Skin Electroporation: Effects on Transgene Expression, DNA Persistence and Local Tissue Environment

BACKGROUND: Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. CONCLUSIONS/SIGNIFICANCE: This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance

In Vivo Electroporation Enhances the Immunogenicity of an HIV-1 DNA Vaccine Candidate in Healthy Volunteers

DNA-based vaccines have been safe but weakly immunogenic in humans to date.We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines.This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate.ClinicalTrials.gov NCT00545987

Rapid identification of bovine MHCI haplotypes in genetically divergent cattle populations Using Next-Generation Sequencing

The major histocompatibility complex (MHC) region contains many genes that are key regulators of both innate and adaptive immunity including the polymorphic MHCI and MHCII genes. Consequently, the characterisation of the repertoire of MHC genes is critical to understanding the variation that determines the nature of immune responses. Our current knowledge of the bovine MHCI repertoire is limited with only the Holstein-Friesian breed having been studied in any depth. Traditional methods of MHCI genotyping are of low resolution and laborious and this has been a major impediment to a more comprehensive analysis of the MHCI repertoire of other cattle breeds. Next-generation sequencing (NGS) technologies have been used to enable high throughput and much higher resolution MHCI typing in a number of species. In this study we have developed a MiSeq platform approach and requisite bioinformatics pipeline to facilitate typing of bovine MHCI repertoires. The method was validated initially on a cohort of Holstein-Friesian animals and then demonstrated to enable characterisation of MHCI repertoires in African cattle breeds, for which there was limited or no available data. During the course of these studies we identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire

Transcriptional analysis of the bovine herpesvirus 1 Cooper isolate

Blot hybridization analysis of infected bovine herpesvirus 1 (BHV-1) cellular RNA isolated at various times post infection and after treatment with specific metabolic inhibitors was used to characterize transcription of the BHV-1 Cooper isolate. Synthesis of BHV-1 RNA was detected as early as 3 h post infection and reached a maximum at six to eight hours post infection. The most transcriptionally active area of the genome was between map units 0.110 to 0.195, within the Hin dIII I fragment. From the entire genome a total of 59 transcripts ranging in size from approximately 0.6 to 10 kilobases were characterized as belonging to one of three distinct classes. Using the protein synthesis inhibitor cycloheximide, three immediate-early transcripts were identified as originating from the internal inverted repeat region between map units 0.734 and 0.842, corresponding to the Hin dIII D fragment. Using phosphonoacetic acid to prevent virus DNA synthesis by inhibition of the BHV-1 DNA polymerase, 28 early transcripts were recognized. The remaining 28 transcripts, classified as late RNA, were detected without the use of metabolic inhibitors at 6 to 8 h post infection. Transcription of early and late RNA was not restricted to any specific area of the genome. Eighty percent of the transcripts from both the Hin dIII A fragment, between map units 0.381 to 0.537 within the unique long segment, and the Hin dIII K fragment, between map units 0.840 to 0.907 of the unique short segment, were designated as belonging to the early class.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41672/1/705_2005_Article_BF01316744.pd