Adenovirus vector-specific T cells demonstrate a unique memory phenotype with high proliferative potential and coexpression of CCR5 and integrin α4_4β7_7

Background: The Step Study was a randomized trial to reduce HIV infection through vaccination with an adenovirus type 5-based gag/pol/nef construct; analysis following early cessation of the trial revealed an excess of HIV seroconversion in Ad5 seropositive men. This led to the suggestion that the Ad based vector may boost the number of CD4$^+$ CCR5$^+$ T-cells, target cells for HIV infection. Objectives: We sought to determine the immunophenotype and proliferative capacity of Ad5-specific T cells in the peripheral blood of adult donors to determine whether stimulation with replication defective Ad5 vectors could result in the significant expansion of a CD4$^+$ CCR5$^+$ T cell subset. Methods: Ad5 specific T cells were identified in the peripheral blood of healthy donors by IFN-$_y$ secretion assay and proliferative response was measured by CFSE labelling. Cells were analysed by flow cytometry to determine T cell differentiation marker, CCR5 and α$_4$β$_7$ expression on memory and proliferated cells. Results: Ad5-specific CD4$^+$ T cells within healthy adult donors exhibit a unique minimally differentiated memory phenotype with co-expression of CD45RA, CD45RO and CCR7. Stimulation with Ad vector leads to rapid expansion in vitro and a switch to an effector memory phenotype. Both short-term reactivated and proliferating Ad5-specific CD4+ T-cells express the HIV co-receptor CCR5 and the HIV gp120-binding integrin α$_4$β$_7$. Conclusion: Ad5-specific T cells demonstrate a phenotype and proliferative potential that would support HIV infection; these results are pertinent to the findings of the Step Study and future use of Ad5 as a vaccine vector

Quantum dot labelling of adenovirus allows for highly sensitive single cell flow and imaging cytometry

A quantum dot method for highly efficient labelling of single adenoviral particles is developed. The technique has no impact on viral fitness and allows the imaging and tracking of virus binding and internalisation events using a variety of techniques including imaging cytometry and confocal microscopy. The method is applied to characterise the tropism of different adenoviral vectors