**5. Generation of resistance to small molecule CCR5 inhibitors**

The most parsimonious escape pathway from the selection pressure of a CCR5 inhibitor would be coreceptor switching to CXCR4 use, with its well documented devastating consequences on the rate of CD4+ T cell depletion and disease progression of patients [22, 53, 67]. Both *in vitro* and *in vivo* studies have nevertheless shown that this is not the preferred escape pathway by HIV-1 despite the short distance in sequence space (few mutations) that separate R5 from X4 variants. The reduced replication fitness or the suboptimal coreceptor use, as indicated by the increased sensitivity to the selecting CCR5 antagonist of viral intermediates in the R5 to X4 evolution process compared to baseline virus, may account for this apparently paradoxical phenomenon; the requirements of inserting charged amino acids at specific locations and the strong bias in favor of G-to-A substitutions rather than random mutations are thought to impose additional constraints on the transition [115, 116]. Whatever the reasons may be, when R5 viruses escape from the selection pressure of CCR5 inhibitors they tend to retain the R5 phenotype.

Several factors diversify the pathways of resistance development to CCR5 inhibitors compared to those to conventional antiretroviral drugs [22]. The seemingly facile, for HIV, escape pathway of mutating the active site of a viral enzyme in order to prevent the binding of an antiviral would simply not work in the case of CCR5 antagonists that target a hostrather than a virus-encoded gene. Instead, the virus must devise a strategy to recognize and use the coreceptor to gain access into its target cells despite the presence of the inhibitor. The highly variable nature of the *env* gene certainly facilitates the escape process; however, *env* is also under the constant selection pressure from neutralizing antibodies [34]. Thus, during the development of resistance to CCR5 inhibitors the HIV-1 Env complex must accommodate sequence changes that enable the virus to escape both from the selection pressure of the inhibitors as well as from the humoral immune response, without violating the structural constraints of the Env protein.

#### **5.1.** *In vitro* **resistance: No coreceptor switch to CXCR4 and no uniform genotypic signature**

128 Immunodeficiency

for new CCR5 entry inhibitors [113].

inhibitors they tend to retain the R5 phenotype.

the structural constraints of the Env protein.

preformed ligand-CCR5 complexes with an efficiency that correlates with their ability to act as inverse agonists [112]. The identification of residues mandatory for gp120 binding in the predicted CCR5 dimer interface suggests that receptor dimerization might represent a target

AK530 and AK317, two CCR5 inhibitors containing spirodiketopiperazine scaffolds, lodge in a hydrophobic cavity located between the upper TM domain and the ECL2 of CCR5 [114]. The interaction profile of the inhibitors with ECL2 residues S180 and K191 was found to be an important determinant of antiviral potency; furthermore, amino acid residues in the betahairpin structural motif of ECL2 were critical for HIV-1-elicited fusion and binding of these spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but it also prohibits the virus from utilizing the "inhibitor-bound" CCR5 for cellular entry - a

mechanism of HIV-1's resistance to CCR5 inhibitors, as discussed in the next section.

The most parsimonious escape pathway from the selection pressure of a CCR5 inhibitor would be coreceptor switching to CXCR4 use, with its well documented devastating consequences on the rate of CD4+ T cell depletion and disease progression of patients [22, 53, 67]. Both *in vitro* and *in vivo* studies have nevertheless shown that this is not the preferred escape pathway by HIV-1 despite the short distance in sequence space (few mutations) that separate R5 from X4 variants. The reduced replication fitness or the suboptimal coreceptor use, as indicated by the increased sensitivity to the selecting CCR5 antagonist of viral intermediates in the R5 to X4 evolution process compared to baseline virus, may account for this apparently paradoxical phenomenon; the requirements of inserting charged amino acids at specific locations and the strong bias in favor of G-to-A substitutions rather than random mutations are thought to impose additional constraints on the transition [115, 116]. Whatever the reasons may be, when R5 viruses escape from the selection pressure of CCR5

Several factors diversify the pathways of resistance development to CCR5 inhibitors compared to those to conventional antiretroviral drugs [22]. The seemingly facile, for HIV, escape pathway of mutating the active site of a viral enzyme in order to prevent the binding of an antiviral would simply not work in the case of CCR5 antagonists that target a hostrather than a virus-encoded gene. Instead, the virus must devise a strategy to recognize and use the coreceptor to gain access into its target cells despite the presence of the inhibitor. The highly variable nature of the *env* gene certainly facilitates the escape process; however, *env* is also under the constant selection pressure from neutralizing antibodies [34]. Thus, during the development of resistance to CCR5 inhibitors the HIV-1 Env complex must accommodate sequence changes that enable the virus to escape both from the selection pressure of the inhibitors as well as from the humoral immune response, without violating

**5. Generation of resistance to small molecule CCR5 inhibitors** 

To gain insights into what may happen *in vivo* if or when small molecule CCR5 inhibitors are used more extensively, resistance is first studied *in vitro*. To generate resistant variants, primary R5 isolates are cultured with physiological target cells that are permissive for replication of both R5 and X4 viruses in the presence of increasing concentrations of the inhibitor under study. Despite the intrinsic difficulty associated with the selection process in each case that proved to require prolonged periods of culture in the presence of these compounds, several such studies have been published (reviewed in [22, 72, 117]). An alternative *in vitro* system that allows for the selection of escape mutants to CCR5 inhibitors over a relatively short period of time is the construction of V3 loop libraries of R5 infectious clones (reviewed in [118]). The results of these studies collectively show that the escape mutants tend to retain usage of CCR5 despite the availability of CXCR4. The mechanism that allows resistant viruses to continue to use CCR5 in the presence of the inhibitor is discussed in the next section. X4 viruses have been observed in CCR5 inhibitor-selection experiments, either in the control culture alone [119] or in both the selection and the control cultures [110], but their emergence was probably due to adaptation to growth in culture rather than the inhibitor escape process. Conditional recognition of CXCR4 in specific cell lines has also been reported for one VCV-resistant virus [109]. HIV-1 can be "forced" to escape CCR5 drug pressure through coreceptor switch under certain experimental conditions, which may not be very relevant physiologically [120].

To date, no clear signature resistance mutations have been identified for CCR5 antagonists and different genetic pathways can lead to the same resistance phenotype (Table 2).

The determinants of resistance commonly map to the V3 region of gp120, a localization that is consistent with the highly variable nature of this region as well as with its role in coreceptor engagement [22, 45]. Four V3 substitutions (K305R, H308P, A316V and G321E) were, for example, responsible for the resistant phenotype of isolate CC101.19 when the CCR5-using, subtype B virus CC1/85 [62] was cultured with the small molecule CCR5 inhibitor AD101, a preclinical precursor of VCV [107, 108]. A much rarer route to resistance was undertaken by viruses derived from the same CC1/85 lineage in an AD101/VCV selection culture [109]; the resulting D101.12 escape mutants contained at least one substitution in V3 (H308P) as well as either of the following combinations of substitutions within and downstream of the gp41 fusion peptide (FP): G514V+V535M (Pattern I) or M518V+F519L+V535M (Pattern II) [127]. We have also described a unique VCV escape pathway for viruses from the D1/85.16 lineage that involved no V3 changes; instead, resistance mapped to three conservative substitutions in the FP: G516V, M518V and F519I [126]. As shown recently [131], the G516V change is critical to VCV resistance, although it must be accompanied by either M518V or F519I to have a substantial impact phenotypically. Nevertheless, introducing the three FP changes together into a heterologous virus, JR-FL, did not create a VCV-resistant variant [126]. CCR5 inhibitor resistance is usually dependent upon the Env genetic context and it is not transferred when introduced into other viruses [124, 126, 132], but that is not always the case [129].


Chemokine Receptors as Therapeutic Targets in HIV Infection 131

inhibitor enfuvirtide) [117]. Small molecule CCR5 inhibitors also retain sensitivity to anti-

**Viral fitness.** When HIV-1 develops resistance to conventional antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. To investigate whether the *in vitro* development of resistance to small molecule CCR5 inhibitors has an associated fitness cost, we developed a growth-competition assay involving dual infections with molecularly cloned viruses that are essentially isogenic outside the *env* genes under study. Real-time TaqMan quantitative PCR (QPCR) was used to quantify each competing virus individually via probes specific to different, phenotypically silent target sequences engineered within their *vif* genes. Head-to-head competition assays of *env* clones derived from the AD101 and VCV resistant isolate, the inhibitor-sensitive parental virus, and a passage control virus showed that CCR5 inhibitor resistance was not associated with a fitness loss [133]. This observation is consistent with the retention of the resistant phenotype when the escape mutant virus CC101.19 was cultured for a total of 20 passages in the absence of the selecting compound [133]. Amino acid substitutions in the V3 region of gp120 that confer complete resistance cause a fitness loss when introduced into an inhibitorsensitive, parental clone; however, in the resistant isolate, changes elsewhere in *env* that occurred prior to the substitutions within V3, appear to compensate for the adverse effect of the V3 changes on replicative capacity. Hence, a decrease in fitness resulting from CCR5 inhibitor resistance may not be as common as with the fitness loss observed with resistance

The *in vivo* situation may differ, but further studies are needed to explore the fitness impact of HIV-1 with acquired resistance to CCR5 antagonists. In one case, pre-treatment V3 sequences reemerged in a subtype C-infected patient following VCV discontinuation, implying that VCV resistance has associated fitness costs [122]. Nonetheless, all the VCVresistant isolates we have studied (CC101.19, D101.12 and D1/85.16) have highly stable phenotypes in peripheral blood mononuclear cells (PBMC), in that they do not rapidly revert to sensitivity when cultured without the inhibitor [108, 126, 127, 133]. During reversion experiments of individual resistant clones in the absence of the inhibitor, we discovered that M518V, which has little or no effect on VCV sensitivity on its own, appears to be under no pressure to revert either [131]. Val-518 is a naturally occurring FP polymorphism that becomes enriched during VCV or AD101 selection [126]. However, a resistant clone D1/85.16 cl.23 (= R) reverted to a sensitive phenotype by five weekly, VCVfree passages in PBMC culture. Interestingly, the complete cluster of the three FP changes persisted within the genetic context of the gp120 derived from the resistant clone R; however, the three FP changes were accompanied by a Thr-to-Ala substitution at position 244 of the second conserved region (C2) of gp120 that appears to counter the VCV-resistance phenotype created by the FP substitutions. This residue is located in strand β7, part of a βsandwich structure in the inner domain that is involved in CD4-induced conformational changes within gp120 and the interaction with gp41 [134-137]. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence

CCR5 MAbs that act by a dissimilar mechanism.

to almost all other antiretroviral drugs (reviewed in [72]).

both HIV-1 entry and resistance to CCR5 inhibitors.

**ND**, Not determined; **I322aV** occurs at an amino acid not present in HXB2 located between residues 322 and 323 and is designated 322a.

**Table 2.** Genetics of HIV-1 escape from the selection pressure of small molecule CCR5 inhibitors

The findings of these studies have important implications for the clinical use of small molecule inhibitors, particularly for the generation of genotypic algorithms and phenotypic assays to predict the emergence of resistant viruses and the likelihood of cross-resistance to other members of this drug class. Cross-resistance to other CCR5 antagonists may or may not arise with these molecules that retain sensitivity to all other drug classes (e.g. nucleoside and non-nucleoside reverse transcriptase inhibitors, protease and integrase inhibitors) as well as to other entry inhibitors acting at different stages of the entry process (e.g. the fusion inhibitor enfuvirtide) [117]. Small molecule CCR5 inhibitors also retain sensitivity to anti-CCR5 MAbs that act by a dissimilar mechanism.

130 Immunodeficiency

 **Parental virus gp120** 

TAK-779 JR-FLV3Lib <sup>B</sup>I304V, H305N, I306M,

AD101 CC1/85 B K305R, H308P, A316V,

TAK-652 KK Unknow

isolate <sup>C</sup>

Clinical isolate S91

CC101.6 (CC1/85 passage 6 AD101 selection)

B

isolate B P/T308H, T320H,

MVC JR-FLV3Lib <sup>B</sup>I304V, F312W, T314A,

I322aV

E317D, I318V

VCV Clinical

VCV

VCV

AVC Clinical

MVC Clinical

designated 322a.

**Resistance mutations** 

<sup>n</sup>ND ND ND [119]

E328K, G429R (C4)

None

D407G, ΔN386 (V4)

T199K, T275M (C2)

F312L, E317D None None [121]

G321E None None [107, 108]

None None [122]

G514V, V535M (Pattern I) or M518V, F519L, V535M (Pattern II)

None [123]

F519I [109, 126]

None [129]

None [130]

[109, 127]

**Drug Name Subtype V3 Elsewhere gp41 References** 

VCV RU570 G K305R, R315Q, K319T P437S (C4) None [124, 125]

isolates UnknownSeveral Several Several [128]

MVC CC1/85 B A316T, I323V ND ND [110] MVC RU570 G ΔQAI (315-317) ND ND [110]

**ND**, Not determined; **I322aV** occurs at an amino acid not present in HXB2 located between residues 322 and 323 and is

The findings of these studies have important implications for the clinical use of small molecule inhibitors, particularly for the generation of genotypic algorithms and phenotypic assays to predict the emergence of resistant viruses and the likelihood of cross-resistance to other members of this drug class. Cross-resistance to other CCR5 antagonists may or may not arise with these molecules that retain sensitivity to all other drug classes (e.g. nucleoside and non-nucleoside reverse transcriptase inhibitors, protease and integrase inhibitors) as well as to other entry inhibitors acting at different stages of the entry process (e.g. the fusion

**Table 2.** Genetics of HIV-1 escape from the selection pressure of small molecule CCR5 inhibitors

K305R, T307I, F316I, T318R, G319E and

VCV CC1/85 B None None G516V, M518V,

H308P and one or more of: K305R, A316V, G321E

S306P

D Q315E, R321G

**Viral fitness.** When HIV-1 develops resistance to conventional antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. To investigate whether the *in vitro* development of resistance to small molecule CCR5 inhibitors has an associated fitness cost, we developed a growth-competition assay involving dual infections with molecularly cloned viruses that are essentially isogenic outside the *env* genes under study. Real-time TaqMan quantitative PCR (QPCR) was used to quantify each competing virus individually via probes specific to different, phenotypically silent target sequences engineered within their *vif* genes. Head-to-head competition assays of *env* clones derived from the AD101 and VCV resistant isolate, the inhibitor-sensitive parental virus, and a passage control virus showed that CCR5 inhibitor resistance was not associated with a fitness loss [133]. This observation is consistent with the retention of the resistant phenotype when the escape mutant virus CC101.19 was cultured for a total of 20 passages in the absence of the selecting compound [133]. Amino acid substitutions in the V3 region of gp120 that confer complete resistance cause a fitness loss when introduced into an inhibitorsensitive, parental clone; however, in the resistant isolate, changes elsewhere in *env* that occurred prior to the substitutions within V3, appear to compensate for the adverse effect of the V3 changes on replicative capacity. Hence, a decrease in fitness resulting from CCR5 inhibitor resistance may not be as common as with the fitness loss observed with resistance to almost all other antiretroviral drugs (reviewed in [72]).

The *in vivo* situation may differ, but further studies are needed to explore the fitness impact of HIV-1 with acquired resistance to CCR5 antagonists. In one case, pre-treatment V3 sequences reemerged in a subtype C-infected patient following VCV discontinuation, implying that VCV resistance has associated fitness costs [122]. Nonetheless, all the VCVresistant isolates we have studied (CC101.19, D101.12 and D1/85.16) have highly stable phenotypes in peripheral blood mononuclear cells (PBMC), in that they do not rapidly revert to sensitivity when cultured without the inhibitor [108, 126, 127, 133]. During reversion experiments of individual resistant clones in the absence of the inhibitor, we discovered that M518V, which has little or no effect on VCV sensitivity on its own, appears to be under no pressure to revert either [131]. Val-518 is a naturally occurring FP polymorphism that becomes enriched during VCV or AD101 selection [126]. However, a resistant clone D1/85.16 cl.23 (= R) reverted to a sensitive phenotype by five weekly, VCVfree passages in PBMC culture. Interestingly, the complete cluster of the three FP changes persisted within the genetic context of the gp120 derived from the resistant clone R; however, the three FP changes were accompanied by a Thr-to-Ala substitution at position 244 of the second conserved region (C2) of gp120 that appears to counter the VCV-resistance phenotype created by the FP substitutions. This residue is located in strand β7, part of a βsandwich structure in the inner domain that is involved in CD4-induced conformational changes within gp120 and the interaction with gp41 [134-137]. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

Chemokine Receptors as Therapeutic Targets in HIV Infection 133

A B C **Figure 4. Resistance mechanisms for viral escape from CCR5 inhibitors.** Model inhibition curves are displayed for sensitive and resistant viruses (solid grey and dashed black curves, respectively). The atypical dose-response curves of resistant viruses with gp41 fusion peptide (FP) mutations are shown in blue. **A.** Competitive resistance results in a shift in the IC50 of an inhibitor to a higher concentration, with complete inhibition potentially achievable at a high enough inhibitor concentration. **B.** In noncompetitive resistance, the IC50 values are typically equivalent to those for the sensitive virus, but the extent of inhibition is incomplete for the resistant virus. The maximum percent inhibition (MPI) value, at a saturating inhibitor concentration, reflects the efficiency with which the inhibitor-CCR5 complex is used relative to free CCR5, thereby providing a measure of the degree of resistance of the HIV-1 variant under study. MPI values vary by cell type for inhibition by VCV and related compounds. **C.** In the novel paradigm of noncompetitive resistance, resistant FP mutant viruses display reduced

**Noncompetitive or "allosteric" resistance.** Noncompetitive resistance (Figure 4B) is defined as resistance that results in saturable inhibition, such that increasing concentrations of the inhibitor have no effect on the virus; the EC50 required to reach this saturation level is the same as the IC50 for the fully sensitive virus [142]. Phenotypically, resistance manifests as a plateau in the maximum achievable suppression of viral replication. The implication of this mechanism is that the resistant Env has adapted to use the inhibitor bound form of CCR5 as a coreceptor as well as, but not instead of, the free coreceptor [110, 142]. The level of residual inhibition, termed the "plateau" or the maximum percent inhibition (MPI), once inhibition has reached saturation reflects the efficiency with which the inhibitor-CCR5 complex is used relative to free CCR5; the higher the MPI value, the less efficiently a resistant virus uses the inhibitor-CCR5 complex [110, 142]. This mechanism is consistent with the allosteric mode of action of small molecule CCR5 inhibitors; these compounds lock the extracellular domains of CCR5 in a conformation that can be recognized only by resistant variants -in addition to the natural coreceptor-, but not by wild-type HIV-1 [117]. In a few instances, the preferred use of the inhibitor-bound CCR5 over the unbound receptor by some CCR5 antagonistresistant viruses leads to modestly increased replication of resistant viruses in the presence of the inhibitor *in vitro* [142]. The cell type influences the mode in which resistance to small molecule CCR5 inhibitors is manifested, particularly MPI values [110, 124, 126, 127, 131, 142]. Moreover, the differences between the MPI values of VCV-resistant and sensitive viruses we have studied are generally much smaller in cell lines engineered to express CCR5, such as TZM-bl or U87.CD4.CCR5 (that serves as the basis of the PhenoSense and

MPI and IC50 values. Adapted from [143].

Trofile tropism assays), than in PBMC [126, 127, 131].

**Figure 3.** Mapping residue 244 on the structure of the gp120 of the representative clone, CC1/85 cl.7, of the parental, inhibitor-sensitive virus CC1/85. Residue 244 (magenta) is located in strand β7, part of a βsandwich structure in the inner domain of gp120 that is thought to be involved in interactions with gp41. The CC1/85 cl.7 gp120 core structure was modeled based on the V3-containing structure of gp120 [138] with Swiss-Model [139] using 2B4C.pdb as template and visualized using PyMOL [140].

#### **5.2. Mechanisms of resistance to small molecule CCR5 inhibitors**

As portrayed graphically in Figure 4, resistance to small molecule CCR5 inhibitors may likely develop via two, not necessarily exclusive, mechanisms: competitive resistance and noncompetitive or allosteric resistance (reviewed in [22, 72, 117, 118, 141]), not to be confused with the competitive or not, properties of the inhibitors themselves [142]. Either mechanism may be effective at overcoming inhibition for some CCR5 inhibitors, while some HIV-1 escape mutants may undertake a combination of the two mechanisms. Understanding how resistance arises is relevant to how it is detected and quantified.

**Competitive resistance.** Competitive resistance (Figure 4A) is defined as resistance that results in a shift in the IC50 of an inhibitor to a higher concentration; complete inhibition can still be achieved at sufficient inhibitor concentrations [142]. Competitive resistance could arise from more efficient utilization of inhibitor-free CCR5 to gain access into target cells; in turn, this could arise either from a greater affinity of Env for the coreceptor, or from an increase in the kinetics of Env-mediated fusion after CCR5 engagement. Either scenario would allow the virus to better compete with the inhibitor for the coreceptor, reflecting its adaptation to scavenge low levels of inhibitor-free CCR5 [142]. A partially (~5-fold) AD101 resistant isolate had this ability [108]; however, this mechanism alone cannot count for complete resistance to CCR5 antagonists [117].

#### Chemokine Receptors as Therapeutic Targets in HIV Infection 133

132 Immunodeficiency

**Figure 3.** Mapping residue 244 on the structure of the gp120 of the representative clone, CC1/85 cl.7, of the parental, inhibitor-sensitive virus CC1/85. Residue 244 (magenta) is located in strand β7, part of a βsandwich structure in the inner domain of gp120 that is thought to be involved in interactions with gp41. The CC1/85 cl.7 gp120 core structure was modeled based on the V3-containing structure of gp120

As portrayed graphically in Figure 4, resistance to small molecule CCR5 inhibitors may likely develop via two, not necessarily exclusive, mechanisms: competitive resistance and noncompetitive or allosteric resistance (reviewed in [22, 72, 117, 118, 141]), not to be confused with the competitive or not, properties of the inhibitors themselves [142]. Either mechanism may be effective at overcoming inhibition for some CCR5 inhibitors, while some HIV-1 escape mutants may undertake a combination of the two mechanisms.

**Competitive resistance.** Competitive resistance (Figure 4A) is defined as resistance that results in a shift in the IC50 of an inhibitor to a higher concentration; complete inhibition can still be achieved at sufficient inhibitor concentrations [142]. Competitive resistance could arise from more efficient utilization of inhibitor-free CCR5 to gain access into target cells; in turn, this could arise either from a greater affinity of Env for the coreceptor, or from an increase in the kinetics of Env-mediated fusion after CCR5 engagement. Either scenario would allow the virus to better compete with the inhibitor for the coreceptor, reflecting its adaptation to scavenge low levels of inhibitor-free CCR5 [142]. A partially (~5-fold) AD101 resistant isolate had this ability [108]; however, this mechanism alone cannot count for

[138] with Swiss-Model [139] using 2B4C.pdb as template and visualized using PyMOL [140].

Understanding how resistance arises is relevant to how it is detected and quantified.

**5.2. Mechanisms of resistance to small molecule CCR5 inhibitors** 

complete resistance to CCR5 antagonists [117].

**Figure 4. Resistance mechanisms for viral escape from CCR5 inhibitors.** Model inhibition curves are displayed for sensitive and resistant viruses (solid grey and dashed black curves, respectively). The atypical dose-response curves of resistant viruses with gp41 fusion peptide (FP) mutations are shown in blue. **A.** Competitive resistance results in a shift in the IC50 of an inhibitor to a higher concentration, with complete inhibition potentially achievable at a high enough inhibitor concentration. **B.** In noncompetitive resistance, the IC50 values are typically equivalent to those for the sensitive virus, but the extent of inhibition is incomplete for the resistant virus. The maximum percent inhibition (MPI) value, at a saturating inhibitor concentration, reflects the efficiency with which the inhibitor-CCR5 complex is used relative to free CCR5, thereby providing a measure of the degree of resistance of the HIV-1 variant under study. MPI values vary by cell type for inhibition by VCV and related compounds. **C.** In the novel paradigm of noncompetitive resistance, resistant FP mutant viruses display reduced MPI and IC50 values. Adapted from [143].

**Noncompetitive or "allosteric" resistance.** Noncompetitive resistance (Figure 4B) is defined as resistance that results in saturable inhibition, such that increasing concentrations of the inhibitor have no effect on the virus; the EC50 required to reach this saturation level is the same as the IC50 for the fully sensitive virus [142]. Phenotypically, resistance manifests as a plateau in the maximum achievable suppression of viral replication. The implication of this mechanism is that the resistant Env has adapted to use the inhibitor bound form of CCR5 as a coreceptor as well as, but not instead of, the free coreceptor [110, 142]. The level of residual inhibition, termed the "plateau" or the maximum percent inhibition (MPI), once inhibition has reached saturation reflects the efficiency with which the inhibitor-CCR5 complex is used relative to free CCR5; the higher the MPI value, the less efficiently a resistant virus uses the inhibitor-CCR5 complex [110, 142]. This mechanism is consistent with the allosteric mode of action of small molecule CCR5 inhibitors; these compounds lock the extracellular domains of CCR5 in a conformation that can be recognized only by resistant variants -in addition to the natural coreceptor-, but not by wild-type HIV-1 [117]. In a few instances, the preferred use of the inhibitor-bound CCR5 over the unbound receptor by some CCR5 antagonistresistant viruses leads to modestly increased replication of resistant viruses in the presence of the inhibitor *in vitro* [142]. The cell type influences the mode in which resistance to small molecule CCR5 inhibitors is manifested, particularly MPI values [110, 124, 126, 127, 131, 142]. Moreover, the differences between the MPI values of VCV-resistant and sensitive viruses we have studied are generally much smaller in cell lines engineered to express CCR5, such as TZM-bl or U87.CD4.CCR5 (that serves as the basis of the PhenoSense and Trofile tropism assays), than in PBMC [126, 127, 131].

**Resistant FP mutants: A novel paradigm of noncompetitive resistance.** The VCV-resistant viruses carrying FP changes represent a novel paradigm of noncompetitive resistance (Figure 4C); apart from the plateau effect, these FP mutants paradoxically have lower IC50 values than wild-type viruses, particularly in certain cell types (e.g. TZM-bl cells). Hence, although by one measure (MPI) the FP-mutant viruses display a modest level of resistance, by another (IC50) they appear to be hypersensitive [126, 127, 131]. To explain this paradox, we created a theoretical model of resistance [126]. The mathematical formulation of the model is presented in detail in the Supporting Information (SI) section of [126]. The model is based on the assumption that distinct forms of CCR5, with varying affinities (ranging from low to high) for small molecule CCR5 inhibitors, are present in different proportions on disparate cell types; these coreceptor forms, which could be conformers (conformational isomers) or differentially Tyr-sulfated forms, are used selectively by resistant HIV-1 variants when ligated with an inhibitor. According to the model, wild-type virus can use both forms of unligated CCR5 for entry, possibly with a preference between them, but it cannot enter via the inhibitor-coreceptor complexes. The resistant virus, on the other hand, uses preferentially the unoccupied high-affinity coreceptor and the occupied low-affinity coreceptor. Applying this model led to the generation of theoretical inhibition curves that closely mimic the experimental data for resistant viruses in each case [126, 127, 131]. Using the model, we have recently identified the third FP mutation, F519I, (of the V3-independent or FP only- genetic pathway to resistance) as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5 [131]. Modeling the inhibition data from two cell types, PBMC and TZM-bl cells, suggested that D101.12, which harbors both V3 and gp41 substitutions, discriminates between high- and low-affinity forms of CCR5 less than D1/85.16, the resistant virus with three FP substitutions [127].

Chemokine Receptors as Therapeutic Targets in HIV Infection 135

CCR5 interactions are nonetheless characterized by considerable complexity. Thus, a subtype D, VCV-resistant patient isolate recognizes the drug-bound form of CCR5 more efficiently but still uses both the Nt and ECL2 [123]. A recently proposed model suggests that broad cross-resistance to multiple inhibitors is associated with an increased dependence on the N-terminus, while a more specific pattern of resistance to individual compounds involves more subtle changes in how the virus interacts with both the Nt and ECL2 [129].

**5.3.** *In vivo* **resistance: Potential expansion of pre-existing, CXCR4-using viruses** 

The limited information currently available from published results of clinical studies suggests that the escape process from small molecule CCR5 inhibitors *in vivo* bears similarities to that observed *in vitro*; clinical resistance is also likely to map to the V3 region of gp120, but the lack of consistency of observed mutations renders impossible the prediction of *in vivo* resistance by sequence analysis at present (reviewed in [72, 77, 117, 141]). As mentioned above, two pathways of virological escape from the selection pressure of CCR5 antagonists have been identified: continued use of CCR5 via selection of R5 virus that can use drug-bound CCR5 for entry in addition to free coreceptor and expansion of pre-existing minority populations of dual-tropic or X4 virus (collectively called "CXCR4-using" viruses). The first pathway of R5 virological failure with reduced susceptibility to CCR5 antagonists is not the most common mechanism of failure. Virologic failure most commonly involves expansion of pre-existing, CXCR4-using viruses that are insensitive to CCR5 inhibitors. This outcome underscores the importance of developing tropism assays with improved ability to

detect minor or archived virus populations capable of using the CXCR4 coreceptor.

phenotypic assays, much like the genotypic resistance methods.

**6. Conclusions and future directions** 

The Trofile assay has been the assay of choice for clinical trials of CCR5 inhibitors. However, the sensitivity of the original commercially available assay was compromised with respect to the detection of low abundance (minority) CXCR4-using variants. As a result, a number of subjects were misjudged to have R5 virus at screening, whereas they actually harbored Dual/Mixed (D/M) virus populations when they entered clinical trials of CCR5 inhibitors; these patients appeared to have a blunted virologic response and high rates of early virologic failure [152-154]. Application of an enhanced-sensitivity Trofile assay or "deep sequencing" and re-analysis of the data showed that virologic failure in a significant proportion of these patients stemmed from the expansion of pre-existing minority CXCR4 using variants that went undetected during the first round of testing [155-157]. Accurate determination of coreceptor usage is therefore necessary to optimize treatment strategies before the initiation of and during therapy with CCR5 inhibitors. Techniques such as deep V3 sequencing may be useful for identifying treatment-experienced individuals who could benefit from CCR5-antagonist-containing regimens [158]. Ongoing improvements in genotypic methodologies and algorithms may eventually render them a viable alternative to

The identification of CCR5 as the principal HIV coreceptor for cellular entry over a decade ago combined with the observed effects of the CCR5Δ32 polymorphism (natural resistance

Although derived from a common parent, CC1/85, two resistant viruses that we have described, CC101.19 and D1/85.16, followed different genetic pathways to reach the same phenotypic endpoint: CC101.19 acquired four substitutions (K305R, H308P, A316V and G321E) in the V3 region [107], while the key determinants of resistance in D1/85.16 were three changes in the gp41 FP (G516V, M518V and F519I) [126] (Table 2). Both resistant variants acquired the ability to use the VCV–CCR5 complex for entry [107, 108, 126, 142]. However, unlike a clone derived from CC101.19 (V3 mutations), the D1/85.16 cl.23 virus (FP mutations) did not have an increased dependency on the CCR5 Nt, and its CCR5 binding site was not obviously more exposed [144]. The FP-mutant was, however, atypically sensitive to a CCR5 MAb that stains discrete cell surface clusters of CCR5 that might correspond to lipid rafts [145]. The precise molecular mechanism by which the FP changes confer resistance remains to be determined.

The V3 changes that confer resistance to CCR5 inhibitors have been suggested to render the virus more dependent on the CCR5 Nt, to compensate for impaired interactions between the V3-crown and ECL2 [144]. Accordingly, substantial deletions in V3 from both HIV-1 and HIV-2 confer complete resistance to coreceptor antagonists, presumably by disrupting the interaction between V3 and ECL2 [146-150]. Any adverse effect the V3 sequence changes have at the CCR5-binding stage may be compensated for by increases in the affinity of resistant viruses for CD4 and/or in the kinetics of virus entry [147, 151]. The altered virus–

CCR5 interactions are nonetheless characterized by considerable complexity. Thus, a subtype D, VCV-resistant patient isolate recognizes the drug-bound form of CCR5 more efficiently but still uses both the Nt and ECL2 [123]. A recently proposed model suggests that broad cross-resistance to multiple inhibitors is associated with an increased dependence on the N-terminus, while a more specific pattern of resistance to individual compounds involves more subtle changes in how the virus interacts with both the Nt and ECL2 [129].

#### **5.3.** *In vivo* **resistance: Potential expansion of pre-existing, CXCR4-using viruses**

The limited information currently available from published results of clinical studies suggests that the escape process from small molecule CCR5 inhibitors *in vivo* bears similarities to that observed *in vitro*; clinical resistance is also likely to map to the V3 region of gp120, but the lack of consistency of observed mutations renders impossible the prediction of *in vivo* resistance by sequence analysis at present (reviewed in [72, 77, 117, 141]). As mentioned above, two pathways of virological escape from the selection pressure of CCR5 antagonists have been identified: continued use of CCR5 via selection of R5 virus that can use drug-bound CCR5 for entry in addition to free coreceptor and expansion of pre-existing minority populations of dual-tropic or X4 virus (collectively called "CXCR4-using" viruses). The first pathway of R5 virological failure with reduced susceptibility to CCR5 antagonists is not the most common mechanism of failure. Virologic failure most commonly involves expansion of pre-existing, CXCR4-using viruses that are insensitive to CCR5 inhibitors. This outcome underscores the importance of developing tropism assays with improved ability to detect minor or archived virus populations capable of using the CXCR4 coreceptor.

The Trofile assay has been the assay of choice for clinical trials of CCR5 inhibitors. However, the sensitivity of the original commercially available assay was compromised with respect to the detection of low abundance (minority) CXCR4-using variants. As a result, a number of subjects were misjudged to have R5 virus at screening, whereas they actually harbored Dual/Mixed (D/M) virus populations when they entered clinical trials of CCR5 inhibitors; these patients appeared to have a blunted virologic response and high rates of early virologic failure [152-154]. Application of an enhanced-sensitivity Trofile assay or "deep sequencing" and re-analysis of the data showed that virologic failure in a significant proportion of these patients stemmed from the expansion of pre-existing minority CXCR4 using variants that went undetected during the first round of testing [155-157]. Accurate determination of coreceptor usage is therefore necessary to optimize treatment strategies before the initiation of and during therapy with CCR5 inhibitors. Techniques such as deep V3 sequencing may be useful for identifying treatment-experienced individuals who could benefit from CCR5-antagonist-containing regimens [158]. Ongoing improvements in genotypic methodologies and algorithms may eventually render them a viable alternative to phenotypic assays, much like the genotypic resistance methods.

#### **6. Conclusions and future directions**

134 Immunodeficiency

**Resistant FP mutants: A novel paradigm of noncompetitive resistance.** The VCV-resistant viruses carrying FP changes represent a novel paradigm of noncompetitive resistance (Figure 4C); apart from the plateau effect, these FP mutants paradoxically have lower IC50 values than wild-type viruses, particularly in certain cell types (e.g. TZM-bl cells). Hence, although by one measure (MPI) the FP-mutant viruses display a modest level of resistance, by another (IC50) they appear to be hypersensitive [126, 127, 131]. To explain this paradox, we created a theoretical model of resistance [126]. The mathematical formulation of the model is presented in detail in the Supporting Information (SI) section of [126]. The model is based on the assumption that distinct forms of CCR5, with varying affinities (ranging from low to high) for small molecule CCR5 inhibitors, are present in different proportions on disparate cell types; these coreceptor forms, which could be conformers (conformational isomers) or differentially Tyr-sulfated forms, are used selectively by resistant HIV-1 variants when ligated with an inhibitor. According to the model, wild-type virus can use both forms of unligated CCR5 for entry, possibly with a preference between them, but it cannot enter via the inhibitor-coreceptor complexes. The resistant virus, on the other hand, uses preferentially the unoccupied high-affinity coreceptor and the occupied low-affinity coreceptor. Applying this model led to the generation of theoretical inhibition curves that closely mimic the experimental data for resistant viruses in each case [126, 127, 131]. Using the model, we have recently identified the third FP mutation, F519I, (of the V3-independent or FP only- genetic pathway to resistance) as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5 [131]. Modeling the inhibition data from two cell types, PBMC and TZM-bl cells, suggested that D101.12, which harbors both V3 and gp41 substitutions, discriminates between high- and low-affinity forms of CCR5 less than

Although derived from a common parent, CC1/85, two resistant viruses that we have described, CC101.19 and D1/85.16, followed different genetic pathways to reach the same phenotypic endpoint: CC101.19 acquired four substitutions (K305R, H308P, A316V and G321E) in the V3 region [107], while the key determinants of resistance in D1/85.16 were three changes in the gp41 FP (G516V, M518V and F519I) [126] (Table 2). Both resistant variants acquired the ability to use the VCV–CCR5 complex for entry [107, 108, 126, 142]. However, unlike a clone derived from CC101.19 (V3 mutations), the D1/85.16 cl.23 virus (FP mutations) did not have an increased dependency on the CCR5 Nt, and its CCR5 binding site was not obviously more exposed [144]. The FP-mutant was, however, atypically sensitive to a CCR5 MAb that stains discrete cell surface clusters of CCR5 that might correspond to lipid rafts [145]. The precise molecular mechanism by which the FP changes

The V3 changes that confer resistance to CCR5 inhibitors have been suggested to render the virus more dependent on the CCR5 Nt, to compensate for impaired interactions between the V3-crown and ECL2 [144]. Accordingly, substantial deletions in V3 from both HIV-1 and HIV-2 confer complete resistance to coreceptor antagonists, presumably by disrupting the interaction between V3 and ECL2 [146-150]. Any adverse effect the V3 sequence changes have at the CCR5-binding stage may be compensated for by increases in the affinity of resistant viruses for CD4 and/or in the kinetics of virus entry [147, 151]. The altered virus–

D1/85.16, the resistant virus with three FP substitutions [127].

confer resistance remains to be determined.

The identification of CCR5 as the principal HIV coreceptor for cellular entry over a decade ago combined with the observed effects of the CCR5Δ32 polymorphism (natural resistance to HIV infection in homozygotes and delayed disease progression in heterozygotes) in the absence of any overt pathology, sparked great interest in its pharmaceutical blockade. The orally bioavailable small molecule CCR5 inhibitors are perhaps the most promising drugs that have been developed to block CCR5. In 2007, Maraviroc (MVC) became the first member of this new class of antiretrovirals, which target host- rather than virus-encoded structures, to receive regulatory approval for clinical use in the treatment of HIV infection. The theoretical virological concern that use of CCR5 antagonists in therapy may accelerate the evolution towards the more pathogenic CXCR4-using viruses has not been observed in clinical studies to date. MVC and other CCR5 antagonists have the potential for use in a variety of other clinical situations, such as the prevention of HIV transmission, intensification of HIV treatment and prevention of rejection in organ transplantation (reviewed in [159]). Moreover, CCR5 antagonists may be used in combination with other entry inhibitors or with agents such as rapamycin which downregulates CCR5 receptors thus decreasing CCR5 density [159]. New drugs that promote CCR5 and CXCR4 internalization, independent of cellular signaling, might also provide clinical benefits with minimum side effects [13].

Chemokine Receptors as Therapeutic Targets in HIV Infection 137

*Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras,* 

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**VCV** Vicriviroc **V3** Variable loop 3 **X4 HIV** CXCR4-tropic HIV **ZFNs** Zinc-finger nucleases

**Author details** 

*Patras, Greece* 

**7. References** 

872-877.

Cleo G. Anastassopoulou

**Acknowledgement** 

220(4599): 868-871.

HIV Research 2006; 4(3): 365-373.

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States of America 1995; 92(19): 9004-9008.

#### **Abbreviations**



#### **Author details**

136 Immunodeficiency

minimum side effects [13].

**AIDS** Acquired immunodeficiency syndrome

**ART** Antiretroviral therapy

**CNS** Central nervous system

**ECL2** Second extracellular loop **Env** Envelope glycoprotein

**MAb** Monoclonal antibody

**Nt** Amino terminus

**R5 HIV** CCR5-tropic HIV **R5X4 HIV** Dual-tropic HIV **RNAi** RNA interference **SDF-1** Stromal derived factor-1

**FP** Fusion peptide

**MVC** Maraviroc

**CCR5** C-C chemokine receptor type 5

**CXCR4** C-X-C chemokine receptor type 4

**GALT** Gut-associated lymphoid tissue **GPCRs** G protein-coupled receptors **HIV** Human immunodeficiency virus

**MIP-1**α Μacrophage inflammatory protein-1-alpha **MIP-1**β Μacrophage inflammatory protein-1-beta

**RANTES** Regulated on activation, normal T cell expressed and secreted

**PBMC** Peripheral blood mononuclear cells

**SIV** Simian immunodeficiency virus

**7-TM** Seven-transmembrane

**Abbreviations** 

to HIV infection in homozygotes and delayed disease progression in heterozygotes) in the absence of any overt pathology, sparked great interest in its pharmaceutical blockade. The orally bioavailable small molecule CCR5 inhibitors are perhaps the most promising drugs that have been developed to block CCR5. In 2007, Maraviroc (MVC) became the first member of this new class of antiretrovirals, which target host- rather than virus-encoded structures, to receive regulatory approval for clinical use in the treatment of HIV infection. The theoretical virological concern that use of CCR5 antagonists in therapy may accelerate the evolution towards the more pathogenic CXCR4-using viruses has not been observed in clinical studies to date. MVC and other CCR5 antagonists have the potential for use in a variety of other clinical situations, such as the prevention of HIV transmission, intensification of HIV treatment and prevention of rejection in organ transplantation (reviewed in [159]). Moreover, CCR5 antagonists may be used in combination with other entry inhibitors or with agents such as rapamycin which downregulates CCR5 receptors thus decreasing CCR5 density [159]. New drugs that promote CCR5 and CXCR4 internalization, independent of cellular signaling, might also provide clinical benefits with

Cleo G. Anastassopoulou *Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Patras, Greece* 
