PhD - Pathology / The entry stage of the HIV-1 viral life cycle has become a prime target for preventing HIV-1 infection. This has led to the development of a new class of anti-retroviral agents termed entry inhibitors, which have proven effective in vitro and in the clinic. These new agents target three different stages of entry, namely CD4 binding, coreceptor interaction with either CCR5 or CXCR4 and the fusion process. Here we studied isolates from patients with HIV-1 subtype C infection and the effectiveness of different coreceptor and fusion inhibitors in vitro. Further we examined resistance profiles to the first licensed entry inhibitor, T-20.
In Chapter 2 we examined coreceptor usage of HIV-1 subtype C isolates and their sensitivity to CCR5 and CXCR4 inhibitors. Twenty-nine viral isolates with different coreceptor usage profiles were isolated from patients with advanced AIDS. The CCR5-specific agents, PRO140 an anti-CCR5 monoclonal antibody and RANTES, the natural ligand for CCR5 inhibited all 24 R5 isolates, while the two X4 and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor, AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells (PBMC) that did not express CCR5 confirming their ability to use CXCR4 on primary cells. When tested using coreceptor-transfected cell lines, one R5 virus was also able to use CXCR6, and another R5X4 virus could use CCR3, Bob/GPR15 and CXCR6. The R5X4 and X4 viruses contained more diverse V3 loop sequences with a higher overall positive charge, compared to the R5 viruses. Hence, HIV-1 subtype C viruses are able to use CCR5, CXCR4 or both for entry, and they are sensitive to specific inhibitors of entry via these coreceptors.
In Chapter 3 we analyzed isolates from 10 acutely infected individuals, who were followed longitudinally for up to three years. Two of these individuals (Du151 and Du179) underwent a coreceptor switch and were studied more intensively. The other eight individuals retained CCR5 usage throughout the duration of the study. The initial 4 isolates from Du151 were able to utilize CCR5 but the later isolates were able to use both CCR5 and CXCR4 (R5X4). Du179 used both CCR5 and CXCR4 (R5X4) initially, but the later isolate was found to be monotropic and used CXCR4 (X4) exclusively. Viral isolates were tested for their sensitivity to small molecule inhibitors of CCR5, CXCR4 and the fusion process in a PBMC assay. All of the R5 isolates were sensitive to RANTES and PRO140 and insensitive to the two CXCR4 coreceptor inhibitors AMD3100 and T-140. There was a tendency for later isolates to become slightly less sensitive to the CCR5 inhibitors and more sensitive to the CXCR4 entry inhibitors. None of the R5X4 Du179 isolates were effectively inhibited by PRO140 and RANTES, but the X4 isolate of Du179 became sensitive to CXCR4 entry inhibitors. Both Du151 and Du179 underwent amino acid changes in their V3 sequences that included an increased charge associated with CXCR4 usage. This indicates that coreceptor switching can occur in subtype C infections and is associated with changes in the V3 loop. However, both Du151 and Du179 were subsequently found to be dually infected with another subtype C strain, which may account for some of the phenotypic and genotypic changes seen in these individuals including the appearance of CXCR4-virus variants.
In Chapter 4 we explored two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors for entry (i.e. coreceptors other than CCR5 or CXCR4) on transfected cell lines. These isolates were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. Both isolates were from patients with cryptococcal meningitis, a severe AIDS defining condition. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. This isolate grew in D32/D32 CCR5 PBMC in the presence of AMD3100, indicating that it used a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, Bob/GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is thus currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6 and CCR3 on transfected cells and was able to replicate in the presence of AMD3100 in D32/D32 CCR5 PBMC. It was insensitive to vMIP-1, eotaxin and I309 used individually, but was inhibited completely when vMIP-1 or I309, the ligand for CCR8, were combined with AMD3100. These results strongly suggest that this isolate can use CCR8 on primary cells. Collectively these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry using coreceptor inhibitors.
In Chapter 5 we examined the effectiveness of T-20 to inhibit HIV-1 subtype C isolates. T-20 blocks the fusion stage of the viral entry cycle and it is the first entry inhibitor to be licensed for clinical use. T-20 consists of 36 amino acids and was designed based on the HR-2 region of HIV-1 subtype B. A total of 23 HIV-1 subtype C isolates were tested for their ability to replicate in the presence and absence of T-20. This included five isolates with multiple genotypic drug resistance mutations to reverse transcriptase and protease inhibitors. Among the 23 subtype C isolates there were 10-16 amino acid changes in the 36 amino acid region corresponding to T-20. However, all isolates were effectively inhibited by T-20 at 1 mg/ml, including the 5 isolates resistant to other anti-retroviral drugs. The gp41 region was sequenced and the HR-1 and HR-2 amino acids analyzed. All isolates had the amino acids GIV at positions 36-38 in gp41, which are associated with sensitivity to T-20. One X4 had a GVV motif but this did not affect its sensitivity. Thus, T-20 inhibited subtype C viruses despite significant genetic differences in the HR-2 regions of subtypes B and C. These data suggest that T-20 would be highly effective in patients with HIV-1 subtype C infection including those failing existing anti-retroviral drug regimens.
In Chapter 6 we examined the in vitro resistance patterns of HIV-1 subtype C to T-20. Resistance to T-20 is a consequence of persistent exposure to the antiretroviral peptide. To establish if patterns of resitance to T-20 were similar to resistance mutations occurring in subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of T-20. The subtype C isolates showed varying levels of replication at 1 mg/ml T-20 by day 18, but by day 29 all replicated efficiciently at 10 mg/ml T-20. All isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to T-20 that included G36S/E/D, I37T, V38M/A/L/E, N42D, N43K/S, L45R/M and A50T/V. Five viruses had compensatory changes in the HR-2 region, which corresponds to the T-20 sequence, and two isolates had changes in the V3 region. Mutational patterns among the 4 subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to T-20 develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for T-20 is similar for HIV-1 subtype B and C isolates.
Observations from these studies indicate that HIV-1 subtype C predominantly use the CCR5 coreceptor to enter cells. CXCR4 usage is rare compared to other subtypes, although such isolates are found in patients with advanced AIDS. The two cases of coreceptor switching reported here were dually infected. Subtype C isolates were sensitive to coreceptor and fusion inhibitors except for two isolates able to utilize alternate coreceptors. However, alternate coreceptor usage is very rare and unlikely to impact on the utility of coreceptor inhibitors. Given the propensity for CCR5 usage this may imply that CCR5 coreceptor inhibitors may be more effective in countries where HIV-1 subtype C predominate. Entry inhibitors may be useful for prevention and treatment strategies and have the potential to provide sterilizing immunity. These agents could be used as microbicides and as an adjunct to existing antiretroviral therapies for use in HIV-1 subtype C infected individuals. However resistance to entry inhibitors can emerge and should be used in combination with other antiretrovirals to minimize this outcome. While entry inhibitors provide a new line of defence against HIV-1, their cost may prevent their use in developing countries in the immediate future. Nevertheless, this is the first comprehensive study of the sensitivity of HIV-1 subtype C isolates to entry inhibitors providing a data-driven rationale for their use in individuals infected with HIV-1 subtype C.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/176 |
| Date | 09 February 2006 |
| Creators | Cilliers, Reginald Anthony |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 1400299 bytes, application/pdf, application/pdf |
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