Helen Tighe

Immunostimulatory DNA Sequences Necessary for Effective Intradermal Gene Immunization

Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-α, interferon-β, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

Gene vaccination: plasmid DNA is more than just a blueprint

Abstract Despite the popularity of using plasmid DNA for vaccination, it is only recently that the basic mechanisms that drive the immune response to the encoded antigen have begun to unfold. Here, Helen Tighe and colleagues outline the characteristics of the immune response induced by gene vaccination and describe the multifaceted properties of DNA in initiating and determining the process.

Inhibition of IgE antibody formation by plasmid DNA immunization is mediated by both CD4+ and CD8+ T cells.

BACKGROUND We previously showed that immunization of mice with plasmid DNA (pDNA) encoding the Escherichia coli beta-galactosidase gene (pCMV-LacZ) induces a Th1 response, whereas beta-galactosidase (beta-gal) in saline or alum induces a Th2 response. Furthermore, the Th1 response dominates over the Th2 response and downregulates preexisiting IgE antibody formation. Here, we determined by passive transfer of CD4+ or CD8+ lymphocytes and by immunizing beta2-microglobulin knockout (beta2-M KO) mice whether CD4+ and/or CD8+ cells from pDNA-immunized mice suppress IgE antibody production. METHODS BALB/c mice were injected with either CD4+ or CD8+ lymphocytes from naive beta-gal-in-alum or pCMV-LacZ-immunized mice, then immunized with beta-gal in alum, and the IgE antibody formation was determined. Second, C57BL/6 wild-type (WT) or beta2-M KO mice were immunized with beta-gal orpCMV-LacZ, and the IgE antibody production was assessed. RESULTS Passive transfer of both CD4+ and CD8+ lymphocytes from pDNA-immunized mice suppressed the IgE antibody response by 90% compared to transfer of CD4+ T cells from naive or beta-galin-alum immunized mice. beta2-M KO mice produced 3 times more IgE than the WT control mice both in the primary and secondary response. CONCLUSION Both CD4+ and CD8+ subsets of T cells from pDNA-immunized mice can suppress IgE antibody production by affecting the primary response and/or by propagating the Th1 memory response in a passive cell transfer system. Immunization with pDNA-encoding allergens may be an effective new form of immunotherapy for atopic diseases.

Inhibition of allergic inflammation in the lung by plasmid DNA allergen immunization.

The nature of the immune response (Th1/Th2) in mice to protein antigens or allergens was compared to that of immunization with pDNA encoding the same antigens. pDNA immunization induced a Th1 response and no IgE antibodies whereas the proteins induced a Th2 response and IgE antibodies. Furthermore, the pDNA induced Th1 response dominated over the protein elicited Th2 response in a secondary immune response. In particular, a preexisting Th2 response (as is the case in allergic patients) did not prevent a new Th1 response to an allergen-pDNA booster injection. The major reason why pDNA immunization induced a Th1 response to allergens was the presence of immunostimulatory non-coding DNA sequences (ISS) in the plasmid constructs having a CpG motif. These ISS caused antigen presenting cells to secrete INF-alpha, INF-beta and IL-12, all cytokines that induce naive T cells to differentiate into CD4+ Th1 cells and CD8+ Tc1 cells. Passive transfer of both Th1 and Tc1 cells from pDNA immunized mice into naive mice inhibited a Th2 response and IgE antibody formation to a subsequent injection of allergen in alum. pDNA immunization or ISS-oligonucleotide injection prior to allergen challenge reduced both immediate type airway sensitivity and late phase allergen induced eosinophil filtration of the lung. Allergen-pDNA immunization may provide a novel type of immunotherapy for the treatment of allergic diseases in man. Since only small amounts of allergen are secreted by the allergen-pDNA transformed cells, allergen-pDNA immunotherapy will unlikely carry the risk of the anaphylactic reactions that are associated with classical allergen injection immunotherapy.

Transgenic Expression of a Human Polyreactive Ig Expressed in Chronic Lymphocytic Leukemia Generates Memory-Type B Cells That Respond to Nonspecific Immune Activation

We generated transgenic mice, designated SMI, expressing unmutated H and L chain Ig genes encoding a low-affinity, polyreactive human (h)IgM/κ rheumatoid factor. These animals were compared with control AB29 transgenic mice expressing a hIgM/κ rheumatoid factor specific for human IgG, with no detectable reactivity with mouse proteins. SMI B cells expressed significantly lower levels of surface hIgM/κ than did the B cells of AB29 mice, but still could be induced to proliferate by surface Ig cross-linking in vitro and could be deleted with anti-Id mAb in vivo. Transgene-expressing B cells of AB29 mice had a B-2 phenotype and were located in the primary follicle. In contrast, a relatively high proportion of hIgM-expressing B cells of SMI mice had the phenotype of B-1 B cells in the peritoneum or marginal zone B cells in the spleen, where they were located in the periarteriolar sheath, marginal zone, and interfollicular areas that typically are populated by memory-type B cells. Although the relative proportions of transgene-expressing B cells in both types of transgenic mice declined with aging, SMI mice experienced progressive increases in the serum levels of IgM transgene protein over time. Finally, SMI transgene-expressing B cells, but not AB29 transgene-expressing B cells, were induced to secrete Ab when cultured with alloreactive T cells. These results indicate that expression of polyreactive autoantibodies can allow for development of B cells that are neither deleted nor rendered anergic, but instead have a phenotype of memory-type or Ag-experienced B cells that respond to nonspecific immune activation.

Plasmid DNA vaccination: mechanism of antigen presentation

Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived immune responses to the antigen encoded by the gene vaccine. While the intradermal route of administration appears to be the most efficient, there is evidence that either route leads to production of antibody and the activation of major histocompatibility complex (MHC) class I-restricted, antigen-specific cytotoxic T lymphocytes (CTL) and MHC class II-restricted CD4+ T cells secreting Thl-type cytokines such as interferon-γ (IFN-γ) [1–9]. Plasmid DNA immunization has potential advantages compared to traditional protein vaccination due to the strong CTL and Thl responses induced, the prolonged antigen expression, and the resistance of the antigen source to antibody-mediated clearance. As a consequence, gene vaccination has potential applications in the fields of infectious diseases, allergy and cancer.

Protein-Immunostimulatory DNA-Conjugate

The original finding made by Tokunaga and colleagues (1,2) demonstrated that DNA from Mycobacterium bovis Bacillus Calmette Guerin has antitumor activities and induces natural killer (NK) cells to secrete interferon-γ (IFN-γ). Over the last 10 yr, this finding has stimulated much research on the immunostimulatory properties of bacterial DNA. It was shown that bacterial immunostimulatory DNA sequences (ISS) consist of palindromic sequences of the motif purine-purine-CpG-pyrimidine-pyrimidine (3,4). This motif occurs about 20 times more often in bacterial DNA than in mammalian DNA (5). Furthermore, the cytosine of the crucial CpG sequence in ISS is usually methylated in mammalian DNA and methylated CpG motifs do not show the immuno-stimulatory properties of ISS (6). ISS not only activate NK cells to secrete IFN-γ, but also activate both murine and human antigen presenting cells (APC) to secrete type 1 cytokines such as, IFN-a, β, IL-6, IL-12, and IL-18 (4,7,8), which direct the immune response toward a Th 1 immune response. ISS also induce costimulatory molecules on APC (9) and cause human B-cell proliferation and IgM secretion (10). Another property of ISS is the ability to induce “cross-priming,” the phenomenon of priming CD8+ cytotoxic lymphocytes (CTL) against foreign protein antigens (11,12).