16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

Effects of epitopes combination and adjuvants on immune responses to anti-Alzheimer disease DNA vaccines in mice.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuropathological hallmarks including deposits of the beta-amyloid peptide (AssP). Studies have shown that immunization with Abeta42 peptide reduces both the spatial memory impairments and Alzheimer disease-like neuropathologic changes in Alzheimer disease transgenic mice, but can cause side effect of a cell-mediated autoimmune meningoencephalitis. Recently, some studies showed that DNA vaccination could be used to generate an antibody response to Abeta without the adverse cell-mediated immune effect. In the current study, we generate four DNA vaccine plasmids (pV-GE1, pV-GE2, pV-GE3, and pV-GE4) against Alzheimer disease by separately fusing Abeta epitope sequences (coding for EFGH, DAEFGH, EFGH+EFGH, and EFGH+DAEFGH) with IgG heavy chain coding region of mouse. Meanwhile, the full-length gene Abeta encoding plasmid (pV-Abeta), empty vector (pVAX) and synthetic AssP were also included as control. The sera of BALB/c mice immunized via intramuscular with plasmids and peptide were tested by indirect ELISA for auto-AssP immunoreactivity. The results showed that all the DNA vaccine plasmids induced AssP-specific antibodies; moreover pV-GE2 and pV-Abeta constructs elicited higher antibody titers than other constructs (P < 0.05). To further enhance the immune response, GM-CSF encoding plasmid (pGM-CSF) and purified BCG-DNA were used as molecular adjuvants. BCG-DNA could enhance humoral and cellular immune responses simultaneously and did not alter the phenotype of the immune responses, whereas pGM-CSF showed no obvious effect on immune response. These results suggest that this immunization strategy of using Abeta epitope encoding plasmid plus BCG-DNA adjuvant may serve as the basis for developing anti-Alzheimer disease vaccines.

Tissue distribution of a plasmid DNA containing epitopes of foot-and-mouth disease virus in mice.

It is known that only the minority of plasmid DNAs effect a cure or prevention after intramuscular injection into host. But what is the fate of the majority? And indeed how many of the injected DNAs work? Till now, little is known about it. To answer these questions, two methods including PCR and autoradiography were used in distribution study in mice that had received a single muscular inoculation of plasmid DNA containing antigenic epitopes of foot-and-mouth disease virus. The results showed that the plasmid DNAs were distributed by blood circulation and degraded soon. The degradation ratio of super coiled plasmid DNA was 20.9% in 10 min, 34.1% in 1h, 86.8% in 1 day and 97.8% in 1 week in sera in vivo. And over a half of the whole were output in urine and faeces. The rest resided most in muscles as 'antigen pool', next in immune organs, kidney, liver, heart, lung and little in brain or gonad. About 40% or 0.5% of total plasmid DNAs, inferring to be effective, resided in muscles or immune organs, respectively. Collective results suggested that 'nude' DNA, as water injection, was characterized as quick absorbent, extensive distribution, but low utilization rate. Finally, the immune mechanism for the DNA vaccine was discussed.

Immune response in mice inoculated with plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus.

This paper focuses on the development of candidate DNA vaccine encoding antigenic epitopes of type O foot-and-mouth disease virus (FMDV). A series of plasmids encoding different combinations of B cell epitopes and a T cell epitope were constructed and characterized by inoculating BALB/c mice. The specific antibodies were only detectable in the mice inoculated with plasmids encoding the T cell epitope and B cell epitopes from sites 5 and 1, within which site 5 includes residues 135-167 of VP1 and site 1 includes 141-160 region (G-H loop) and carboxyl terminus of VP1. Stronger cellular immune responses were also observed in these mice using T cell proliferation assay.