John Van Uden

IFN-αβ Promote Priming of Antigen-Specific CD8+ and CD4+ T Lymphocytes by Immunostimulatory DNA-Based Vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-αβ and -γ, TNF-α, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8+ and CD4+ lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-αβ pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8+ CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4+ Th cells by ISS-based vaccines.

Immunostimulatory DNA-Based Vaccines Elicit Multifaceted Immune Responses Against HIV at Systemic and Mucosal Sites

Immunostimulatory DNA sequences (ISS, also known as CpG motifs) are pathogen-associated molecular patterns that are potent stimulators of innate immunity. We tested the ability of ISS to act as an immunostimulatory pathogen-associated molecular pattern in a model HIV vaccine using gp120 envelope protein as the Ag. Mice immunized with gp120 and ISS, or a gp120:ISS conjugate, developed gp120-specific immune responses which included: 1) Ab production; 2) a Th1-biased cytokine response; 3) the secretion of β-chemokines, which are known to inhibit the use of the CCR5 coreceptor by HIV; 4) CTL activity; 5) mucosal immune responses; and 6) CD8 T cell responses that were independent of CD4 T cell help. Based on these results, ISS-based immunization holds promise for the development of an effective preventive and therapeutic HIV vaccine.

DNA-based immunotherapeutics for the treatment of allergic disease.

Summary: Allergic diseases are a growing health concern in industrialized countries. Despite a number of effective therapeutic options for the prevention and treatment of the pathophysiologic responses which characterize allergic diseases, the induction of true allergen desensitization remains an elusive therapeutic goal. Only immunotherapy (IT) has been shown to have any effect on the underlying hypersensitivities which mediate allergic reactions, and traditional protein‐based allergen IT has a limited scope of efficacy. However, a number of reagents collectively termed DNA‐based immunotherapeutics have proven highly effective in both the prevention and reversal of Th2‐mediated hypersensitivity states in mouse models of allergic disease. Four basic DNA‐based immunotherapeutic modalities have been used for these studies. These include immunization with gene vaccines, allergen mixed with immunostimulatory oligodeoxynucleotide (ISS‐ODN), and physical allergen–ISS‐ODN conjugates (AIC), as well as immunomodulation with ISS‐ODN alone. Results from many laboratories have generated guarded optimism that DNA‐based immunotherapeutics may be effective for the reversal of allergic hypersensitivity states in humans, and several clinical trials have already been initiated. This review will focus on our present understanding of the biological activities of DNA‐based immunotherapeutics and their application to the treatment of allergic diseases.

Enhancement of Innate Immunity against Mycobacterium avium Infection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase

ABSTRACT Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course ofMycobacterium avium infection. M. aviumgrowth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, and M. avium growth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-α/β), and IFN-γ. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.

Immunostimulatory DNA and applications to allergic disease

The vertebrate immune system reacts to certain sequences of DNA with a strong T(H)1-inducing innate response. These sequences, termed immunostimulatory DNA sequences, are not fully defined but generally consist of a central nonmethylated CG dinucleotide, flanked by less highly conserved sequences (hence the alternate name CpG motifs). These sequences seem to be rare in vertebrates but relatively common in many lower organisms, including bacteria and viruses. It is likely that these sequences represent a danger signal to the immune system; a powerful T(H)1 response is induced against colocalized foreign antigen. This can be used to modify an allergic response away from a pathogenic T(H)2-dominated immune profile toward a nonpathogenic and even protective T(H)1 profile.

Type I interferon is required to mount an adaptive response to immunostimulatory DNA

Immunostimulatory DNA sequences (ISS, CpG motifs) potently stimulate Th1 and cytotoxic T lymphocyte (CTL) responses to antigens and have thus generated considerable interest due to their potential use in immunotherapeutics. An array of cytokines are produced in response to ISS exposure, but the relative importance of each of these mediators in the stimulation of innate and adaptive ISS‐induced immunity has yet to be fully investigated. To address this issue, we measured immune responses in mice with targeted deletions of the ISS‐induced genes encoding IL‐12 (IL‐12–/–), IFN‐γ (IFN‐γ–/–), the IFN‐γ receptor (IFN‐γR–/–), and the IFN‐α/β receptor (IFN‐α/βR–/–) after immunization with ISS‐containing oligodeoxynucleotides and model antigens. IL‐12–/– and IFN‐α/βR–/– mice were compromised in their ability to develop a cross‐primed CTL response, whereas IFN‐γ–/– and IFN‐γR–/– mice were not. In addition, lymphocytes from immunized IFN‐α/βR–/– mice had defective IFN‐γ responses to antigen restimulation. Antigen nonspecific ISS‐induced B cell proliferation was normal in the four deficient strains; however, innate IL‐6 production was reduced in IFN‐γ–/– and IFN‐γR–/– splenocytes and eliminated in IFN‐α/βR–/– cells. While IL‐12 production was defective in only the IFN‐γ–/– splenocytes, innate natural killer cell IFN‐γ synthesis was virtually absent in the IL‐12–/– and IFN‐α/βR–/– mice. Thus, while IFN‐α/β, IFN‐γ, and IL‐12 each play important and distinct roles in the development of the innate and adaptive immune responses to ISS, IFN‐α/β is a particularly crucial and currently under‐appreciated factor in this system.

A protective role of the low density lipoprotein receptor-related protein against amyloid beta-protein toxicity.

In order to delineate the neuroprotective role of the low density lipoprotein receptor-related protein (LRP) against amyloid β-protein toxicity, studies were performed in C6 cells challenged with amyloid β-protein in the presence or absence of activated α2-macroglobulin. Toxicity was assessed via two cell viability assays. We found that this endocytic receptor conferred protection against amyloid β-protein toxicity in the presence of activated α2-macroglobulin and its down-regulation via inhibition by receptor-associated protein or transfection of cells with presenilin 1, increased susceptibility to amyloid β-protein toxicity. Increased surface LRP immunoreactivity in response to amyloid β-protein challenge was associated with increased translocation of LRP from the endoplasmic reticulum to the surface, rather than from increased mRNA or protein expression. Furthermore, this translocation of LRP to the surface was mediated by a calcium/calmodulin protein kinase II-dependent signaling pathway. These studies provide evidence for a protective role of LRP against amyloid β-protein toxicity and may explain the aggressive nature of presenilin-1 mutation in familial Alzheimers disease.

DNA Immunotherapeutics: New Potential Treatment Modalities for Allergic Disease

Genetic immunization is a relatively new approach to vaccination, one that has generated considerable interest for its potential to prevent or treat a number of types of disease processes. Although initial work focused on potential applications in infectious disease, it was soon recognized that the antigen–specific Th1 response typically generated by genetic immunization protocols could also be useful for the treatment of allergic disease. In this review we present a summary of genetic vaccination and the related topic of immunostimulatory DNA sequences – which we collectively designate DNA immunotherapeutics – and discuss their potential for the prevention and therapy of allergic disease.

Immunostimulatory DNA Sequences

The biochemical and genetic properties of DNA have been thoroughly investigated, yet only recently has it been appreciated that DNA carries more information than simply a blueprint for the regulation and construction of proteins. Indeed, the immune systems of vertebrates appear to have evolved the ability to distinguish the foreign DNA of bacteria and certain viruses from the self-DNA of the host, a new twist on the self vs non-self detection system already well-known for foreign proteins. Specifically, the frequency of unmethylated CpG motifs (CpG denotes covalently linked CG dinucleotides, not C∶G base pairs) is extensively suppressed in vertebrates, including mammals (by at least 20-fold [1]), whereas it is found at the usual frequency (1/16) in most bacterial and viral DNA. There have now been several reports (detailed in Subheadings 2. and 4. ) that bacterial DNA or synthetic oligodeoxyribo-nucleotides (ODNs) containing bacterially derived sequences, stimulate the immune systems of mice and humans to first mount innate, and then antigen-specific (when foreign antigen is present), Th(1)-type responses. This adjuvant effect of bacterial immunostimulatory DNA sequences (ISS) appears to be important for the robust Th(1)-type immune response usually seen in genetic vaccination (2). Although the terms CpG motif and ISS are generally used synonymously in this field, CpG motifs are defined structurally, whereas ISS are defined functionally (and therefore include non-CpG sequences that have been found to be stimulatory).