRTI resistance and replication ca pacity, suggest that the insertion is well-tolerat ed and confers a selective advantage in the presen ce of NRTI-containing ART. The remaining par t of the thesis focused on the coreceptor ant agonists, as the recent approval of the CCR5¨ antagonist maraviroc for clinical use offers¨ extra therapeutic options. Maraviroc is only ¨eligible for patients infected with HIV-1 R5 viru ses. To identify these patients, HIV tropism testi ng is recommended before prescribing such a corece ptor antagonist. In chapter 1, we reviewed and dis cussed the state-of-the-art on the efficacy and re sistance profile of the different coreceptor antag onists. The large genetic diversity of the viral e nvelope hampers the quest for markers of in vitro drug resistance and of reduced virol ogical in vivo response to coreceptor ant agonists. This prompted the questions whether the¨ prevalence of R5X4 and X4 variants differed betwee n subtypes and whether existing interpretation sys tems for coreceptor use were applicable to ot her subtypes. In chapter 4, we used genotypi c and phenotypic testing to investigate the in vitro coreceptor use and coreceptor ant agonist resistance levels of subtype G clinical is olates. When focusing on genotypic data, our¨ study revealed that subtype G is characterized by¨ a high prevalence of R5 variants (≥96%), even i n patients with an advanced immunological profile. ¨For subtype G viral variants with phenotypic ¨results, 98.2% were R5, and this was not always i n concordance with the genotypic prediction. Of the different algorithms used, the 11/25 rule a nd the 11/24/25 rule were the only genotypic tropism prediction tools that were fully concordan t with the obtained phenotypic results. However, l arger studies are needed to accurately determine t he tropism prevalence and to fully assess the accu racy of current genotypic tropism prediction tools . In conclusion, this work provides new information and insights into drug resistance, valuabl e for the use of drug resistance testing in clinic al practice.