Quantification of oligodeoxynucleotides in human plasma with a novel hybridization assay offers greatly enhanced sensitivity over capillary gel electrophoresis.

Capillary gel electrophoresis using UV detection (CGE-UV) has been used to quantify oligodeoxynucleotides (ODN) in human plasma. Although the sensitivity of this method is adequate to detect antisense ODN, which are administered in daily doses up to 10 mg/kg, CGE-UV is not sensitive enough to detect the much lower quantities of ODN administered for other purposes, such as immune stimulation by CpG ODN. We have developed a very sensitive colorimetric hybridization assay that increases the sensitivity of detection by more than four logs compared with CGE-UV. The hybridization assay uses sequence-specific capture and detection ODN probes complementary to portions of the ODN sequence. Herein we provide a prototype for assay development and validation using a 24- mer immunostimulatory phosphorothioate ODN. Probes were locked nucleic acids (LNA), resulting in increased sensitivity and specificity. The linear range of the assay is 7.8-1000 pg/ml, with a 7.8 pg/ml lower limit of quantification (LLOQ) and a detection limit of 2.8 pg/ml. This translates to detection of 40 attamoles. Intraassay and interassay precision were < or =5.0% CV and < or =12.9% CV, respectively, for quality control samples. The assay is suitable for a variety of matrices, including monkey and rat plasma, allowing application to toxicokinetic samples. The methodology is highly specific, with the ability to distinguish almost all single-base mismatched ODN. The assay detects 100% of the parent as well as some metabolites up to N-4, which are known to be the primary metabolites forming in the first hours after in vivo administration and are physiologically active with in vitro assays.

CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study.

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) act as potent Th1-like immune enhancers with many antigens in animal models. We have extended these observations to the first clinical evaluation of the safety, tolerability and immunogenicity of CPG 7909 when added to a commercial HBV vaccine. In a randomized, double-blind phase I dose escalation study, healthy volunteers aged 18-35 years were vaccinated at 0, 4 and 24 weeks by intramuscular injection with Engerix-B (GlaxoSmithKline). The regular adult dose of 20 microg recombinant hepatitis B surface antigen (HBsAg) adsorbed to alum was administered mixed with saline (control) or with CPG 7909 at one of three doses (0.125, 0.5 or 1.0 mg). HBsAg-specific antibody responses (anti-HBs) appeared significantly sooner and were significantly higher at all timepoints up to and including 24 weeks in CPG 7909 recipients compared to control subjects (p< or = 0.001). Strikingly, most CpG 7909-vaccinated subjects developed protective levels of anti-HBs IgG within just two weeks of the priming vaccine dose. A trend towards higher rates of positive cytotoxic T cell lymphocyte responses was noted in the two higher dose groups of CPG 7909 compared to controls. The most frequently reported adverse events were injection site reactions, flu-like symptoms and headache. While these were more frequent in CPG 7909 groups than in the control group (p<0.0001), most were reported to be of mild to moderate intensity regardless of group. In summary, CPG 7909 as an adjuvant to Engerix-B was well-tolerated and enhanced vaccine immunogenicity. CPG 7909 may allow the development of a two-dose prophylactic HBV vaccine.

Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine.

CPG 7909, a 24-mer B-Class CpG oligodeoxynucleotide (ODN), was tested for safety, tolerability and its ability to augment the immunogenicity of a commercial trivalent killed split influenza vaccine (Fluarix containing A/Beijing/262/95, A/Sydney/5/97 and B/Harbin/7/94; SmithKline Beecham) in a phase Ib blinded, randomized, controlled clinical trial. Sixty healthy volunteers were recruited in two consecutive cohorts of 30 subjects, who were randomly assigned to receive Fluarix plus 1mg CPG 7909 or Fluarix plus saline control (15 subjects each). Vaccines were administered by intramuscular injection on a single occasion with subjects in the first cohort receiving a 1/10th dose of Fluarix and those in the second cohort receiving the full-dose. All safety measures including physical evaluation, laboratory blood assays, and assays for DNA autoimmunity were within normal values except for transient and clinically inconsequential decreases in total white blood cell counts in groups receiving CPG 7909. All vaccines were found to be generally well tolerated with similar frequency and intensity for most adverse reactions for groups receiving CPG 7909 as controls. Exceptions were injection site pain and headache, which were reduced in frequency in subjects receiving the 1/10th Fluarix dose without CpG, compared to the frequency in all other groups. There was a lack of pre-existing immunity, defined as hemagglutinin inhibition (HI) activity < or =20, for all subjects to the influenza strains A/Beijing/262/95 and B/Harbin/7/94 and for some subjects to A/Sydney/5/97. Post-vaccination humoral immune responses, as determined 2 and 4 weeks later by assay of HI activity and ELISA to detect antibodies against hemagglutinin (anti-HA) were similar for both full and reduced Fluarix doses but the cellular immune responses (measured as PBMC antigen-specific IFN-gamma secretion) were reduced in the 1/10th Fluarix dose group. Humoral responses were not significantly enhanced by the addition of CPG 7909, except in individuals with pre-existing immunity to A/Sydney/5/97 strain (baseline HI activity titre >20), where there was a trend to higher HI activity with CPG 7909 (P = 0.06). The addition of CPG 7909 to the 1/10th dose of Fluarix did however result in significantly higher levels of IFN-gamma secretion from peripheral blood mononuclear cells recovered at 4 weeks and restimulated ex vivo with A/Beijing/262/95 (P = 0.048) and B/Harbin/7/94 (P = 0.0057), restoring these to the level seen with full-dose vaccine. These results suggest that addition of CPG 7909 to Fluarix may allow the use of reduced vaccine doses without reduced immunogenicity.

Designing gene therapy vectors: avoiding immune responses by using tissue-specific promoters.

Attempts to correct genetic disorders by gene therapy have been hindered by various problems including unwanted immune responses against the gene product. It has been shown that immune responses with DNA vaccines after i.m. injection of antigen-encoding plasmid DNA are primed solely by professional antigen-presenting cells (APC), even though myocytes are the primary type of cell transfected. This possibly involves direct transfection of some APC in regional lymph nodes draining the injected muscle. Here we have used plasmid DNA vaccines that express hepatitis B surface antigen (HBsAg) to evaluate the possibility of abrogating these immune responses by use of a tissue-specific promoter that does not drive expression in APC. We show that HBsAg-specific humoral or cell-mediated responses are not induced in mice when the muscle-specific human muscle creatine kinase promoter is used in place of the ubiquitous cytomegaloviral promoter to drive expression of HBsAg. This may have significance in the field of gene therapy where one aims to achieve stable expression of the desired gene product without interference from the host immune response.

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs.

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5' side or a G on the 3' side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.