[CANCER RESEARCH 61, 7861–7867, November 1, 2001] Eradication of Established Tumors by Vaccination with Venezuelan Equine Encephalitis Virus Replicon Particles Delivering Human Papillomavirus 16 E7 RNA 1 Markwin P. Velders, 2 Sue McElhiney, Maria Cristina Cassetti, Gretchen L. Eiben, Terry Higgins, Gerald R. Kovacs, Amira G. Elmishad, W. Martin Kast, 3 and Larry R. Smith 3 Cardinal Bernardin Cancer Center, Loyola University-Chicago, Maywood, Illinois 60153 [M. P. V., G. L. E., A. G. E., W. M. K.], and Wyeth-Lederle Vaccines, Pearl River, New York 10965 [S. M., M. C. C., T. H., G. R. K., L. R. S.] ABSTRACT The etiological role of human papillomaviruses (HPV) in cervical and other cancers suggests that therapeutic vaccines directed against requisite viral antigens may eradicate tumors or their precursors. A Venezuelan equine encephalitis (VEE) alphavirus vector delivering the HPV16 E7 RNA was evaluated for antitumor efficacy using a murine E7  tumor model. Vaccination with VEE replicon particles expressing E7 (E7-VRP) induced class I-restricted CD8  T-cell responses as determined by IFN- enzyme-linked immunospot (ELISPOT), tetramer, and cytotoxicity as- says. E7-VRP vaccination prevented tumor development in all of the mice and effectively eliminated 7-day established tumors in 67% of tumor- bearing mice. The induction of protective T-cell responses was dependent on CD8  , but not CD4  T cells. Long-lasting T-cell memory responses developed in E7-VRP-vaccinated mice as determined by complete protec- tion from tumor challenge 3 months after the final vaccination. These promising results highlight the potent CD8  T-cell-mediated antitumor effects elicited by VEE replicon-based vectors and support their further development toward clinical testing against cervical intraepithelial neo- plasia or carcinoma. INTRODUCTION Approximately 15% of human cancers worldwide are associated with viruses, the majority of which are attributed to HPV 4 (1). HPV infection and persistence confer risks of developing cervical carci- noma (2, 3), the most prevalent HPV-associated cancer. HPV DNA is detectable in 99% of cervical carcinomas (4) and to varying degrees in other cancers, collectively implicating HPV as the causative agent in 10% of female cancers (1). The oncogenic potential of the “high risk” HPV genotypes 16, 18, and others is attributed in part to their E6 and E7 genes, which possess transforming and immortalizing activi- ties in vitro (5, 6). Sustained E6 and E7 expression is required for maintenance of the malignant phenotype (7, 8) and is evident in cervical carcinomas and their CIN precursors (9, 10). These observa- tions suggest that E6 and E7 may be appropriate targets for eradicat- ing HPV-associated tumors or their precursors by therapeutic vacci- nation. Cell-mediated immune responses are important determinants of HPV-associated disease outcome (11). Protective CTL responses against a MHC class I- restricted E7 epitope have been demonstrated in E7 + murine tumor models (12, 13). HPV16 E6- and E7-derived peptides capable of binding specific human leukocyte antigen (HLA) class I alleles have been identified and have successfully primed CTL responses in HLA-A2.1 transgenic mice and in vitro from normal human donors (14). Memory CTL responses against some of these HLA-restricted peptides from E6 and E7 are detectable in some patients with CIN and cervical carcinoma but not in normal subjects (14). HLA-A2.1-restricted E7 peptides (15) and E6-E7 expressing recombinant vaccinia viruses (16) have been evaluated in Phase I trials with end stage cervical cancer patients and have revealed CTL responses in some cases but poor clinical responses (14). An encour- aging recent HPV16 E7 peptide trial in high-grade CIN patients has revealed both increases in CTL activity and lesion regression in many of the vaccinees (17). Whereas vaccine intervention at earlier stages of disease may be one aspect of improving clinical efficacy, more powerful strategies for inducing memory CTLs, preferably against a broader array of HPV epitopes, will be critical for priming or boosting antitumor responses. Several HPV vaccine candidates have been tested against murine tumors constitutively expressing HPV16 E6 and E7 genes (18). The most practical and efficacious vaccine approaches tested include pep- tides (12, 13), recombinant fusion proteins (19), chimeric virus-like particles (20), viral vectors (21), and plasmid DNA (22, 23). Limita- tions currently exist with several vectored approaches such as vaccinia virus and plasmid DNA because of preexisting immunity or vaccine take in humans, whereas peptide vaccines are limited to predeter- mined target HLA alleles. Although several candidates have not yet been tested clinically, more effective strategies should continue to be investigated in preclinical models. One viral vector under consider- ation is derived from VEE virus. VEE and other enveloped, positive- stranded RNA AVs like Sindbis and SFV have been engineered as replication-incompetent viral-delivery vectors or replicons (24). Rep- licon vectors are generated by removing the structural genes of the virus and replacing them with a foreign gene; they contain AV replicase genes, which mediate RNA replication and high-level pro- tein expression but produce no progeny virus. The replicon-recombi- nant RNA encoding the foreign gene of interest in lieu of the VEE structural genes can be packaged into VRPs on provision of the structural RNAs in trans (25). VRPs encoding several viral genes have been shown to be immunogenic and protective in murine, guinea pig, and primate models (25–29). AVs in general, and VEE in particular, are attractive vaccine vectors for several reasons: (a) there is no widespread preexisting anti-VEE immunity in humans; (b) repeated immunization appears to be possible (25); (c) replicating RNA directs high-level heterologous protein expression (30); (d) double-stranded RNA is produced after AV infection and apoptosis is induced, which may be highly condu- cive for inducing immune responses (31); and (e) the envelope gly- coproteins of VEE, in contrast to those of other AVs, confer dendritic cell tropism (32). These features, as well as the cytoplasmic transcrip- tion of VEE RNA and the unlikelihood of RNA integration, suggest Received 6/6/01; accepted 8/28/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 M. P. V. is a fellow of the Cancer Research Institute (CRI). 2 To whom requests for reprints should be addressed, at Cardinal Bernardin Cancer Center, Room 211, Loyola University Medical Center, 2160 South First Avenue, May- wood, IL 60153. 3 W. M. K. and L. R. S. made equal contributions as senior authors. 4 The abbreviations used are: HPV, human papillomavirus; AV, alphavirus; CIN, cervical intraepithelial neoplasia; GFP, green fluorescent protein; PE, phycoerythrin; SFV, Semliki Forest virus; VEE, Venezuelan equine encephalitis virus; VRP, VEE replicon particle(s); ATCC, American Type Culture Collection; MVA, modified vaccinia virus Ankara; MOI, multiplicity/multiplicities of infection; ELISPOT, enzyme-linked immu- nospot. 7861 Research. on February 6, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from