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).

Preparation and enzymatic recognition of guanine lesions induced by nitrogen oxides.

Xanthine (Xan) and oxanine (Oxa) are the major deamination products of guanine formed by the treatment with nitrogen oxides (e.g., NO and HNO2). In this study, 2'-deoxyribonucleoside 5'-triphosphates of Xan and Oxa were prepared by the NaNO2 treatment of dGTP. These modified nucleotides were incorporated into oligonucleotides by DNA polymerase reactions. The repair activities of various DNA N-glycosylases for Xan and Oxa were examined using these substrates.

Enzymatic repair of 5-formyluracil. I. Excision of 5-formyluracil site-specifically incorporated into oligonucleotide substrates by alka protein (Escherichia coli 3-methyladenine DNA glycosylase II).

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. (1)H and (13)C NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

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-gamma 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.