Despite Absence of Measurable Immune Responses against Adenovirus in the First 100 Days After Unrelated Umbilical Cord Blood Transplant in Vitro amplification Strategies Can Unveil Hexon and Penton-Specific Immunity.

Abstract 4640

Unrelated cord blood transplantation (UCBT) has become a life saving modality of hematopoietic cell transplantation (HCT) for those cancer patients who lack HLA-matched sibling donors. However, success after UCBT is limited by the high incidence of opportunistic infections (OI), most of which are viral and typically detected < 3 months after UCBT. A minority of UCBT patients with evidence of viral infection/reactivation post-transplant actually resolve their disease without antiviral therapy. Since these infections are largely controlled by T-cell immunity this suggests that in vivo T-cell priming may occur despite immunosuppressive drugs and functional T-cell deficits and protective antiviral immunity can be attained once a threshold number of antiviral T-cells have been achieved. To determine whether this was the case we collected PBMCs from 10 UCBT patients who underwent myeloablative regimens and were diagnosed with adenovirus infection (defined as positive PCR at least twice and/or viral culture in blood and/or stool and/or nasopharynx). Samples were collected a median of 88 days (range 37-209 days) post-transplant at first indication of Adv positivity (blood PCR + in 5/10 patients at a mean of ∼110,000 copies/ml, median of 350, range 100-1×10⁶ copies/ml, 4 patients positive in stool only, one patient + in respiratory tract only). The median age of patients was 4.6 years (range 2.0-16.6y). Eight (8) out of ten (10) patients had grade 2-4 acute GVHD prior to the studies, while the other two patients had grade I.

At baseline we were consistently unable to detect T-cells with reactivity against the immunodominant adenoviral virion proteins hexon and penton by IFN-g ELIspot [hexon - median 1.75 spot forming cells (SFC)/2×10⁵ PBMC (range 1-12.5), penton – median 1 SFC)/2×10⁵ PBMC (range 0.5-1)], even though the patients had evidence of active adenovirus infections. However, in 5/10 patients adenovirus-specific T cells could be amplified by a single in vitro stimulation with donor-derived mononuclear cells transduced with a recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber (MOI 200 virus particles (vp) per cell) and culture in the presence of IL4 (1000u/ml). The median hexon-specific T cell frequency in the reactivated cytotoxic T lymphocyte (CTL) lines was 62.5 SFC/2×10⁵ (range 34.5-299.5), and in one patient hexon epitope mapping revealed reactivity against at least four distinct peptides. Penton was also recognized by the CTL lines with a median T-cell frequency of 30 SFC/2×10⁵ (range 26.5-164), with no recognition of control pepmixes. In contrast we were unable to expand adenovirus-reactive T cells from the remaining five patients. Two of three patients with positive ELIspot results from the CTL cultures against both hexon and penton are alive and adenovirus negative without any antiviral agents used at a median of 446 days post-UCBT (range 422-470). Four (4) patients died despite receiving antiviral treatment with Cidofovir. Two of these patients had no detectable antiviral immunity, while the others had only restricted or weak reactivity (reactivity against only one antigen or reactivity below the median SFC values of the responder cohort).

To our knowledge this is the first study that analyzes the anti-adenoviral immune response in UCBT recipients with active infection in the early post-transplant period. It appears that in the first 100 days after UCBT, during a time of active immunosuppression, adenovirus-specific T-cells in peripheral blood are consistently (10/10 patients) below the level of detection of conventional IFN-g ELIspot assays. However, dormant immune responses can be amplified in vitro using a single stimulation in the presence of IL4. Furthermore, detection of functional CTL against hexon and penton proteins may be a useful marker to distinguish between those with effective antiviral T cell immunity and those at risk of developing progressive disease.