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.

Student Number : 0114163J - PhD thesis - Faculty of Health Sciences / HIV-1 gains entry into host cells by binding to CD4 and a coreceptor, predominantly CCR5 or CXCR4. Viruses that use CCR5 are termed R5, those able to use CXCR4 are termed X4 while viruses able to use both coreceptors are referred to as R5X4. Accelerated CD4 decline and disease progression within an infected HIV-1 subtype B infected individual is often associated with the emergence of viruses able to use CXCR4. However, CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses, the most predominant strain circulating globally, including South Africa. The aim of this study was to investigate the genetic determinants of CXCR4 usage in HIV-1 subtype C isolates. The V3 region of the envelope glycoprotein is the major determinant of coreceptor usage. In Chapter 2, 32 subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed using a subtype C specific V3-heteroduplex tracking assay. Results indicated that there were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4 (both R5X4 and X4). In general, R5 isolates had a mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). Thus, the rare occurrence of CXCR4-using viruses in subtype C may be due to the highly conserved nature of the GPGQ crown that may limit the potential for the development of X4 viruses. The usefulness of available genotype-based methods for predicting viral phenotypes in subtype C was explored in Chapter 3. Results indicated that commonly used prediction methods could detect R5 viruses, but were not very sensitive at identifying X4 viruses. We therefore developed a subtype C specific predictor based on position specific scoring matrices (PSSM). Similar methodology, as used in developing the subtype B PSSM, was applied on a training set of 280 subtype C sequences of known phenotype (229 NSI/CCR5 and 51 SI/CXCR4). The C-PSSM had a specificity of 94% (C.I. [92%-96%]) and sensitivity of 75% (C.I. [68%-82%]), indicating that the C-PSSM had improved sensitivity in predicting CXCR4 usage. This method also highlighted amino acid positions within V3 that could contribute differentially to phenotype prediction in subtypes B and C. A reliable phenotype prediction method, such as the C-PSSM, could provide a rapid and less expensive approach to identifying CXCR4 variants, and thus increase our knowledge of subtype C coreceptor usage. In Chapter 4 we examined the genetic changes in full-length gp160 envelope genes of 23 sequential isolates from 5 patients followed for two to three years. Three of the patients' isolates used CCR5 at all time points while 2 patients underwent a coreceptor switch with disease progression. The genetic changes observed over time indicated changes in length of variable loops particularly the V1, V4 and V5 and shifting N-glycosylation sites, particularly in the 2 patients that used CXCR4. Changes in the V3 were only noted in the 2 patients’ that used CXCR4 which included substitutions of specific amino acids including those in the crown and increased amino acid charge in the V3 region. Both of these patients were dually infected suggesting that recombination may contribute to the rapid emergence of X4 viruses. The in vitro and in vivo development of CXCR4 usage was analysed in a pediatric patient that experienced a coreceptor switch during disease progression (Chapter 5). Biological and molecular clones were generated and the V1-V5 regions sequenced. Analyses of the V3 region indicated that the evolution to CXCR4 usage happens in a step-wise manner that included increased charge and changes in the crown motif. The intermediate variants with predicted dualtropism were also associated with increased V1-V2 lengths, suggesting that other regions may contribute to coreceptor switching. Furthermore, the development of CXCR4 usage within this patient was due to two mutational pathways, in which one resulted in R5X4 viruses and the other X4 variants. In Chapter 6, the impact and treatment of acute TB on HIV-1 diversity in co-infected patients was investigated, specifically to determine the genetic characteristics of the viral populations present before, during and after TB treatment. Plasma samples from 18 HIV-1 infected patients were analysed using the C2V3 region, six of whom showed a high degree of variation using a V3-HTA and were selected for further analyses. All patients were predicted as R5 with no evidence of coreceptor switching over time. There was no correlation between the degree of genetic diversity and viral load, although both showed fluctuations over time. Phylogenetic and pairwise genetic distance analysis indicated that there was amplification of existing variants in 3 patients while in the other 3 patients there were dramatic shifts in viral populations suggesting selection of viral sub-populations over time. Thus in some co-infected patients, TB can affect HIV-1 genetic heterogeneity although there was no evidence of a shift towards CXCR4 usage despite the presence of an AIDS defining illness. Observations in this study have shown that the V3 region is the major determinant of coreceptor usage within HIV-1 subtype C, similar to HIV-1 subtype B. Characteristics such as increased charge length variability of the V3 region and loss of the glycosylation site within this region are associated with CXCR4 usage. The limited number of X4 viruses in subtype C does suggest some restricting mechanisms for CXCR4 usage. In this study we looked at genetic determinants and found that the rare occurrence of CXCR4-using viruses in subtype C, may be due to the highly conserved nature of the GPGQ crown that may limit the potential for the development of subtype C X4 viruses. Furthermore, the development of CXCR4 usage happened in a step-wise manner, with R5X4 viruses intermediates, in which an increased V1-V2 was observed suggesting that other regions within the envelope protein do contribute to coreceptor usage. Thus, regions such as V1-V2 and V4-V5 did contribute to coreceptor usage, but the V3 region remained the most important determinant of coreceptor usage in HIV-1 subtype C isolates. Collectively these findings have provided important data on the genetic determinants of CXCR4 usage in HIV-1 subtype C and an understanding of how they might evolve within a patient.