Project Summary/Abstract: HPV-associated cancer incidence is significantly elevated in cervical and at other sites in HIV+ patients. HIV+ patients acquire more frequent multi-type infections, including many genotypes infrequently seen in healthy individuals, and not targeted by the current HPV preventive vaccines. We previously developed a candidate therapeutic and preventive HPV vaccine, pNGVL4a-CRTE6E7L2 (CRTE6E7L2), which comprises a DNA vector encoding the heat shock protein calreticulin fused genetically with HPV16 E6 and E7 (that are obligately expressed in HPV malignancies) as well as the L2 capsid protein (a broadly protective antigen). We showed that fusion with calreticulin (CRT) profoundly enhances the potency of DNA vaccines in generating HPV antigen-specific CD8+ T cell mediated immune responses even in CD4-depleted animals. In addition, vaccination with the CRTE6E7L2 DNA vaccine induces both L2-specific neutralizing antibodies and protection from experimental vaginal challenge. These features make the CRTE6E7L2 DNA vaccine particularly promising for use in HIV+ patients, a challenging group to treat, and to prevent multiple types of HPV infections. Although DNA vaccines are relatively safe and well suited for multiple administrations, they generally exhibit suboptimal immunogenicity when administered by conventional intramuscular needle injection, likely reflect inefficient host cell transduction. We have previously shown that electroporation is a much more effective DNA vaccine administration method to generate HPV-specific CD8+ T cell immune responses as compared to conventional intramuscular injection or epidermal delivery via gene gun. Thus, the goal of this proposal is to use the Ichor TriGrid™ Delivery System Electroporation Device, which has been used in multiple clinical trials, for intramuscular administration of the CRTE6E7L2 DNA vaccine at escalating doses in both HIV– and HIV+ patients with HPV16-associated high-grade cervical intraepithelial neoplasia grades 2 and 3 (CIN2/3), and to examine the safety, virologic, and disease outcomes. The Specific Aims of this study are to (1) evaluate the safety and toxicity of CRTE6E7L2 administered via electroporation in HIV– and HIV+ patients with HPV16+ CIN2/3;; (2) characterize the HPV16 E6/E7-specific cell-mediated and humoral immune responses in HIV– and HIV+ patients with HPV16+ CIN2/3 vaccinated with CRTE6E7L2 via electroporation;; (3) characterize L2-specific humoral immune responses in HIV– and HIV+ patients with HPV16-associated CIN2/3 upon vaccination with CRTE6E7L2 DNA vaccine via electroporation;; and (4) determine the HPV load and histopathological changes in the lesion and its microenvironment in HIV– and HIV+ patients with HPV16-associated...