Gary Van Nest

Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions.

Recent reports have identified two major classes of CpG motif‐containing oligodeoxynucleotide immunostimulatory sequences (ISS): uniformly modified phosphorothioate (PS) oligodeoxyribonucleotides (ODNs), which initiate B cell functions but poorly activate dendritic cells (DCs) to make interferon (IFN)‐α, and chimeric PS/phosphodiester (PO) ODNs containing runs of six contiguous guanosines, which induce very high levels of plasmacytoid DC (PDC)‐derived IFN‐α but poorly stimulate B cells. We have generated the first reported ISS, C274, which exhibits very potent effects on all human immune cells known to recognize ISS. C274 is a potent inducer of IFN‐γ/IFN‐α from peripheral blood mononuclear cells and exhibits accelerated kinetics of activity compared with standard ISS. This ODN also effectively stimulates B cells to proliferate, secrete cytokines, and express costimulatory antigens. In addition, C274 specifically activates PDCs to undergo maturation and secrete cytokines, including very high levels of IFN‐α. Sequence variation studies based on C274 were used to identify the general motif requirements for this novel and distinct class of ISS. In contrast, chimeric PO/PS CpG‐containing ODNs with polyguanosine sequences exert a differential pattern of ISS activity compared with C274, perhaps in part as a result of their greatly different structural nature. This pattern is composed of high IFN‐α/IFN‐γ induction and low DC maturation in the absence of B cell stimulation. In conclusion, we have generated a novel class of ISS that transcends the limitations ascribed to classes described previously in that it provides excellent stimulation of B cells and simultaneously activates PDCs to differentiate and secrete large amounts of type I IFN.

A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant

Certain oligodeoxynuclotides with CpG motifs provide enhanced immune response to co-delivered antigens. We performed a phase I, observer-blinded, randomized study in healthy anti-hepatitis B surface antigen (anti-HBsAg) antibody negative adults to explore safety and immunogenicity of co-injection of recombinant HBsAg combined with an immunostimulatory DNA sequence (ISS) 1018 ISS. Four ISS dosage groups (N=12 per group) were used: 300, 650, 1000 or 3000 microg. For each group, two controls received 20 microg HBsAg alone, two controls received ISS alone, and eight subjects received ISS+20 microg HBsAg. Subjects received two doses 8 weeks apart. Injection site reactions (tenderness and pain on limb movement) were more frequent at higher ISS+HBsAg doses but were mainly mild and of short duration. Higher anti-HBsAg antibody levels were associated with higher ISS doses. Four weeks after the first dose, a seroprotective titer (>or=10 mIU/ml) was noted for 0, 25, 75, and 87.5% of subjects by increasing ISS dose group (P<0.05) for those who received ISS+HBsAg; 1 month after the second dose this increased to 62.5, 100, 100, and 100%, respectively. Geometric mean anti-HBsAg antibody levels by increasing ISS+HBsAg dose were 1.22, 5.78, 24.75, and 206.5 mIU/ml after the first dose and 65.37, 877.6, 1545, and 3045 mIU/ml after the second dose. We conclude that 1018 ISS+HBsAg was well tolerated and immunogenic in this phase I study in healthy adults and may offer the potential for enhancement of hepatitis B virus (HBV) immunization and protection after one or two doses or in individuals who fail to respond to the standard vaccine regimen.

CpG-C Immunostimulatory Oligodeoxyribonucleotide Activation of Plasmacytoid Dendritic Cells in Rhesus Macaques to Augment the Activation of IFN-γ-Secreting Simian Immunodeficiency Virus-Specific T Cells

There are two principle subsets of dendritic cells (DCs); CD11c+CD123− myeloid DCs (MDCs) and CD11c−CD123+ plasmacytoid DCs (PDCs). DC activation via TNF-TNFRs (e.g., CD40L) and TLRs (e.g., immunostimulatory oligodeoxyribonucleotides (ISS-ODNs)) is crucial for maximal stimulation of innate and adaptive immunity. Macaque DC biology is being studied to improve HIV vaccines using the SIV macaque model. Using lineage (Lin) markers to exclude non-DCs, Lin−HLA-DR+CD11c+CD123− MDCs and Lin−HLA-DR+CD11c−CD123+ PDCs were identified in the blood of uninfected macaques and healthy macaques infected with SIV or simian-human immunodeficiency virus. Overnight culture of DC-enriched Lin-depleted cells increased CD80 and CD86 expression. IL-12 production and CD80/CD86 expression by MDC/PDC mixtures was further enhanced by CD40L and ISS-ODN treatment. A CpG-B ISS-ODN increased CD80/CD86 expression by PDCs, but resulted in little IFN-α secretion unless IL-3 was added. In contrast, a CpG-C ISS-ODN and aldrithiol-2-inactivated (AT-2) SIV induced considerable PDC activation and IFN-α release without needing exogenous IL-3. The CpG-C ISS-ODN also stimulated IL-12 release (unlike AT-2 SIV) and augmented DC immunostimulatory activity, increasing SIV-specific T cell IFN-γ production induced by AT-2 SIV-presenting MDC/PDC-enriched mixtures. These data highlight the functional capacities of MDCs and PDCs in naive as well as healthy, infected macaques, revealing a promising CpG-C ISS-ODN-driven DC activation strategy that boosts immune function to augment preventative and therapeutic vaccine efficacy.

Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha.

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon‐γ (IFN‐γ) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS‐induced IFN‐γ from NK cells is primarily dependent upon IFN‐α release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC‐released soluble factor(s) may contribute to IFN‐γ induction. Indeed, tumour necrosis factor‐α (TNF‐α) was identified as a significant contributor to ISS‐mediated activation of NK cells and was observed to act in an additive fashion with IFN‐α in the induction of IFN‐γ from NK cells and to up‐regulate CD69 expression on NK cells. This activity of TNF‐α, however, was dependent upon the presence of PDC‐derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF‐α.

Immunostimulatory DNA as a vaccine adjuvant

Immunostimulatory DNA containing unmethylated CpG motifs is recognized by Toll-like receptor 9, resulting in the activation of innate immune responses that subsequently amplify the adaptive-immune response. Advances in the characterization of Toll-like receptor 9 signaling have identified immunostimulatory sequences (ISS) with distinct biological activities. Numerous animal models have demonstrated that synthetic ISS are effective adjuvants that enhance both humoral and cellular immune responses in diverse indications, ranging from infectious disease to cancer and allergy. An added benefit supporting the use of ISS as a vaccine adjuvant is that the specific activation of a pathway critical to the regulation of the immune response results in minimal toxicity. To date, clinical testing has largely affirmed the potency and safety of ISS-adjuvanted vaccines.

A minimal human immunostimulatory CpG motif that potently induces IFN‐γ and IFN‐α production

Recent reports have shown that immunostimulatory sequences (ISS) containing CpG motifs have minimal length requirements (≥12 bases) for the exertion of immune‐enhancing function upon mammalian cells. Herein we demonstrate that short ISS (5–7 bases), which exhibit no activity on their own, induce IFN‐γ and IFN‐α secretion from human peripheral blood mononuclear cells when adsorbed to the surface of cationic poly(D,L‐lactide‐co‐glycolide) microparticles (cPLGA). Utilizing this technique, we discovered a minimal ISS sequence for induction of IFN‐γ and IFN‐α from human cells: 5′‐TCGXX‐3′. These short ISS/cPLGA formulations targeted PDC in similar fashion to longer ISS ODN, the activity of which does not require (but is enhanced by) cPLGA. PDC stimulated with short ISS/cPLGA responded with enhanced uptake of ISS and elevated production of cytokines, including IFN‐α. However, ISS‐responsive B cells did not respond to short ISS/cPLGA, underlining the plasmacytoid dendritic cell selectivity of this formulation. These results describe a novel technique for formulating active, but very short, ISS oligodeoxynucleotide that allows for the dissection and characterization of minimal immunostimulatory CpG motifs.

Negative regulation of TLR9‐mediated IFN‐α induction by a small‐molecule, synthetic TLR7 ligand

Toll‐like receptors (TLRs) are a family of molecules that function as sensors for the detection of foreign pathogens through the recognition of nonvariable microbial motifs. Although numerous studies have focused on singular TLRs, less attention has been focused on how simultaneous signaling of multiple TLRs may result in counter‐regulation of the effects of each. Here, we examine the counter‐regulation that occurs during simultaneous stimulation of TLR7 and TLR9 on human plasmacytoid dendritic cels (PDCs) and B cells. Interestingly, we observed that the capacity for potent IFN‐α‐induction by TLR9 ligands like CpG‐C and CpG‐A is markedly reduced by concurrent small molecule TLR7 stimulation. However, this inhibition is specific to particular CpG motif‐containing immunostimulatory sequence (ISS) functions such as IFN‐α induction and BDCA‐2 down‐regulation. Other ISS activities such as PDC expression of CD80/CD86, secretion of IL‐6, and B cell proliferation are not altered by the presence of TLR7 ligands (TLR7Ls). In concordance with the ability of TLR7Ls to decrease IFN‐α secretion induced by ISS, we also find that the expression of interferon regulatory factor‐7 (IRF‐7), a transcriptional factor critical for IFN‐α expression, is reduced. Furthermore, down‐regulation of TLR9 mRNA expression is accelerated after TLR7 stimulation. These data indicate that TLR7 and TLR9 costimulation do not combine synergistically for IFN‐α induction and demonstrate that, instead, a negative feedback mechanism has evolved, possibly to prevent levels of IFN‐α secretion potentially detrimental to the host.

Evolving strategies for the prevention of influenza infection: potential for multistrain targeting.

Approved influenza vaccines based on the induction of antibodies to hemagglutinin are strain specific and cumbersome to manufacture. Several alternative vaccine strategies based on the induction of humoral responses against the external domain of the M2 protein, as well as cellular responses against nucleoprotein, have the potential to target multiple strains of influenza. A universal vaccine would be a major advancement in the prevention of influenza infection as it would alleviate the need for tailored vaccines to control seasonal influenza epidemics while simultaneously providing a level of protection against potential pandemic strains.

Encapsulating Immunostimulatory CpG Oligonucleotides in Listeriolysin O-Liposomes Promotes a Th1-Type Response and CTL Activity

Immunostimulatory sequences (ISS) are short DNA sequences containing unmethylated CpG dimers that have multiple effects on the host immune system, including the ability to stimulate antigen-specific cytotoxic T lymphocytes (CTLs) and drive Th1-type immune responses. Listeriolysin O (LLO)-containing pH-sensitive liposomes have been shown to efficiently deliver macromolecules to the cytosol of APCs and efficiently stimulate CTLs. We hypothesized that encapsulating ISS-oligodeoxyribonucleotides (ODNs) in this delivery system would enhance the cell-mediated immune response and skew Th1-type responses in protein antigen-based vaccination utilizing LLO-liposomes. In vitro studies indicated that coencapsulation of ISS in LLO-liposomes engendered activation of the NF-κB pathway while maintaining the efficient cytosolic delivery of antigen mediated by the coencapsulated LLO. Antigen-specific CTL responses monitored by using the model antigen ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA and ISS-ODN-containing LLO-liposomes compared with those immunized with OVA-containing LLO-liposomes. The enhanced immune responses were of the Th1-type as monitored by the robust OVA-specific IgG2a induction and the OVA CD8 peptide-stimulated IFN-γ secretion. Our study suggests that including ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type.

Local and Systemic Effects of Intranodally Injected CpG-C Immunostimulatory-Oligodeoxyribonucleotides in Macaques

Immunostimulatory CpG-C oligodeoxyribonucleotides (ISS-ODNs) represent a promising strategy to enhance vaccine efficacy. We have shown that the CpG-C ISS-ODN C274 stimulates macaque blood dendritic cells (DCs) and B cells and augments SIV-specific IFN-γ responses in vitro. To further explore the potential of C274 for future vaccine studies, we assessed the in vivo effects of locally administered C274 (in naive and healthy infected macaques). Costimulatory molecules were marginally increased on DCs and B cells within cells isolated from C274-injected lymph nodes (LNs). However, cells from C274-injected LNs exhibited heightened responsiveness to in vitro culture. This was particularly apparent at the level of CD80 (less so CD86) expression by CD123+ plasmacytoid DCs and was further boosted in the presence of additional C274 in vitro. Notably, cells from C274-injected LNs secreted significantly elevated levels of several cytokines and chemokines upon in vitro culture. This was more pronounced when cells were exposed to additional stimuli in vitro, producing IFN-α, IL-3, IL-6, IL-12, TNF-α, CCL2, CCL3, CCL5, and CXCL8. Following C274 administration in the absence of additional SIV Ag, endogenous IFN-γ secretion was elevated in LN cells of infected animals, but SIV-specific responses were unchanged. Endogenous and SIV-specific responses decreased in blood, before the SIV-specific responses rebounded by 2 wk after C274 treatment. Elevated IFN-α, CCL2, and CCL5 were also detected in the plasma after C274 injection. Thus, locally administered C274 has local and systemic activities, supporting the potential for CpG-C ISS-ODNs to boost immune function to enhance anti-HIV vaccine immunogenicity.