A 49y-old HIV-infected male patient received unmodified HSCT from a 10/10 CCR5-d32 donor in Feb 2013 because of acute myeloid leukemia (AML) while in 2nd complete remission (CR). At time of HSCT proviral HIV DNA load was 1.45 log10cop/Mio PBMCs. All anticipated antibodies were detected by western blot. HIV coreceptor-usage was predicted R5 (Sanger: FPR 44.5%; NGS: 0.14% X4 at 3.5% FPR, geno2pheno), confirmed by phenotypic testing (TropChase). He had a 2nd relapse of AML in Jun/13 but after 8 courses of 5-azaC and 4 donor lymphocyte infusions CR was achieved. Immunosuppression was stopped in Oct/17. During HSCT the patient remained on ART until Nov/18 with undetectable plasma viral load.
PBMC and tissues were analysed by ddPCR, qPCR and in situ hybridization in several laboratories as well as humoral and T-cell responses. Infectious virus was analysed on CD4+T-cells (qVOA, MVOA). Patient was registered to IciStem as #19.
Almost all PBMC samples were negative for proviral HIV-DNA by qPCR/ddPCR at multiple time points. CSF (Jul/14), rectum (Apr/15, Mar/16), ileum (Mar/16) and bone marrow (Aug/15) were negative. Further testing with 0.1 Mio cells from ileum showed in 1/4 replicates one positive droplet with LTR-, but none with gag-primers. There was also a signal in TCM 0.2 Mio cells (ddPCR 1 positive droplet, qPCR neg) and in TEM 0.36 Mio cells (qPCR 5cop/Mio cells, ddPCR neg) while all other T-cell subsets were negative in ddPCR & qPCR. No HIV-DNA could be detected by PCR in lymph nodes of May/17, but in situ hybridization assays (RNAscope, DNAscope) detected few positive signals. Viral outgrowth assays (qVOA) in Feb/16, Mar/16 and May/16 were negative (23 Mio CD4+T-cells, IUPM<0.031/Mio cells CD4+T-cells). Mouse VOA (Apr/16: Rag2-/-γc-/-, Apr/17: NOD-SCID IL2gR-/-) were also negative. Gp160 was the only remaining band on the blot. Peptide stimulation assays revealed the presence of CCR5-negative HIV-specific CTL recognizing HLA-A2-restricted RT-epitope YV9 and HLA-B7-restricted Gag-p6 epitope YL9.
Despite low signals in ultrasensitive assays no virus could be detected in qVOA/mVOA in the Duesseldorf patient. Taking into account the homozygous CCR5-d32 status we consider a viral rebound to be unlikely. An ATI is the only way to find out whether HIV has been eradicated by allogeneic CCR5-d32 HSCT. Therefore ART was stopped in Nov/18 after thorough discussion with the patient. Despite all plasma samples being negative after ATI longer surveillance is essential.