For leukemia vaccine generation, high-efficiency gene transfer is required to express immunomodulatory molecules that stimulate potent antileukemic immune responses. In this context, herpes simplex virus type-1 (HSV-1)-derived vectors have proven to be a promising tool for genetic modification of lymphoblastic leukemia cells. Yet, vector-associated viral protein expression might inadvertently modulate vaccine efficacy facilitating both immune evasion and immune stimulation. To explore the issue of immune-stimulation versus immune-suppression in immature lymphoblastic leukemia cells, two types of HSV-1 amplicon vectors, helper virus-dependent and helper virus-free that express the immunomodulatory molecules CD70 and IL-2, were compared with regard to their vector-associated immunomodulatory potential. We first established that lymphoblastic cell lines and primary acute lymphoblastic leukemia (ALL) cells express HSV receptor genes. Lymphoblastic cell lines were transduced with high efficiency, and in primary ALL cells high gene transfer rates of 47+/-15 and 42+/-14% were obtained with helper virus-dependent and -free HSV-1 amplicon vectors, respectively. The efficacy of the two amplicon vectors to induce antineoplastic responses was assessed in a vaccine setting in mice with pre-existing highly malignant lymphoblastic disease. Treatment of mice with vaccine cells transgenically expressing CD70+IL2 significantly suppressed lymphoblastic cell proliferation and improved survival. Of note, when helper virus-dependent HSV-1 amplicon vectors were used for vaccine preparation, the high immunogenic potential of the vector itself, in the absence of transgenic CD70+IL2 expression, seemed to be sufficient to mediate protection comparable to the antineoplastic response achieved by expression of immunomodulatory molecules. Thus for vaccine generation in B lymphoblastic leukemia, the immunogenic potential of HSV-1 helper virus-dependent amplicon vectors does provide additional benefit to the high transduction efficiency of HSV-1-derived vectors.