Kenji Hamajima

Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene.

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

Adjuvant effect of multi-CpG motifs on an HIV-1 DNA vaccine

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs trigger an immune response characterized by the activation of B cells, NK cells and monocytes/macrophages. Based on evidence that the immunogenicity of DNA vaccines can be augmented by the addition of CpG motifs, 5-20 additional CpG motifs were cloned into a pUC-derived plasmid. Treating bone-marrow derived dendritic cells (BM-DCs) with CpG-enriched plasmids in vitro boosted their expressions of MHC class II molecules, the CD40 and CD86 activation markers. Co-administering the CpG-enriched plasmids with a DNA vaccine encoding the envelope glycoprotein of HIV to BALB/c mice significantly increased HIV-specific cell mediated and humoral immunity. A significant boost was observed when the CpG plasmid was administered either 2 or 4 days after DNA vaccination. Plasmids containing 20 CpG copies were the most effective immune enhancers both in vitro and in vivo. These results suggest that plasmids containing multiple CpG motifs may improve the immunogenicity of DNA vaccines.

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.

HIV-1-specific cell-mediated immune responses induced by DNA vaccination were enhanced by mannan-coated liposomes and inhibited by anti-interferon-gamma antibody.

The adjuvant effect of mannan‐coated liposomes on human immunodeficiency virus type‐1 (HIV‐1) DNA vaccine and the mechanism of this enhancement were studied. Coating of cationic liposomes with mannan significantly enhanced the ability of this vaccine to induce an HIV‐specific delayed‐type hypersensitivity (DTH) response. HIV‐specific cytotoxic T‐cell (CTL) activity elicited by DNA vaccination was also significantly enhanced with the mannan‐liposome cocktail. This mannan‐liposome‐mediated activity was greatly inhibited by in vivo injection of anti‐interferon (IFN)‐γ antibody, which suggests that IFN‐γ plays an important role in this HIV‐specific immune response. The results of both isotype‐specific antibody and cytokine analysis revealed that mannan‐liposome‐mediated DNA vaccination enhances Th1‐mediated immunity.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Protection against influenza virus challenge by topical application of influenza DNA vaccine.

We studied the use of a DNA vaccine expressing the matrix (M) gene of the influenza virus A/PR/8/34. Mice were immunized by painting the DNA vaccine three times on the skin after removal of its keratinocytic layers. Immunization by this method produced M-specific antibodies and cytotoxic T lymphocyte (CTL) response, and acquired resistance against influenza virus challenge. This protection was abrogated by the in vivo injection of anti-CD8 or anti-CD4 monoclonal antibody. We further found that simultaneous topical application (t.a.) of GM-CSF expression plasmid (pGM-CSF) or liposomes plus mannan produced stronger immune response competence and enhanced the protective effect against influenza virus challenge. The present study revealed that administering DNA vaccine by topical application can elicit both humoral and cell-mediated immunity (CMI).

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.

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.

Oral Administration of Recombinant Adeno-Associated Virus Elicits Human Immunodeficiency Virus-Specific Immune Responses

Oral vaccines can induce both systemic and mucosal immunity. Mucosal immunity, especially regional cell-mediated immunity, plays an important role in protecting individuals from infectious diseases such as acquired immunodeficiency syndrome. In this study, a recombinant adeno-associated virus vector expressing human immunodeficiency virus type 1 env gene (AAV-HIV) was orally administered to BALB/c mice. Systemic and regional immunity was induced in the mice. Furthermore, the immunization significantly reduced viral load after an intrarectal challenge with a recombinant vaccinia virus expressing HIV env gene. Moreover, we also show that dendritic cells might contribute to the AAV-HIV vector-induced immune responses.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.