Acquired and transmitted drug resistance in HIV-1 subtype C : implications of novel mutations on replication capacity, cleavage and drug susceptibility.

Gag-Pro mutations associated with altered replication capacity in HIV-1 subtype C-infected individuals failing a comprehensive protease inhibitor treatment regimen. The prevalence of low-frequency TDR mutations in a cohort of acute HIV-1 subtype C infection was also investigated.

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW

Introduction

Sub-Saharan Africa accounts for most infections globally (approximately 24.7 million HIV infections), followed by Southeast Asia (3.4 million HIV infections) and the Americas (3.2 million HIV infections). In addition, the structure of HIV-1, the protein components of the virus and their function, and the viral replication cycle are detailed.

Figure 1-1 Global distribution of HIV . Sub-Saharan Africa accounts for the most infections globally (approximately 24.7 million HIV infections), this is followed by south-east Asia (3.4 million HIV infections) and the Americas (3.2 million HIV infection

The history of HIV

Classification and origins of HIV

Several studies suggest that the growth of cities in Africa, urbanization, migration and the improved ease of travel in the mid-twentieth century contributed to the exponential growth of the HIV epidemic and the uneven global distribution of subtypes and CRF' is. Figure 1.3). For the purposes of this thesis, the following sections will focus on aspects of HIV-1 infection.

Figure 1-2 Origins of HIV-1 showing SIV in several old world monkeys which crossed the species barrier to infect chimpanzees, gorillas and humans

Global distribution of HIV-1

Structure of HIV-1

The genomic organization of HIV-1
HIV-1 proteins and their function
Structure of HIV-1

Viral protein R is a second accessory protein that functions primarily to enhance viral pathogenicity via: enhancing LTR transcription in infected cells, orchestrating the import of the reverse transcription complex (RTC)4 into the host cell nucleus, and induce cell cycle arrest. The matrix protein (MA) is attached to the inner surface of the viral lipoprotein membrane.

Replication cycle of HIV-1

Virus entry
Reverse transcription
Uncoating
Nuclear import
Integration
Transcription and nuclear export
Assembly
Virion release
Maturation

The first step in viral entry is the adsorption of the virion to the host cell. HIV-1 transcription is mediated by an individual promoter located in the 5' LTR of the provirus.

Figure 1-4 Genomic organization, virion structure and replication cycle of HIV-1. (a) Nine genes of HIV-1, flanked on either end by an LTR, within their respective reading frames

HIV-1 pathogenesis

Acute infection
Chronic HIV-1 infection
Advanced HIV-1 disease (AIDS)

Studies have shown that nearly 80% of CD4+ T lymphocytes in the GALT are depleted within the first three weeks of HIV-1 infection (199). In addition, infected individuals may present with non-AIDS comorbidities including: cardiovascular disease, atherosclerosis, neurocognitive impairment, and liver disease during this phase of HIV-1 infection (203).

Figure 1-6 Illustration of the three stages of HIV-1 infection and diagram highlighting the Fiebig stages of infection

HIV pathogenesis and antiretroviral therapy

Antiretroviral therapy

Such countries typically have access to only three classes of HIV-1 drugs (ie: NRTIs, NNRTIs, and PIs) that are offered as standardized treatment regimens (229). The first regimen consists of two NRTIs and one NNRTI, delivered as a fixed-dose combination (FDC) 15 pill.

Figure 1-7 Site of action of antiretroviral drugs. The target site of: fusion inhibitors, CCR5 inhibitors, integrase strand transfer inhibitors (InSTI’s), non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide transcriptase inhibito

Drug resistance

In the absence of ARV drugs, the dominant population of HIV-1 is WT virus. By doing so, viral replication is more effectively controlled and the chances of replication of mutant viruses and transmission of drug-resistant strains of HIV-1 are less likely.

Overview of antiretroviral drugs

There are currently four NNRTIs used in the treatment of HIV-1, including: Efavirenz (EFV), Nevirapine (NVP), Rilpivirine (RPV), and Etravirine (ETR) (175). Drug resistance mutations associated with NNRTIs generally have minimal effect on the replicative capacity of a mutant virus and have long return times in the absence of drug, making them easily transmissible (228). Similarly, the p1/p6 splice site mutation P452K only causes PI resistance in the presence of the I84V/L90M mutation in PR (315).

Mutations in Gag CS and non-CS can also occur as primary mutations that confer PI resistance in the absence of PR mutations.

Figure 1-8 Crystal structure of reverse transcriptase with an illustration on the mechanism of action of NRTI’s and NNRTI’s

Drug resistance testing

Genotypic drug resistance testing
Phenotypic drug resistance testing

Like conventional sequencing, phenotypic testing uses viral RNA extracted from patient plasma for PCR to generate amplicons of the gene of interest. The proliferation of the recombinant construct in the presence/absence of ARVs can be measured using a single-cycle phenotypic assay or a multi-cycle phenotypic assay. 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 detect variations in drug susceptibility of the PR, RT and part of the Gag measure none.

Replication capacity and viral fitness

Viruses used in replication capacity assays
The use of primary cells versus T-cell lines
Measuring viral replication capacity
Single cycle replication capacity assays
Multiple cycle replication capacity assays

A single clone or amplified pools of virus from a clinical sample can be used to generate recombinant viruses. Recombinant viruses can be constructed 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. Whole viral isolates can be applied to either PBMCs or cell lines to measure replication capacity.

If cell lines designed to express reporter genes are used, measurement of replication capacity can be done via flow cytometry (in the case of GFP).

The current study: Rationale, aims and objectives

Study Rationale
Aims and objectives

The focus of this study was to identify mutations in Gag-Protease that were associated with PI resistance/exposure and to determine the impact of these mutations on replication capacity, drug sensitivity, and polyprotein cleavage, in a cohort of subtype C patients. of HIV-1 that fail. an inclusive PI treatment regimen from KwaZulu-Natal, South Africa. The first aim was to examine the hypothesis that mutations in both the amino and carboxy terminus of Gag contribute to decreased PI drug sensitivity and altered viral fitness in HIV-1 subtype C-infected participants who are failing a regimen comprehensive PI treatment. Identify mutations in Gag-Protease associated with PI exposure or resistance in 80 HIV-1 subtype C-infected participants who were failing a PI-containing treatment regimen.

Determine the effect of Gag-Protease mutations on replication capacity in 80 participants infected with HIV-1 subtype C who did not receive a treatment regimen that included a PI.

Structure of thesis

Determine the presence of low frequency mutations associated with TDR in a cohort of acutely infected participants. UDPS of a subset of these samples (n=14) was performed to detect low frequency mutations in RT, PR and INT. Exploratory analysis was performed to determine the impact of low frequency mutations on treatment outcomes.

This article discusses the prevalence of TDR mutations in an acute cohort and includes an exploratory analysis of the impact of low-frequency TDR mutations on treatment outcomes.

Role of Matrix Protein in Virion Binding of the Virion Envelope Glycoprotein of Immunodeficiency Virus Type 1. Variability in Human Immunodeficiency Virus Type 1 Subtype C Protease Cleavage Sites: An Indicator of Viral Fitness. Genotype and phenotype patterns of human immunodeficiency virus type 1 resistance to enfuvirtide during long-term treatment.

Reduced fitness of human immunodeficiency virus type 1 variants with high resistance to protease inhibitors. Novel Gag-Pol frameshift site in human immunodeficiency virus type 1 variants resistant to protease inhibitors. Gag determinants of fitness and drug sensitivity in protease inhibitor-resistant human immunodeficiency virus type 1.

Introduction

Currently, there are only two studies that have investigated Gag mutations associated with PI resistance in HIV-1 subtype C ( 31 , 32 ). The authors showed that most Gag mutations associated with PI resistance occurred in the carboxy terminal of Gag ( 31 ). This present study aimed to identify Gag-Protease mutations associated with PI resistance/exposure in an HIV-1 subtype C cohort of 80 patients who failed a PI inclusive treatment regimen between from KwaZulu-Natal, South Africa .

Briefly, the Gag-Protease region from a total of 80 participants infected with HIV-1 subtype C who failed a PI-containing treatment regimen was genotyped.

Methods

Study participants/ sequences
RNA Extraction
Amplification of the Gag-Protease region of HIV-1
Sanger sequencing of HIV-1 Gag-Protease amplicons
Data analysis
Statistical analysis

Control groups consisting of sequences from: HIV-1 subtype C acutely infected individuals, HIV-1 subtype C treatment-naive individuals, and HIV-1 subtype B treatment-naive individuals as discussed below. 54 Gag-Protease sequences from HIV-1 subtype C acutely infected individuals were obtained from Drs. Jaclyn Mann (HIV Pathogenesis Programme, University of Kwa-Zulu Natal). The frequency of variants at each codon was compared among sequences from: participants not on a PI-inclusive treatment regimen (PCS cohort), HIV-1 subtype C treatment-naïve individuals, and HIV-1 subtype C acutely infected individuals.

The first compared the viral signature patterns between the PCS cohort and the HIV-1 subtype C treatment-naïve cohort.

Figure 2-1 Overview of study participants and control groups utilized in this study. Study samples comprised of 80 participants failing a PI inclusive treatment regimen (PCS cohort), whilst the control group comprised of: 54 sequences from HIV-1 subtype

Results

Participant characteristics
Identification of Protease DRMs in the PCS cohort
Identification of known Gag mutations associated with PI resistance
Identification of novel Gag mutations associated with PI resistance
Assessing frequency of Gag mutations in sequences without PR RAMs

Eight out of 11 Gag mutations known to be associated with PI resistance were detected in the PCS cohort (Table 2.1 and Figure 2.5). Exploratory analysis was performed to identify novel Gag (nGag) mutations associated with PI resistance/exposure in HIV-1 subtype C, using the RegaDB sequence analysis tool and the VESPA tool. The Q69K and I256V Gag mutations occurred at significantly higher frequencies in the PCS cohort compared to the treatment-naïve (P<0.001) and acute (p<0.0001) cohorts, suggesting that these mutations may be associated with PI exposure/resistance (Figure 2.8 and Figure 2.7).

PID Gag mutations identified in the literature New Gag mutations. nGag) Associated with PI exposure. Associated with PI resistance (rGag).

Figure 2-3 Neighbor joining tree (Paup 4.0) for PCS cohort participants. All sequences from the PCS cohort clustered with HIV-1 subtype C reference sequences indicating that all sequences are representative of HIV-1 subtype C infection

Discussion

These data showed that for the PCS group, PR RAMs always occurred in association with gag mutations, however gag mutations also occurred without PR RAMs. The P453L Gag mutation was found to co-occur with the following PR RAMs in the present study: M46I, 50V, I54V, and V82A. However, all of these participants carried mutations associated with rGag and/or PI exposure, which have been previously reported to increase PI resistance or viral replication in the presence of PR RAMs.

This could suggest that Gag mutations in HIV-1 subtype C contribute to PI resistance in the absence of PR-RAMs.

Conclusion

In the second study, Gatanaga et al., (2002) reported that the H219Q Gag mutation preceded the development of PR RAMs and suggested that it may facilitate the development of PR RAMs (2002). However, there is much debate whether Gag or PR mutations appear first with further investigations needed to elucidate the mechanisms of Gag-Protease co-evolution in PI resistance. The high prevalence of rGag CS mutations occurring in association with PR RAMs may thus suggest that Gag and Protease co-evolve during PI resistance.

Finally, four novel Gag mutations associated with PI resistance have been identified, their role in PI resistance requires further investigation and will be discussed in Chapter 3.

Non-cleavage site mutations contribute to full recovery of viral fitness in human immunodeficiency virus type 1 protease inhibitor-resistant mutations. protease selected from indinavir, ritonavir and/or saquinavir. Selection of resistance in protease inhibitor-experienced human immunodeficiency virus type 1-infected subjects failing lopinavir- and ritonavir-based therapy: mutation patterns and baseline correlates.

Genotypic changes in human immunodeficiency virus type 1 protease associated with reduced susceptibility and virologic response to the protease inhibitor tipranavir.

Introduction

As part of the current study, we sought to identify Gag codon substitutions significantly associated with altered replication capacity in HIV-1 subtype C-infected patients naïve to PI-containing regimens. In addition, we investigated the impact of these codons and four novel Gag mutations (nGag; defined in Chapter 2) on replication capacity and drug sensitivity. A codon-by-codon analysis tool was used to identify mutations in Gag associated with significantly altered replication capacity.

Furthermore, the replication capacity of viruses with nGag mutations, occurring with or without RAM PR, was compared to investigate the role of nGag mutations in PI resistance.

Methods

Generation of Gag-Protease recombinant virus stocks
Virus titration and replication capacity assays
Virus titration and phenotypic drug susceptibility testing
Data analysis

Cell growth was monitored every second day and when not used for experiments, 80% of the cell culture was removed and replaced with fresh, pre-warmed R10. The diluted virus was added to the respective wells of the 24-well culture plate, which was incubated for 24 hours at 37ºC and 5% CO2. Then, 10 ml of pre-warmed DMEM was added and the wall of the T25 flask containing the cell monolayer was rinsed repeatedly to loosen the cells.

The lower FC cutoff for LPV and DRV was calculated using the 99th percentile of the mean IC50 for the reference virus NL43-WT, which was known to be susceptible to LPV and DRV (50).

Figure 3-2 Example of the gating strategy used to distinguish between live and dead GXR cells and infected versus uninfected GXR cells

Results

Replication capacity assay
Phenotypic drug susceptibility

The relationship between the number of mutations in Gag and PR and replication capacity was assessed. For this analysis, replication capacity was compared between: viruses harboring PR-RAMs and rGag mutations (n=34), viruses with only rGag mutations (n=33), and viruses without PR-RAMs and rGag mutations (n=13). Thus, the 335R substitution is associated with reduced replication capacity both in the absence and presence of PR-RAMs (Figure 3.13a).

Viruses with 431V and PR RAMs (n=24) occurring in combination with the 69K (n=16) nGag mutation had significantly higher replication capacity than viruses with 431V and.