Transduction of human B lymphocytes using adenoviral vectors of various serotypes

• Maria M. Sukhova
• Maria G. Byazrova
• Alina S. Bandelyuk
• Artem A. Mikhailov
• Maksim M. Shmarov
• Alexander V. Filatov

Abstract

Introduction. B cells are an attractive target for gene therapy. With the help of gene transfer to B cells, it is possible to treat diseases associated with genetically determined dysfunction of B cells, as well as lymphoproliferative and autoimmune pathologies. In recent years, adenovirus (Ad)-based vectors have become increasingly popular for gene transfer. This is facilitated by such properties of Ad as high transgene capacity, the ability to produce the vector in high titers, and the ability to transduce terminally differentiated cells.

The aim of the study is to determine the efficiency of transduction of human B cell lines, as well as activated primary B lymphocytes after infection using adenoviral vectors of various serotypes.

Material and methods. Mononuclear cells were isolated from the peripheral blood of healthy donors (n = 11) by Ficoll-Verografin density gradient centrifugation with further enrichment of B lymphocytes using negative selection using magnetic microspheres. B cells were stimulated in vitro with a mixture of lymphokines, which included IL-2, IL-4, BAFF, IL-21 and multimerized CD40L. After 24 h, stimulated B lymphocytes were infected with recombinant adenoviral vectors carrying the green fluorescent protein (GFP) reporter gene. Three serotypes were tested: Ad5, sAd23 and Ad26, as well as a chimeric Ad5 vector containing the corresponding domain of the Ad35 serotype (Ad5/35) at the C-terminus of its fiber. At 48 h postinfection, the per center of transduced B cells and the average fluorescence intensity of the GFP reporter protein were determined. Adenoviral transduction was determined in four B cell lines: NALM-6, Ramos, IM-9, and RPMI-8226, and also in primary B lymphocytes. For comparison with B cells the non-lymphoid line HEK293 was used.

Results. Adenoviruses in low and medium doses had a moderate toxic effect on cells, while the viability remained at least 70 %. The most susceptible to adenovirus infection were RPMI-8226 cells, in which, after transduction, more than 90 % of cells expressed GFP, and in this indicator they were close to non-lymphoid HEK293 cells. The cells most resistant to adenovirus infection were NALM-6, which were transduced to some extent with the Ad26 and Ad35 vectors (65 and 66 %), slightly with the sAd23 vector (18 %), and minimally with the Ad5 vector. Ramos and IM-9 cells were intermediate in sensitivity to infection, with Ramos being transduced somewhat more successfully than IM-9. When comparing different serotypes, Ad26 turned out to be the most infectious, which, even in minimal doses, transduced more than 90 % of Ramos, IM-9 and RPMI-8226 cells. For stimulated primary B lymphocytes, the Ad26 vector had the greatest transducing efficiency. At a virus dose of 2000 PFU per cell, the median proportion of GFP⁺ cells was 35 % (interquartile range 30–40 %). Ad5/35 showed slightly less activity (median value 16 %, interquartile range 13–20 %). Finally, Ad5 and sAd23 had the lowest transducing activity, which consistently exceeded the threshold level only at the highest doses of virus (1000 to 2000 PFU per cell).

Conclusion. Ad serotypes differ in their ability to transduce human B cells. The most efficient transduction is provided by the Ad26 serotype. The Ad5 vector pseudotyped with Ad35 fiber has a slightly reduced efficiency. Transduction of primary B lymphocytes occurs only after their activation.

Keywords:adenoviruses (Ad); Ad serotypes; gene transfer; B lymphocytes; transduction; GFP; stimulation of B lymphocytes

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