1. CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
1.13 Replication capacity and viral fitness
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Whilst single cycle assays are performed in a shorter time and offer the benefit of accurate representation of the original virus, multiple cycle assays mimic in vivo conditions more closely and thus provide more accurate results.
Both the single and multiple cycle assays measure the change in IC50 of an ARV required to inhibit 50% of viral growth and report variations in drug susceptibility as FC. The FC is calculated by dividing the IC50 of the test sample by the IC50 of the reference strain.
There are currently two commercially available phenotyping kits: Antivirogram (Tibotec- Virco, Mechelen, Belgium) and Phenosense (Virologic, South San Francisco, California) that are used to measure variations in drug susceptibility of the PR, RT and a portion of the Gag gene. Antivirogram is a multiple cycle assay, whilst Phenosense is a single cycle assay (373).
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more fit and hence replicates more than the WT viral strain, the converse however applies to treatment naïve individuals infected with HIV-1 (381, 382).
In most instances the evolution of viral fitness whilst on ARVs can be divided into two phases: (1) the development of primary mutations which drives reduced susceptibility and impacts negatively on replication capacity and (2) the development of secondary mutations (also referred to as accessory or compensatory mutations) which work in conjunction with primary mutations to improve the replication capacity of the virus (332).
1.13.1 Viruses used in replication capacity assays
Replication capacity assays make use of viruses in any one of the following forms: (1) site- directed mutants; (2) recombinant viruses or (3) whole viral isolates. Each of which is discussed below.
1.13.1.1 Site-directed mutants
The impact of a particular mutation or a group of mutations on viral fitness can be measured by introducing a mutation or group of mutations into a laboratory adapted strain of HIV-1 by site-directed mutagenesis (SDM) and comparing replication capacity of the mutant virus to that of a WT strain (383-385). Engineering the laboratory strain with a reporter gene such as; jellyfish green fluorescent protein (GFP, detected by flow cytometry) or firefly luciferase (detected by luminescence) enables easy detection of viral proliferation (386-388). However viral replication can also be measured by direct detection of gene products such as p24 or by measuring RT-activity. A limitation to using site- directed mutants is the oversight of other mutations which may contribute to viral kinetics.
Whilst several mutations can be engineered into the laboratory strain at one time, there is still a chance that some mutations which work together are excluded (380). This holds particularly true for mutations in genomic regions omitted from the site-directed mutant.
1.13.1.2 Recombinant viruses
Recombinant viruses are generated by inserting an entire genomic region of interest into a standard viral backbone. This allows for links to be drawn between the viral region of interest and viral fitness (380).
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A single clone or amplified pools of virus from a clinical sample can be used to generate recombinant viruses. Whilst single clones offer the advantage of using a precise known sequence, the use of amplified virus pools provides a sample that is more representative of in vivo viral diversity (380).
Recombinant viruses can be constructed by using one of four methods: (1) yeast recombination systems; (2) restriction enzymes; (3) homologous recombination of a vector and virus genomic region of interest in a cell line or (4) gene complementation which produces pseudovirions (387, 389-391). Each of these methods has its pitfalls.
Most yeast recombination systems usually require sub-cloning for viruses to be completely infective and are thus time-consuming and laborious (390, 392) . Restriction enzyme systems are limited by the variability in HIV-1 whereby restriction sites may not be available or may be unsuitable for use (390, 393). Homologous recombination systems are time-consuming and can result in poor recombination efficiency especially for eukaryotic samples (390). Gene complementation can result in the introduction of foreign genetic complements into the pseudovirus and generally produces viruses that can only be used in single cycle replication assays (see section 1.12.3) (390).
A limitation of using recombinant viruses is that the incorporated genomic region is not in its natural context and interactions with other genes are not accounted for. For example interactions between Gag and PR would not be accounted for if only PR was included in a recombinant virus. The most reliable fitness results are thus obtained from using whole viral isolates (380, 386).
1.13.1.3 Whole viral isolates
Whole HIV-1 viral isolates can be extracted from patient plasma or peripheral blood mononuclear cells (PBMCs) (394). This however can be costly, time-consuming and extraction in certain strains can be difficult (380).
Whole viral isolates can be applied to either PBMCs or cell lines to measure replication capacity. In PBMCs, replication capacity is measured by quantifying p24 (using ELISA) or by measuring RT-activity (386). If cell lines engineered to express reporter genes are used, measurement of replication capacity can be via flow cytometry (in the case of GFP)
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or detection of luminescence (in the case of luciferase). The use of reporter gene engineered cell lines is less costly and simpler than using PBMCs (183, 395).
1.13.2 The use of primary cells versus T-cell lines
Replication capacity assays can utilize either primary cells (i.e. cells derived directly from human subjects such as PBMCs) or established T-cell lines (such as CEM-GXR cells).
Studies have shown that results between PBMCs and cell-lines can differ (386, 396).
Additionally results between different established cell lines can also differ (385). Whilst PMBCs supposedly offer results most representative of in vivo environments, they cannot be maintained for long times, they can be highly variable between donors and they require stimulation prior to use (183, 386). These limitations can be addressed by using established cell lines.
1.13.3 Measuring viral replication capacity
Viral fitness can be measured by in vivo and in vitro techniques. In vivo techniques involve comparing the quantity of mutant and WT virus detected within in vivo populations with the most commonly used sample being blood (397). Whilst this technique mimics the hosts’
natural environment and provides the best estimate of viral fitness, it is limited by variation in quantities of viral variants in different compartments within the host (398). For example the most dominant quasispecies in the blood may differ from that in the lung. This limits its use in studies involving host genetics, immune response and drug resistance (398).
In contrast to in vivo methods, in vitro techniques do not mimic the natural environment of the host. In vitro techniques employ either HIV-1 isolates or recombinant viruses in a specific controlled environment thereby making the method useful in the study of drug resistance. There are two types of in vitro assays: single cycle assays and multiple cycle assays. Both of which employs the use of recombinant viruses or pseudovirions.
1.13.4 Single cycle replication capacity assays
The single cycle assay involves the infection of a cell line with a recombinant virus, which encodes reporter genes, and the subsequent detection of the reporter gene in the cell line (either by luminescence or fluorescence) between 24–72 hours post infection (380, 383,
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391, 399). Whilst this assay yields results quickly it is unable to measure the entire replication cycle and is thus less sensitive than multiple cycle assays (183).
1.13.5 Multiple cycle replication capacity assays
Multiple cycle replication capacity assays are divided into pairwise growth competition assays and parallel assays, both of which are described below.
1.13.5.1 Pairwise growth competition assays
For growth competition assays, two viral variants are mixed and added to the same experiment. Both viruses are exposed to identical experimental conditions and compete for the same resources (380). During several passages, the fitter virus out-competes the less fit variant to become the predominant population, the proportion of which can be measured by Sanger sequencing, heteroduplex tracking assays or real-time PCR (386). These detection techniques are however expensive, labour intensive and produce data which is not easily analysed (183, 386). In order to overcome these pitfalls, cell lines or backbones of viruses (i.e. only in the case of recombinant viruses) can be engineered to include reporter genes which can be detected by fluorescent antibodies using flow cytometry (388, 400).
1.13.5.2 Parallel assays
In contrast to pairwise growth competition assays, parallel assays involve the quantification of HIV-1 replication in parallel cultures (401, 402). Cell lines or PBMCs are infected with a particular virus; replication capacity is then quantified by measuring p24 levels or RT-activity in the supernatant at various time-points (403, 404). Reporter genes in the backbone may also be used in detection. This method is simpler and less labor intensive than growth competition assays but it does not allow for the identification of subtle differences in replication kinetics between viruses that are tested, since cell populations in the parallel cultures may grow at slightly different rates thereby influencing the calculation of replicative capacity (380). Parallel assays are however more sensitive than single cycle assays.
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1.14 The current study: Rationale, aims and objectives