Title

Rapid rebound of a highly replication competent preexisting CXCR4-tropic HIV variant after allogeneic stem cell transplantation with CCR5Δ32 stem cells

Presenter

Monique Nijhuis

Authors

J. Verheyen^1,2, A. Thielen², N. Lübke³, M. Dirks¹, M. Widera¹, U. Dittmer¹, L. Kordales⁴, M. Däumer², D. de Jong⁵, A. Wensing⁵, R. Kaiser⁶, M. Nijhuis⁵, S. Esser⁷

Institutions

¹University of Duisburg-Essen, University Hospital, Institute of Virology, Essen, Germany, ²Institute of Immunology and Genetics, Kaiserslautern, Germany, ³Heinrich-Heine-University, University Hospital, Institute of Virology, Düsseldorf, Germany, ⁴University Hospital, University of Duisburg-Essen, Department of Bone Marrow Transplantation, Essen, Germany, ⁵University Medical Center Utrecht, Medical Microbiology, Utrecht, Netherlands, ⁶University of Cologne, Institute of Virology, Cologne, Germany, ⁷University Hospital, University of Duisburg-Essen, Clinic for Dermatology, Essen, Germany

Background: To date, the case of the Berlin patient provides the only evidence of an intervention that has been able to cure HIV infection. The procedure involved an allogeneic stem cell transplantation (SCT) with donor cells lacking the CCR5 coreceptor (CCR5Δ32). Interestingly, in the Berlin patient no viral rebound was observed despite the fact that cART was stopped at the day of transplantation. In a similar setting in the Essen patient, cART was stopped before initiation of myeloablative therapy and a rapid viral rebound was observed after SCT. To fully understand the underlying mechanism of viral breakthrough in the Essen patient we retrospectively analyzed the genotypic and phenotypic characteristics of the viral population.
Methods: RNA was isolated from plasma and total DNA was isolated from PBMCs at different time points before (-287d: RNA, -103d: RNA/DNA, -18d: DNA) and after (+20d: RNA, +373d: RNA/DNA) SCT. HIV coreceptor tropism was genotypically assessed (geno2pheno) after deep-sequence analysis of the viral envelope (gp120-V3). The observed gp120-V3 sequences were cloned in our shuttle vector pHXB2-Δgp120-V3 and chimeric viruses were tested for replication capacity and coreceptor usage in primary cells (PBMCs).
Results: Viral breakthrough was observed three weeks after SCT. Every single viral RNA sequence detected with deep-sequence analysis at time of breakthrough was predicted to be CXCR4-tropic (FPR: 0.2%-0.7%). These sequences are genetically distinct from the pre-SCT viral variants predicted to be CCR5-tropic (FRP: 8.5%-10.5%). Interestingly, the most dominant viral variant rebounding after SCT (FPR 0.4%) could already be detected as a minority variant in the proviral DNA 103 days before transplantation. This dominant variant, once cloned in our HIV shuttle vector, demonstrated a high replication capacity in primary cells and is completely dependent on the alternative CXCR4 coreceptor for replication.
Conclusions: In this study we demonstrate that the rapid rebound after SCT was related to a highly replicative CXCR4-tropic HIV variant, which was already present prior to SCT. These data indicate that in-depth HIV coreceptor analysis is essential for future CCR5-based stem cell transplantation and gene therapy studies.

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