Augmentation of Antitumor Activity of a Recombinant Adeno-Associated Virus Carcinoembryonic Antigen Vaccine with Plasmid Adjuvant

Abstract

Recombinant adeno-associated virus 2 (rAAV) vectors have been successfully used for sustained expression of therapeutic genes. The potential of using rAAV as a cancer vaccine vector and the impact of a bacterial plasmid adjuvant on this activity were investigated. C57BL/6 mice received a single intramuscular injection of rAAV expressing the human tumor-associated antigen, carcinoembryonic antigen (CEA). Three weeks later, when CEA expression was optimal, a bacterial plasmid containing methylated DNA motifs was injected into the same muscle. Mice were challenged 1 week later with syngeneic MC38 tumor cells stably expressing CEA. Immunization with rAAV-CEA alone resulted in sustained transgene expression and the elicitation of a humoral immune response to CEA. Cellular immune response, however, was weak, and tumor protection was not significant. In contrast, immunization with rAAV-CEA and the plasmid adjuvant resulted in stronger cellular immune response to CEA and tumor protection. The addition of plasmid adjuvant increased both myeloid dendritic cell recruitment in situ and CEA-specific T-helper-1-associated immune response. These data indicate that robust rAAV transgene expression of a tumor antigen followed by transient plasmid delivery to recruit and activate dendritic cells is an effective method of eliciting antitumor cellular immune responses.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands

“…[4][5][6] In addition, potential applications of AAV as a vector for immunotherapy of infectious diseases and cancer are being developed. 2,7,8,10,12 Although most of the preclinical studies and clinical trials so far have utilized serotype 2 AAV vectors, recent studies suggest that alternate serotype vectors with different host cell receptor specificity could have better utility for gene transfer to rAAV2 refractory target cells. [16][17][18][19] One of the cell types that shows poor transduction efficiency for rAAV2 is DC, mouse DC in particular.…”

“…The construction of recombinant AAV plasmids containing either the b-Gal or the full-length human CEA gene for packaging as a single-stranded DNA has been described. 10 A recombinant AAV plasmid containing CEA for packaging as a double-stranded self-complementary genome was constructed by subcloning a short (70 kDa) version of human CEA 22 in the AAV plasmid pLY4 (kind gift of Dr Arun Srivastava, University of Florida, Gainesville, FL, USA) as described below. Human CEA protein was purchased from either Fitzgerald Industries International Inc. (Concord, MA, USA) or Aspen Bio Inc. (Littleton, CO, USA).…”

“…10 A recombinant AAV plasmid containing the same transgene for packaging as self-complementary structure (sc) was constructed using the plasmid pLY-4 in which the 'D' and terminal resolution site (TRS) sequences at the 5 0 end of the genome were deleted. Packaging and purification of rAAV were carried out by helper virusfree transient transfection.…”

“…[4][5][6] However, recent reports have indicated the potential of rAAV for nongenetic disorders including infectious diseases and cancer. [7][8][9][10] Potential advantages of AAV as vector for genetic immunotherapy are sustained transgene expression and absence of vector genes encoding viral structural proteins thereby minimizing antigenic competition. Most of the current understanding of the potential for rAAV in gene therapy is based upon studies with AAV serotype 2-based vectors.…”

Intraperitoneal gene therapy by rAAV provides long-term survival against epithelial ovarian cancer independently of survivin pathway

“…Vector biodistribution, histopathology and toxicity analyses Unlike the previous studies, which utilized the skeletal muscle or the liver for the production of rAAV transgene proteins, [19][20][21][22][23][24][25] the present study is the first preclinical validation of i.p. transduction of rAAV encoding therapeutic transgenes.…”