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Final Report Summary - SWITCHR5-X4 (Determinants of the switch from R5- to X4-tropic virus in HIV-1 infected individuals)

✓A summary description of the project objectives,
This multi-disciplinary, internationally collaborative project was set up with three main aims: We firstly aimed to investigate the prevalence of CXCR4-utilizing viruses in South African cohorts and the correlation with HIV-1 disease progression, Mycobacterium Tuberculosis (MTB) status and levels of immune activation. In addition we investigated the correlation between immune activation and coreceptor switch in a HIV-1 subtype B (HIV-1B) cohort the Netherlands. As a second main objective we investigated whether target cell affinity would facilitate the coreceptor switch. To do this we employed the MAGI cell system and the inducible affinofile system to analyse the relative binding efficiencies of HIV-1 envelope for its cognate receptors, CD4 and CCR5. Our final and third aim was the validation of the point of care diagnostic Instant Count machine for rapid CD4 counting on HIV-1 positive samples at the UMCU, Utrecht, The Netherlands.

✓A description of the work performed since the beginning of the project and a description of the main results achieved so far,
We investigated the prevalence of X4-tropism in a cross-sectional cohort of 100 antiretroviral therapy (ART)-naïve South African HIV-1 subtype C (HIV-1C) infected individuals and investigated if X4-tropism was correlated with CD4+ T-cell activation, monocyte activation and plasma coreceptor ligand concentration. Plasma and peripheral blood mononuclear cells were isolated. Genotypic prediction of HIV-1 coreceptor usage was performed based on deep sequencing of the envelope region (gp120-V3) and interpretation with the geno2phenocoreceptor algorithm, using a False Positive Rate (FPR) of 3.5%. We report that 24% of the patients harbor X4-tropic virus which is relatively high, but in line with our previous study, were 30% were found to be X4-tropic (Connell et al., 2008) and another study who also detected 30% X4-tropism (Singh et al., 2011).

We speculated that MTB co-infection could be an independent risk factor for coreceptor switching, however found it to have a non-significant correlation with X4-tropism at both an FPR of 3.5% and 10%. In univariate testing the following variables were significantly positively associated with X4-tropism (p<0.1 using the Mann–Whitney–Wilcoxon test (MWW)): RANTES, CD4+ T-cell count, CD169 on classical monocytes, %HLA-DR+ in CD4 cells and %CD38+ HLA-DR+ double positives in CD4+ cells. Negative associations were seen between X4-tropism and the CD4 counts. In the multivariate statistical analysis of principal components, a principal component largely determined by levels of MIP-1β, IP-10 and TNFR2 was significantly associated with X4-tropism (p<0.05).

We also investigated whether immune activation could predispose for X4-tropism in HIV-1B. Longitudinal analysis of samples from 98 HIV-1B ART-naïve patients from the Amsterdam Cohort Studies (ACS) were analyzed for cellular immune markers one and five years after seroconversion and coreceptor tropism was determined in phenotypic assays five years after seroconversion. Interestingly, the level of CD4+ T cell activation (as measured by HLA-DR+ CD38+ expression in CD4+ cells) in the first year post-seroconversion appeared to be predictive for an R5-X4 switch during chronic stages of disease (p = 0.017). For the first time ever, immune activation was shown to predict R5-X4-tropism switch. These data are novel and may have important ramifications in the way we understand HIV-1 pathogenesis and tropism in both HIV-1C and HIV-1B infection. These results have been submitted for publication.

We are currently investigating if markers of immune activation also predict coreceptor switch in HIV-1C infection. Therefore we are currently investigating a longitudinal cohort consisting of 119 HIV-1 / TB positive patients from South Africa.

According to our second aim, we have successfully set up the affinofile system in our laboratory where the quantity of CD4 and that of the CCR5 coreceptor can be simultaneously and independently regulated within a physiological range of surface expression (Johnston et al., 2009). We have optimized CCR5 expression levels because the system was “leaky” and therefore difficult to control using Ponasterone A. We developed a new strategy to use the conformational sensitive antibody 2D7 to mask CCR5 coreceptors rendering them unavailable for HIV-1 binding. Since the detection antibody was labeled with PE we could also quantify the exact level of CCR5 available for HIV-1 binding in each individual experiment. This optimized quantitative affinity profiling system was subsequently used to define the distinct CD4 and coreceptor profiles of HIV-1B viruses obtained before and after coreceptor switch. We showed that the post-switch R5 and X4 isolates are more efficient in replication in those cells expressing CD4 in the physiological relevant range.

In alignment with our third aim, we have successfully tested 105 HIV-1 positive patients from the UMCU Utrecht in the rapid diagnostic test Instant Count Machine. The results have been analysed and compared to the CD4 count in-house gold standard flow cytometric test performed routinely on all HIV-1 patients. The results correlate very well (R2 = 0.93) and form the basis for evaluation and implementation in rural settings.

✓The expected final results and their potential impact and use
We report for the first time that immune activation can predispose the switch from R5 to X4-tropism in drug naïve HIV-1B infected patients and that specific immune activation markers (TNF-R2, IP-10 and MIP-1β) are associated with X4-tropic HIV-1C infection in ART-naïve patients. These data have very significant ramifications for the current knowledge of viral pathogenesis. We are only beginning to understand the underlying mechanisms for HIV-1 coreceptor switch and clearly show that immune activation is one of the driving forces for the switch in coreceptor usage. Ultimately, after coreceptor switch, viruses are selected that have stronger replicative capacity as opposed to the pre-switch ancestors, although the intermediate viral variants may display reduced replication. The post-switch viral isolates also seem to have a higher replication capacity in cells expressing CD4 in the physiological relevant range in the affinofile system.

We also demonstrate that fully printed microfluidic CD4 counting chips with complete on-chip sample preparation can be used as point of care CD4 counting devices in research and clinical settings. The Instant count performed equally well on healthy donors as well as HIV-1 positive patient material and the results made a very good agreement with the gold standard flow cytometric results in both cases. This point of care device will not only help diagnose HIV-1 earlier in very rural settings, but can also be used to monitor how the immune system is responding to therapy.

Figure 1.1 Schematic representation of the project overview.

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