Susan M. Efler

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 μg 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⩽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.

Induction of systemic TH1-Like innate immunity in normal volunteers following subcutaneous but not intravenous administration of CPG 7909, a synthetic B-class CpG oligodeoxynucleotide TLR9 agonist

Subcutaneous injection of normal human volunteers with a B-class CpG oligodeoxynucleotide (ODN) TLR9 agonist, CPG 7909, induced a TH1-like pattern of systemic innate immune activation manifested by expression of IL-6, IL-12p40, IFN-α, and IFN-inducible chemokines. Serum IP-10 was found to be the most sensitive assay for subcutaneous CPG 7909 stimulation; its level was significantly increased in all subjects at all dose levels, including the lowest tested dose of just 0.0025 mg/kg. This pattern of chemokine and cytokine induction was markedly different from that previously reported to be induced by TLR9 stimulation in rodents, most likely reflecting species-specific differences in the cell types expressing TLR9. Subcutaneous CPG 7909 injection induced transient shifts in blood neutrophils, lymphocytes, and monocytes, consistent with the increased chemokine expression. Levels of acute phase reactants such as C-reactive protein were also increased. A second subcutaneous CPG 7909 injection administered 2 weeks after the first elicited similar immune responses, showing little or no tolerance to the effects of repeated in vivo TLR9 stimulation. Subjects developed dose-dependent transient injection site reactions and flu-like symptoms but otherwise tolerated injection well, with no evidence of organ toxicity or systemic autoimmunity. The activation of innate immunity was dependent on the route of ODN administration, since intravenous injection caused no such effects. These studies indicate that in vivo activation of TLR9 by subcutaneous administration of CPG 7909 could be a well-tolerated immunotherapeutic approach for induction of TH1 innate immune activation.

Phase 1B, randomized, double-blind, dose-escalation trial of CPG 10101 in patients with chronic hepatitis C virus†‡

CPG 10101, a synthetic oligodeoxynucleotide (ODN), is a toll‐like receptor 9 (TLR9) agonist with antiviral and immunomodulatory properties that could potentially influence chronic infection with HCV. In this multicenter Phase 1b trial, 60 HCV‐positive patients (50 genotype 1 HCV) were randomized and received either placebo or CPG 10101 at 0.25, 1, 4, 10, or 20 mg subcutaneously (SC) twice weekly for 4 weeks or at 0.5 or 0.75 mg/kg SC once weekly for 4 weeks. Dose‐dependent cytokine induction was observed after administration of CPG 10101. At 24 hours after administering the highest dose of 0.75 mg/kg CPG 10101, interferon (IFN)‐γ‐inducible protein 10 (IP‐10) had a mean increase over baseline levels (±SD) of 15,057 (±9769) pg/ml (P < 0.01, compared to placebo); IFN‐α had a 106 (±63.3) pg/ml increase (P < 0.01); and 2′5′‐oligoadenylate synthetase (OAS) had a 163 (±120.6) pmol/dl increase (P < 0.01). Decreases in HCV RNA also were dose‐dependent, with the greatest group geometric mean maximum reduction of 1.69 ± 0.618 log10 (P < 0.05) observed in the 0.75 mg/kg dose group. Decreases ≥1 log10 were seen in 22 of 40 patients who received ≥1 mg CPG 10101, with 3 patients exceeding a 2.5‐log10 reduction. CPG 10101 was well tolerated, and adverse events were consistent with CPG 10101s mechanism of action. Conclusion: In this Phase 1 study, CPG 10101 was associated with dose‐dependent increases in markers of immune activation and decreases in HCV RNA levels. The data support further clinical studies of CPG 10101 for treating chronic HCV infection. (HEPATOLOGY 2007.)

Immunostimulatory effects of three classes of CpG oligodeoxynucleotides on PBMC from HCV chronic carriers

BackgroundChronic hepatitis C virus (HCV) infection results from weak or absent T cell responses. Pegylated-interferon-alpha (IFN-α) and ribavirin, the standard of care for chronic HCV, have numerous immune effects but are not potent T cell activators. A potent immune activator such as TLR9 agonist CpG oligodeoxynucleotide (CpG) may complement current treatment approaches.MethodsPeripheral blood mononuclear cells (PBMC) obtained from HCV chronic carriers who failed previous treatment and from healthy donors were incubated in vitro with the three main CpG classes (A, B or C), recombinant IFN-α-2b (IntronA) and/or ribavirin. Proliferation and cytokine secretion (IFN-α, IL-10 and IP-10) were evaluated.ResultsCpG induced proliferation and cytokine secretion in patterns expected for each CpG class with similar group means for HCV and healthy donors. IntronA and ribavirin, alone or together, had no detectable effects. IntronA and C-Class CpG together induced more IFN-α than CpG alone in most subjects. IFN-α secretion was proportional to the number of plasmacytoid dendritic cells in PBMC from healthy donors but not HCV donors in whom responses were highly heterogeneous.ConclusionThe strong immune stimulatory effect of CpG on PBMC isolated from treatment-failed HCV patients suggests possible utility alone or in combination with current HCV antiviral treatment.

Development of DNA Vaccines for Aquaculture

Aquaculture is a rapidly growing industry in many countries (Meyer, 1991; Leong & Fryer, 1993). Its development is driven by an increasing demand for fish and shellfish products, and the continuous decline of wild caught harvests (Meyer, 1991; Hanfman, 1993). However, loss due to infectious disease is a major concern for fish farmers. Approximately 10% of all aquatic animals are lost annually due to pathogens (Leong & Fryer, 1993). For various reasons, such as inefficacy, high cost and/or environmental concerns, traditional antigen-based vaccines (i.e., whole killed, live attenuated and subunits) have only been used successfully for the prevention of a limited number of diseases, mainly those of bacterial origin. DNA vaccination could circumvent many of the disadvantages associated with classical methods of immunization. In mammals and birds, they have been shown to induce very potent, long-lasting and protective humoral and cell-mediated immune responses against numerous viral, bacterial or parasitic diseases (Wahren, 1996; Donnelly et al., 1997; Davis & Brazolot Milian, 1997).