Shin Sasaki

Genetic tagging shows increased frequency and longevity of antigen-presenting, skin-derived dendritic cells in vivo

Dendritic cells (DCs) are key regulators of immune responses that activate naive antigen-specific T lymphocytes. In draining lymph nodes, antigen-bearing DCs are reported to be rare and short-lived. How such small numbers of short-lived DCs can activate rare antigen-specific T cells is unclear. Here we show that after immunization of mouse skins by gene gun, the number of antigen-bearing DCs that migrate to draining lymph node is 100-fold higher than previously estimated and that they persist for approximately 2 weeks. The substantial frequency and longevity of DCs in situ ensures ample antigen presentation and stimulation for the rare antigen-specific T cells in draining lymph nodes.

Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases.

Apoptotic bodies can be used to target delivery of DNA-expressed immunogens into professional antigen-presenting cells (APCs). Here we show that antigen-laden apoptotic bodies created by vectors co-expressing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes and mutant caspase genes markedly increased T-cell responses. Both CD8 and CD4 T-cell responses were affected. The adjuvant activity was restricted to partially inactivated caspases that allowed immunogen expression before the generation of apoptotic bodies. Active-site mutants of murine caspase 2 and an autocatalytic chimera of murine caspase 2 prodomain and human caspase 3 induced apoptosis that did not interfere with immunogen expression. The adjuvant activity also enhanced B-cell responses, but to a lesser extent than T-cell responses. The large increases in T-cell responses represent one of the strongest effects to date of a DNA adjuvant on cellular immunity.

IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine

Cytokines are powerful regulators of the immune response. In this study, an HIV-1 envelope DNA vaccine and interleukin 15 (IL-15) expression plasmid were intranasally administered to mice. A significant increase in the HIV-1-specific DTH response and CTL activity, and decrease in the serum IgG/IgG2a ratio was observed in the group which received DNA vaccine and IL-15 expression plasmid compared to DNA vaccination alone. Restimulated immune lymphoid cells from mice which received both agents showed enhanced production of interferon-gamma (IFN-gamma) and reduced secretion of IL-4. However, administration of DNA vaccine with IL-15 and IL-2 or IL-12 expression plasmids did not alter the effect of IL-15 expression plasmid on the DNA vaccine. These results indicate that intranasal administration of DNA vaccine and IL-15 expression plasmid is capable of enhancing the T helper type 1 (Th1) dependent HIV-1-specific cell-mediated immunity, and that the IL-15 and IL-2 or IL-12 expression plasmids may not have a synergistic effect on the immune response induced by DNA vaccine in vivo.

Adjuvant formulations and delivery systems for DNA vaccines.

DNA vaccines have become a reliable and major means to elicit immune responses in the past decade. We and others have attempted to obtain stronger, more long lasting, and optimized immune responses, subsequent to the pioneering works demonstrating the ability of plasmid DNA to raise specific immune responses. Advances in molecular biology and biotechnology allow the application of various adjuvants, immunologic agents that increase the antigenic response, in DNA vaccines. Adjuvants can be broadly separated into two classes based on their origin-genetic and conventional. Genetic adjuvants are expression vectors of cytokines or other molecules that can modulate immune responses when administered with a vaccine antigen. Conventional adjuvants are chemical compounds that enhance, prolong, or modulate antigen-specific immune responses. The use of an appropriate adjuvant is pivotal in optimizing the response to DNA vaccines. Moreover, DNA vaccines themselves possess their own adjuvant activity because of the presence of unmethylated CpG motifs in particular base contents. The route of inoculation is also a critical factor in determining the outcome of vaccination. It is well known that intramuscular injection preferentially induces Th1-type immunity, whereas particle bombardment by gene gun predominantly induces Th2-type response. This article focuses on providing the detailed procedure to construct genetic adjuvant plasmids and prepare DNA vaccines formulated with conventional adjuvants. We also offer a practical guide for the procedure of intramuscular DNA injection.

Transcriptional Regulation of the Human TLR9 Gene

To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5′-PU box, 3′-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPα transcription factors. Other members of the C/EBP family, such as C/EBPβ, C/EBPδ, and C/EBPε, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-κB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPα culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system.

Mycobacterium leprae and Leprosy: A Compendium

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which was discovered by G.H.A. Hansen in 1873. M. leprae is an exceptional bacterium because of its long generation time and no growth in artificial media. Entire sequencing of the bacterial genome revealed numerous pseudogenes (inactive reading frames with functional counterparts in M. tuberculosis) which might be responsible for the very limited metabolic activity of M. leprae. The clinical demonstration of the disease is determined by the quality of host immune response. Th1‐type immune response helps to kill the bacteria, but hosts are encroached upon when Th2‐type response is predominant. The bacteria have affinity to the peripheral nerves and are likely to cause neuropathy. M. leprae/laminin‐α2 complexes bind to α/β dystroglycan complexes expressed on the Schwann cell surface. WHO recommends a chemotherapy protocol [multidrug therapy (MDT)] which effectively controls the disease and contributes to the global elimination program. Leprosy has been stigmatized throughout history, and recent topics regarding the disease in Japan are also discussed.

TRAF4 acts as a silencer in TLR-mediated signaling through the association with TRAF6 and TRIF.

Toll‐like receptors (TLR) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play an essential role in intracellular eradication of engulfed pathogens. Here, we demonstrate the physical and functional association between components of the cytosolic NADPH oxidase and TLR‐mediated signaling molecules. Cytosolic components of NADPH oxidase suppressed TLR‐mediated NF‐κB activation as well as IFN‐β promoter activation. We demonstrate that TNF‐associated factor (TRAF) 4 associates with p47phox, a component of cytosolic NADPH oxidase, and physically interacts and functionally counteracts with TRAF6 and Toll‐IL‐1 receptor (TIR) domain‐containing adaptor‐inducing IFN‐β (TRIF) molecules that critically regulate TLR‐mediated signaling. TRAF4 mRNA expression was elicited in RPMI 8226 cells following LPS or CpG DNA treatment. These results suggest that TRAF4 participates in the molecular mechanism underlying silencing of TLR‐mediated signaling through the interaction with molecules harboring phagosome/endosome membrane.

Macrophage inflammatory protein-1α (MIP-1α) expression plasmid enhances DNA vaccine-induced immune response against HIV-1

CD8+ cell‐secreted CC‐chemokines, MIP‐1α, and MIP‐β have recently been identified as factors which suppress HIV. In this study we co‐inoculated MIP‐1α expression plasmid with a DNA vaccine constructed from HIV‐1 pCMV160IIIB and pcREV, and evaluated the effect of the adjuvant on HIV‐specific immune responses following intramuscular and intranasal immunization. The levels of both cytotoxic T lymphocyte (CTL) activity and DTH showed that HIV‐specific cell‐mediated immunity (CMI) was significantly enhanced by co‐inoculation of the MIP‐1α expression plasmid with the DNA vaccine compared with inoculation of the DNA vaccine alone. The HIV‐specific serum IgG1/IgG2a ratio was significantly lowered when the plasmid was co‐inoculated in both intramuscular and intranasal routes, suggesting a strong elicitation of the T helper (Th) 1‐type response. When the MIP‐1α expression plasmid was inoculated intramuscularly with the DNA vaccine, an infiltration of mononuclear cells was observed at the injection site. After intranasal administration, the level of mucosal secretory IgA antibody was markedly enhanced. These findings demonstrate that MIP‐1α expression plasmid inoculated together with DNA vaccine acts as a strong adjuvant for eliciting Th1‐derived immunity.

Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity

ABSTRACT Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.

Intranasal administration of human immunodeficiency virus type-1 (HIV-1) DNA vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against HIV-1

DNA vaccine against human immunodeficiency virus type‐1 (HIV‐1) can induce substantial levels of HIV‐1‐specific humoral and cell‐mediated immunity. To develop more potent HIV‐1 DNA vaccine formulations, we used a murine model to explore the immunomodulatory effects of an interleukin‐2 (IL‐2) expression plasmid on an HIV‐1 DNA vaccine following intranasal administration of the combination. When the vaccine and expression plasmid were incorporated into cationic liposomes and administered to mice, the HIV‐1‐specific delayed‐type hypersensitivity response and cytotoxic T lymphocyte activity were significantly increased. Restimulated immune lymphoid cells showed enhanced production of both IL‐2 and interferon‐γ and reduced secretion of IL‐4. The level of total antibody to HIV‐1 antigen was not greatly changed by coadministration of the DNA vaccine and IL‐2 expression plasmid. An analysis of serum HIV‐1‐specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid–vaccine combination. These results demonstrate that the IL‐2 expression plasmid strongly enhances the HIV‐1‐specific immune response via activation of T helper type‐1 cells.