**13. Clinical application of tropism**

One property that has been closely correlated with clinical prognosis is the ability to induce syncytia formation in susceptible cells with clinical course of AIDS being associated with a shift from NSI to SI. HIV isolates obtained during early acute infection are generally M-

tropic/NSI whilst T-tropic/ SI are associated with disease progression. The emergence of CXCR4-using HIV-1 variants in a patient is almost invariably associated with a subsequent increase in the rate of decline of circulating CD4+ T cells, an accelerated disease progression and a poor prognosis for survival. While CXCR4-using variants can emerge at any stage of infection, untreated individuals who develop such variants progress to AIDS within an average of two years after their first detection. However, the presence of CXCR4-using variants is not an obligatory prerequisite for disease progression as a significant proportion of individuals' progress to AIDS whilst harbouring exclusive R5 HIV-1 variants. The emergence of SI HIV-1 in a sero-positive individual is now generally regarded as a negative prognostic indicator and thus considerable interest has been focused on the HIV-1 genetic determinants of the SI phenotype. Tropism assays are important for determining HIV phenotype before administering coreceptor antagonists in combination antiretroviral therapy. In view of this, tropism assays must taken by HIV infected people with drug resistance who are considering taking coreceptor antagonists. This new class of antiretroviral targets CCR5-specific HIV during entry into the cell. Thus, knowing the patient's viral tropism can assist and guide the clinician make an informed and effective HIV treatment plan. Consequently, the importance of tropism screening to detect the presence of R5-using HIV may not be over emphasized.

#### **14. Methods for determination of coreceptor usage**

With the recent introduction of HIV-1 chemokine receptor antagonists on the market as components of antiretroviral therapy, it is increasingly important to screen HIV patients' coreceptor phenotype or genotype prior to therapy. Hence simple and efficient methods for routinely characterising and monitoring HIV-1 coreceptor phenotypes or genotypes are of paramount importance. Studies have demonstrated that the ability to induce syncytia is determined by regions of env outside the V3 loop that encompass residues that contribute to the binding of CD4 by gp120. Such observations suggest that areas of the HIV-1 *env* gene contributing to the CD4 binding site may also contribute to the determination of SI and NSI genotypes. HIV-1 tropism can be determined by phenotypic or genotypic based methods.

### **15. Phenotypic methods**

240 Immunodeficiency

ancestor incorporated a chemokine gene into its *env* gene such that this captured chemokine gene rapidly diverge by frequent mutations thereby attaining the ability to effectively interact with various chemokine receptors in a short period of time. Another possible explanation is based on cell division rate where it has been observed that X4 and R5 viruses show preferential tropism for naive and memory T cells, respectively. Since memory T cells divide 10 times more frequently than naive cells, it would be an advantage to have a tropism

Studies have shown that the use of coreceptors is much broader in HIV-2 infections relative to HIV-1. In addition to CCR5 and CXCR4 HIV-2 is able to utilise alternative coreceptors such as GPR15, or CXCR6 and to a lesser extent CCR1, CCR2b or CCR3 in aviremic patients whilst CXCR4 is only used in viremic individuals. It still needs to be explored whether this wide use of coreceptors is the underlying reason behind the less virulent phenotype

One property that has been closely correlated with clinical prognosis is the ability to induce syncytia formation in susceptible cells with clinical course of AIDS being associated with a shift from NSI to SI. HIV isolates obtained during early acute infection are generally M-

for CCR5 coreceptor during the first stages of infection.

**Figure 2.** HIV classification adopted from Moyle G, 2008.

**12. Coreceptor usage in HIV-2** 

associated with HIV-2, compared to HIV-1.

**13. Clinical application of tropism** 

HIV-1 tropism can be assessed using phenotypic assays which are currently the most accurate method based on recombinant viruses. Determination of coreceptor use of HIV-1 isolates is done in cell lines such as U87 and GHOST transfected with CCR5 or CXCR4. Patients' plasma is used to generate pseudoviruses or infectious recombinant viruses of fulllength or partial viral envelopes derived from the patient's viral population. The recombinants are subsequently tested on indicator cell lines expressing CD4 and either CCR5 or CXCR4. The first commercially available tropism assay became available on the market almost at the same time of approval of the CCR5 antagonist, maraviroc. Its brand name of phenotypic assay is Trofile® manufactured by Monogram Biosciences South San

Francisco, USA. The test can detect 10% of X4 variants with 100% sensitivity. More recently, an enhanced Trofile® assay with better sensitivity to improve detection of low level X4-using variants has been developed that can detect 0.3% of these variants with 100% sensitivity. Trofile® phenotypic assay continues to be the only clinically validated assay to identify coreceptor tropism and is considered the gold standard for tropism testing capable of distinguishing pure R5, D/M and pure X4 populations.

Coreceptor Usage in HIV Infection 243

mutations seem to compensate for the harmful V3 mutations. Functional studies have demonstrated that the V3 loop interacts with the N-terminal extra-cellular domain of CCR5 and the extracellular loop 2. Bioinformatics tools based on V3 sequences can be used to predict HIV-1 tropism. The identification of viral genotypic changes associated with different coreceptor usage has led to the development of sequence-based algorithms to predict coreceptor usage. Different rules have been published based on the amino acid sequence of the env V3 region of HIV-gp120, which is known to be the major determinant

At least eight different bioinformatics tools have been used to predict viral tropism in different HIV-1 subtypes which uses phenotypic data to predict the corresponding viral genotype. Studies of genotypic predictors have been retrospective with patient samples selected based on availability of phenotypic tropism determinations. Three of the interpretation systems namely, WetCat, WebPSSM, geno2pheno [coreceptor] are freely available on internet. All three focus on the env-V3 region and only take the amino acid sequence into account. Such genotypic systems provide the possibility for rapid screening of patients who may be administered with CCR5 blockers like maraviroc. On cloned viruses belonging to genetic subtype B, the specificity and sensitivity of most predictive methods exceed 90% and 80%, respectively. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with CCR5 antagonists in populations pre-screened with a phenotypic assay. The first genotypic algorithm designed to predict HIV-1 tropism takes into account only the net charge of amino acids at two key residues located within the V3 loop, amino acids at positions 11 and 25. The most widely used is the 11/25 rule which focuses on identifying sequence patterns within the V3 loop. Predictions using the "11/25 charge rule" are

**19. Correlation between phenotypic and bioinformatics tools in** 

Evaluation of the performances of genotypic tools to predict HIV-1 tropism has been investigated. Paired genotypic and phenotypic determination of HIV-1 coreceptor usage has been performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. Excellent correlations between HIV-1 V3 genotype and phenotype have been observed. Overall, the accuracy of the bioinformatics tools to detect CXCR4-using virus was similar for ES Trofile and Trofile. However, the negative predictive values for genotypic tools with ES Trofile were slightly higher than they were with Trofile. The accuracy of genotypic algorithms for detecting CXCR4-using viruses is high when using Trofile as the reference. The concordance with ES Trofile is better with

**18. Predictive algorithms of HIV-1 coreceptor usage** 

of coreceptor usage.

relatively satisfactory.

**determining HIV coreceptor use** 

Another phenotypic tropism assay, Phenoscript-tropism; Eurofins is also on the market. It was specifically developed to cater for phenotypic test for the evaluation of viral tropism in HIV-1 non-B subtypes. It is worthwhile to appreciate that the HIV-1 env V3 loop is implicated in the determination of phenotypic tropisms
