Comparison of helper containing and helper free HSV amplicon vectors for immune therapy of CLL

HSV-based vectors have several biologic features well suited for gene therapy of leukemia/lymphoma. These include a high transduction efficiency, an ability to infect post-mitotic cells and a large packaging capacity. We have studied Herpes Simplex virus (HSV) based amplicon vectors for transduction of primary B-cell chronic lymphocytic leukemia (CLL). Three HSV amplicon vectors were constructed encoding b-galactosidase (lac), CD80 (B7.1) or CD154 (CD40L) and were packaged using either standard helpervirus packaging methods (HSVlac, HSVB7.1 and HSVCD40L, respectively) or a helper virus-free packaging method (hf-HSVlac, hf-HSVB7.1 and hf-HSVCD40L, respectively), based on the use of a bacterial artificial chromosome (BAC) system. CLL cells were transduced with either HSV or hf-HSV amplicons and studied for the ability to stimulate T-cell proliferation in an allogeneic mixed lymphocyte reaction (MLTR). At a multiplicity of infection (MOI) that transduced 60-90% of the CLL cells, a vigorous T-cell proliferative response was obtained using cells transduced with helper-free HSV amplicon (hf-HSVB7.1 ) but not with helper-containing HSVB7.1. Extrinsic provision of signal one partially restored the T-cell proliferative response to cells transduced with helper-containing HSVB7.1 stocks suggesting that one or more proteins encoded by the helper virus might affect antigen presentation. CLL cells transduced with either HSV-CD40L or hf-HSV-CD40L were compared for their ability to up-regulate B7.1 and function as T-cell stimulators in MLTR. Direct comparison of the two amplicon vectors revealed significantly better B7.1 expression in response to transduction with hf-HSV-CD40L compared to HSV-CD40L. CLL cells transduced with hf-HSV-CD40L were consistently more effective at stimulating allogeneic T-cell proliferative response than those transduced with HSV-CD40L. CLL cells transduced with helper-containing HSV amplicons showed a reduction of MHC-I expression on transduced cells, impaired capacity to respond to CD40 activation and decreased expression of CD 19 compared to cells transduced with hf-HSV amplicons. This may account for the decreased immune stimulatory function of CLL cells transduced with helper-containing HSV amplicons. We conclude that helper-free HSV amplicons represent a logical choice for immune-based clinical trials.