Protection against influenza virus challenge by topical application of influenza DNA vaccine
Introduction
Ulmer et al. and Fynan et al. confirmed the protective effect of influenza DNA vaccine administered to mice by intramuscular (i.m.) injection [1], [2], [3], [4], [5], [6], [7]. Until now, influenza vaccines have been produced with proteins using viral surface antigens such as hemagglutinin (HA) [4], [8], [9] or neuraminidase (NA). The fact that protective immunity against influenza virus appears to be primarily obtained through serum antibody, and the frequent mutation of the HA gene necessitates fresh preparation of the vaccine every year, has been a practical problem. There has consequently been a great desire for the development of a vaccine that uses a conserve region. Ulmer et al. made important progress with both the finding that DNA vaccine enhances cell-mediated immunity (CMI) and thus improves the efficacy of immunization, and the successful creation of a vaccine candidate with nucleoprotein (NP) using a region showing little mutation [5], [6]. The M gene, segment 7 of influenza virus gene, used in the present study was selected as a target because it shows even less mutation than NP, and has a lower molecular weight. It has been discovered that DNA vaccine activates cytotoxic T lymphocytes (CTL) response with a slight elevation of antibody levels [4], [5], [6], [10], [11], [12]. There are also reports that the immunization with matrix (M1) and membrane (M2) antigenic peptides induces protective immunity against influenza virus challenge [13]. In addition, intranasal (i.n.) administration of DNA vaccine induced strong humoral and cell-mediated immunities [14]. We have also reported that injecting DNA vaccine together with a cytokine adjuvant such as IL-12 and GM-CSF enhances the immune responses of Th1 and Th2, respectively [14]. In addition, recently we have been studying the adjuvant activity of mannan-coated liposomes [15], since the surfaces of many infectious agents, such as viruses [16], bacteria, yeast and protozoa [17], are covered with carbohydrate moieties including those rich in mannose, and a number of immune systems use these carbohydrates as activators.
In the present study, we investigated the efficacy of percutaneous immunization with DNA vaccines encoding M or in some case NP gene of influenza antigens against various strains of influenza. We first measured the immunogenicity of the M gene plasmids, and then determined the efficacy against challenge with the influenza virus. It is important for an influenza vaccine to be easily and widely useful for the vaccination of elderly patients and infants. If DNA vaccine can be administered via the skin, we believe that such a method of application can be used with vaccines for other diseases.
Section snippets
Experimental animals
Male BALB/c mice (6–8 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan) and were maintained with free access to sterile food and water in the animal facility of Yokohama City University School of Medicine.
Viral protein expression plasmid and antibodies
pME18S-M expression plasmid was constructed with pME18S expression vector [18], [19], [20] and influenza DNA. The M region cDNA from influenza strain A/PR/8/34 was inserted into the pME18S vector under control of cytomegalovirus immediate-early promotor as described previously
Histology of the skin
At first we studied the histology of the skin after the skin treatment using fast active adhesive glue and a glass slide. As shown in Fig. 1, the loss of keratinocytic layer by the application of fast active adhesive glue on the skin was apparent.
Comparison of immunogenicity by various routes of immunization
To determine the optimal dose of pME18S-M required to induce high titers of influenza M-specific IgG antibody following topical application, 5, 20 and 100 μg DNA vaccine was applied to the skin of the young mice. Before each treatment the keratinocytic
Discussion
The classical or genetic vaccination methods that use needles or various infectious vector systems suffer from the problems including cost of administration and patient compliance. In this study we demonstrated that painting of the DNA vaccine on animal skin induced both cellular and humoral immune responses against influenza M gene products. We previously reported several delivery systems of DNA vaccine, including nasal administration, for inducing an immune response against HIV protein [23],
Acknowledgments
We would like to thank T. Takeishi and A. de la Fuente for their technical and secretarial assistance. We also thank C.W.P. Reynolds for his editorial assistance. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and by a Grant from the Japan Health Sciences Foundation (K-1027).
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