**3. Coreceptor use and HIV tropism**

118 Immunodeficiency

development of CCR5 inhibitors.

initiation of infection.

**2.3. The coreceptors in HIV entry** 

presently accepted molecular mechanism, is shown in Figure 1.

antiretrovirals targeting other steps of the viral life cycle; they could also be administered simultaneously with CCR5 antagonists, even as one compound with high affinity for both receptors, as suggested recently [32]. Down-modulating the expression of CXCR4 may certainly be beneficial for some HIV patients [13]. The pharmacologic targeting of CXCR4 may additionally have therapeutic utility for the treatment of acute viral infections of the central nervous system (CNS) [33]. The rest of this chapter focuses on issues related to the

Conformationally masked by carbohydrate structures and variable amino acid loops that enable it to evade the humoral immune response, the envelope glycoprotein (Env) on virions is organized into trimers of noncovalently associated surface gp120 and transmembrane gp41 heterodimers (reviewed in [34]). The interaction of HIV with host cell surface receptors trigger the fusogenic potential of Env and allow the virus to enter into its target cells by fusion, presumably at the plasma membrane [35]. The process has long been assumed to occur at the cell surface for such pH-independent viruses as HIV [36], although recent studies support the notion that HIV enters disparate cell types through fusion with endosomes [37, 38]. A schematic of the first steps of the HIV entry process, according to the

**Figure 1. Schematic diagram of the first steps of the HIV entry process.** (**A**) HIV entry into target cells is mediated through the interaction of the viral envelope glycoprotein (Env) with host cell surface receptors. (**B**) Surface gp120 first contacts CD4 and then (**C**) a chemokine receptor, typically CCR5 or CXCR4, triggering molecular rearrangements in the gp41 that result in membrane fusion and the

HIV entry begins with the high affinity binding of gp120 to the host cell CD4, which induces a major conformational change in Env that exposes or creates a binding site on gp120 for the coreceptor, typically either CCR5 or CXCR4. Current structural models suggest that the gp120 "bridging sheet", formed between the constant C1, C2, and C4 domains of gp120 after CD4 binding, interacts with the tyrosine (Tyr)-sulfated coreceptor N-terminus (Nt), while the V3 crown interacts principally with the second extracellular loop (ECL2) region of the

#### **3.1. Classification systems of HIV biological phenotype**

Before the identification of the coreceptors, three biological properties were the criteria used to classify the phenotype of HIV variants: (i) the preference for specific target cells (cellular tropism) that distinguished between macrophage (M)-tropic and laboratory T-cell line (T) tropic viruses; (ii) cytopathology, which distinguished between syncytium-inducing (SI) and non-syncytium-inducing (NSI) strains based on the capacity or not to form syncytia (i.e., giant multinucleated cells) through cell fusion in the MT-2 cell line; and (iii) replicative capacity, which mainly considered the *in vitro* growth kinetics of viral strains in culture and distinguished between slow/low (S/L) and rapid/high viruses (R/H) (reviewed in [43]).

The above dichotomies may be explained by the more precise, newly adopted classification system that is based on coreceptor usage; accordingly, viruses that use CCR5 alone or CXCR4 alone for cell entry are currently termed R5 and X4, correspondingly, while variants that can use either coreceptor with comparable efficiency are termed R5X4 [44]. A more appropriate designation for the latter is R5+X4 or D/M (Dual/Mixed), particularly when tropism has not been determined at the clonal level, since many R5X4 isolates are in fact mixtures of R5 plus X4 or, less commonly, either R5 plus dual (R5X4), or even R5 plus dual (R5X4) plus X4, clones [22]. The term R5X4 should thus be reserved for clones that are genuinely capable of using both coreceptors.

#### **3.2. Determinants of HIV biological phenotype and coreceptor tropism assessment**

The determinants of coreceptor choice, and thus of biological phenotype, have been mapped to the surface gp120 subunit of Env and primarily to the V3 domain. Swapping the V3 region alone often suffices to switch the coreceptor tropism of a viral clone from R5 to X4 and vice versa [45]. Furthermore, the charge of the V3 region appears to be an important parameter affecting biological phenotype and, tropism, although it alone cannot be used as a marker for phenotype prediction; in general, however, higher net charges (≥ +5) characterize X4 variants compared to the lower V3 region net charges (≤ +4) of R5 variants typically associated with acute infection (reviewed in [43]). The increase of the overall positive charge of the V3 region that may switch the viral phenotype results from the introduction of basic amino acid residues at one of the two positions 11 and/or 25 of the V3 [46, 47], a property that may not be shared by the uncommon subtype C CXCR4-using strains (reviewed in [48]).

Chemokine Receptors as Therapeutic Targets in HIV Infection 121

whether the declining competence of the host immune system permits the outgrowth of viral strains with increased replicative capacity [43]. The pathogenicity of X4 viruses seems to be greater than that displayed by R5 strains both *in vitro* and probably *in vivo*, as suggested by experiments in macaques [22]. R5 viruses alone can also cause severe damage to the immune system; X4 viruses are never detected in about half of HIV-infected subjects who do progress to AIDS [22, 53]. These late-emerging, R5 viral strains, which have reduced sensitivity to entry inhibitors and increased ability to cause CD4+ T-lymphocyte loss, also possess an improved ability to utilize relatively low levels of CD4 and CCR5 expressed on macrophages (reviewed in [57]). Correlations between increased gp120 net charge, enhanced viral fitness, and augmented cell attachment were disclosed as well for these R5 HIV-1 variants that emerge in an opportunistic manner during severe immunodeficiency [58].

Different CD4+ T cell subpopulations are targeted by the phenotypic variants of HIV: replication of R5 viruses is favored in activated, memory/effector T cells, whereas X4 viruses may additionally infect intrathymic T progenitor cells and naïve T cells in the peripheral lymphoid system [39, 43]. Of note is the massive depletion in the gut-associated lymphoid tissue (GALT) of CCR5-expressing memory CD4+ T cells as a consequence of acute HIV infection (reviewed in [59]). The engagement by gp120 of integrin α4β7, the gut homing receptor, facilitates infection of CD4+ T cells in the earliest stages of transmission [60]. HIV replicates intensively first in the GALT and then systemically in all lymphoid tissues, where it establishes stable viral reservoirs (in the form of latently infected resting CD4+ T cells at the cellular level) that constitute major impediments to virus eradication [59]. The switch in coreceptor usage at the peak of viral diversity expands virus accessibility to circulating naïve CD4+ T cells, thereby increasing viral burden and leading to rapid CD4+ T cell depletion [61, 62]. The regenerative capacity of the immune system is also affected by the X4 emergence or predominance since chronic immune activation is then exacerbated. T cell homeostasis is disrupted, eventually leading to the collapse of the immune system [43].

**4. The development of CCR5 inhibitors as anti-HIV therapeutics – Focus** 

As discussed above, the first clues regarding the potential therapeutic utility of targeting CCR5 came from the observation of the naturally occurring blockade of the coreceptor due to the CCR5Δ32 polymorphism. Recently, the first successful allogeneic stem cell transplantation has been reported in an HIV-positive, acute myeloid leukemia patient from

HIV-infected patient as indicated by the functional reconstitution of his T cell immunity without any signs of viral rebound after the discontinuation of ART [64]. The long-term effects of stem cell transplantation remain nevertheless unknown. Even though it would obviously be impractical to follow such a therapeutic approach in the millions of HIVinfected subjects worldwide, the proof-of-principle case of "the Berlin patient" demonstrates that a functional cure, i.e. a permanent suppression of viral replication in the absence of ART, may be within reach, under certain circumstances, at present; a true sterilizing cure with complete eradication of the virus continues to be the ultimate goal of therapy [6].

*32* homozygous donor [63]. Investigators claimed to have "cured" this

**on small molecules** 

Berlin with a *CCR5*

Δ

Simple genotypic approaches undertaken for predicting coreceptor usage include the detection of positively charged amino acids at positions 11 and 25 (often referred to as the "11/25 rule") and the determination of the total charge of V3, with the cut-off set at +5; heteroduplex tracking assays and various bioinformatics approaches are additional genotypic methods for determining HIV tropism (reviewed in [49-51]). Predictive algorithms of coreceptor usage have been developed based on genetic sequences in the V3 region, but also taking into account genotypic correlates outside the V3, structural information and clinical data [48]. Apart from these genotypic tools, different phenotypic assays have been developed to assess HIV tropism. These phenotypic methods, which are reviewed elsewhere [49-52], include the original MT-2 assay or coreceptor usage assays using reporter cell lines, and cell-based assays that use different methodologies to generate *env*-recombinant or pseudotyped viruses with distinct detection systems. The Trofile assay (Monogram Biosciences, Inc., South San Francisco, CA; now Laboratory Corporation of America [LabCorp]) is the most widely used phenotypic test in clinical practice. In the new era of CCR5 inhibitors, an accurate determination, and perhaps quantification, of coreceptor usage is necessary for the successful clinical management of HIV-infected individuals.

#### **3.3. HIV phenotypes, tropism and disease course**

Regardless of the route of HIV transmission, R5 viruses seem to prevail in the vast majority of primary and early stage HIV infection cases, whilst the X4 phenotype evolves *in vivo* in approximately 40-50% of (subtype B or D) infected subjects, usually 5–7 years after infection (reviewed in [43, 53]). Recent transmitted/founder virus genetic studies confirmed that only R5 (and typically a single R5 virus isolate) and in a few instances R5X4, but not X4 HIV-1, are initially transmitted [54, 55]. Consequently, a "gatekeeper" that nearly always selects transmission of R5 over X4 HIV may well exist. The suppression of X4 transmission has been suggested to result not from one powerful gatekeeper, but from an aggregate of multiple functional gatekeepers, the localization and precise molecular mechanisms of which remain to be determined (reviewed in [56]). CCR5 thus has a pivotal role in viral transmission, but infection by X4 (or R5X4) viruses is not impossible as indicated by the few known cases of HIV acquisition by *CCR5-*∆*32* homozygotes; the lower pre-AIDS viral loads and slower disease progression of *CCR5-*∆*32* heterozygotes, in whom the CCR5 expression levels on target cells are lower, underscore the importance of the CCR5 coreceptor in the early asymptomatic stages of the infection [31, 53].

The emergence of X4 viruses in a proportion of cases correlates with accelerated disease progression [43, 53]. The virological or immunological basis of the selection mechanism is still debated; in other words, it is still unclear whether disease progression is accelerated as a direct consequence of the emergence or predominance of X4 isolates, or, conversely, whether the declining competence of the host immune system permits the outgrowth of viral strains with increased replicative capacity [43]. The pathogenicity of X4 viruses seems to be greater than that displayed by R5 strains both *in vitro* and probably *in vivo*, as suggested by experiments in macaques [22]. R5 viruses alone can also cause severe damage to the immune system; X4 viruses are never detected in about half of HIV-infected subjects who do progress to AIDS [22, 53]. These late-emerging, R5 viral strains, which have reduced sensitivity to entry inhibitors and increased ability to cause CD4+ T-lymphocyte loss, also possess an improved ability to utilize relatively low levels of CD4 and CCR5 expressed on macrophages (reviewed in [57]). Correlations between increased gp120 net charge, enhanced viral fitness, and augmented cell attachment were disclosed as well for these R5 HIV-1 variants that emerge in an opportunistic manner during severe immunodeficiency [58].

120 Immunodeficiency

associated with acute infection (reviewed in [43]). The increase of the overall positive charge of the V3 region that may switch the viral phenotype results from the introduction of basic amino acid residues at one of the two positions 11 and/or 25 of the V3 [46, 47], a property that may

Simple genotypic approaches undertaken for predicting coreceptor usage include the detection of positively charged amino acids at positions 11 and 25 (often referred to as the "11/25 rule") and the determination of the total charge of V3, with the cut-off set at +5; heteroduplex tracking assays and various bioinformatics approaches are additional genotypic methods for determining HIV tropism (reviewed in [49-51]). Predictive algorithms of coreceptor usage have been developed based on genetic sequences in the V3 region, but also taking into account genotypic correlates outside the V3, structural information and clinical data [48]. Apart from these genotypic tools, different phenotypic assays have been developed to assess HIV tropism. These phenotypic methods, which are reviewed elsewhere [49-52], include the original MT-2 assay or coreceptor usage assays using reporter cell lines, and cell-based assays that use different methodologies to generate *env*-recombinant or pseudotyped viruses with distinct detection systems. The Trofile assay (Monogram Biosciences, Inc., South San Francisco, CA; now Laboratory Corporation of America [LabCorp]) is the most widely used phenotypic test in clinical practice. In the new era of CCR5 inhibitors, an accurate determination, and perhaps quantification, of coreceptor usage is necessary for the successful clinical management of HIV-infected individuals.

Regardless of the route of HIV transmission, R5 viruses seem to prevail in the vast majority of primary and early stage HIV infection cases, whilst the X4 phenotype evolves *in vivo* in approximately 40-50% of (subtype B or D) infected subjects, usually 5–7 years after infection (reviewed in [43, 53]). Recent transmitted/founder virus genetic studies confirmed that only R5 (and typically a single R5 virus isolate) and in a few instances R5X4, but not X4 HIV-1, are initially transmitted [54, 55]. Consequently, a "gatekeeper" that nearly always selects transmission of R5 over X4 HIV may well exist. The suppression of X4 transmission has been suggested to result not from one powerful gatekeeper, but from an aggregate of multiple functional gatekeepers, the localization and precise molecular mechanisms of which remain to be determined (reviewed in [56]). CCR5 thus has a pivotal role in viral transmission, but infection by X4 (or R5X4) viruses is not impossible as indicated by the few known cases of HIV acquisition by *CCR5-*∆*32* homozygotes; the lower pre-AIDS viral loads and slower disease progression of *CCR5-*∆*32* heterozygotes, in whom the CCR5 expression levels on target cells are lower, underscore the importance of the CCR5 coreceptor in the

The emergence of X4 viruses in a proportion of cases correlates with accelerated disease progression [43, 53]. The virological or immunological basis of the selection mechanism is still debated; in other words, it is still unclear whether disease progression is accelerated as a direct consequence of the emergence or predominance of X4 isolates, or, conversely,

not be shared by the uncommon subtype C CXCR4-using strains (reviewed in [48]).

**3.3. HIV phenotypes, tropism and disease course** 

early asymptomatic stages of the infection [31, 53].

Different CD4+ T cell subpopulations are targeted by the phenotypic variants of HIV: replication of R5 viruses is favored in activated, memory/effector T cells, whereas X4 viruses may additionally infect intrathymic T progenitor cells and naïve T cells in the peripheral lymphoid system [39, 43]. Of note is the massive depletion in the gut-associated lymphoid tissue (GALT) of CCR5-expressing memory CD4+ T cells as a consequence of acute HIV infection (reviewed in [59]). The engagement by gp120 of integrin α4β7, the gut homing receptor, facilitates infection of CD4+ T cells in the earliest stages of transmission [60]. HIV replicates intensively first in the GALT and then systemically in all lymphoid tissues, where it establishes stable viral reservoirs (in the form of latently infected resting CD4+ T cells at the cellular level) that constitute major impediments to virus eradication [59]. The switch in coreceptor usage at the peak of viral diversity expands virus accessibility to circulating naïve CD4+ T cells, thereby increasing viral burden and leading to rapid CD4+ T cell depletion [61, 62]. The regenerative capacity of the immune system is also affected by the X4 emergence or predominance since chronic immune activation is then exacerbated. T cell homeostasis is disrupted, eventually leading to the collapse of the immune system [43].
