Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice

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Abstract

Background: DNA vaccines have been shown to induce protective immunity against viral infections in different animal models. We have recently demonstrated that DNA vaccine induced protective immunity against influenza A virus and La Crosse virus (LACV) is primarily mediated by humoral immune response. Objective: The goal of this study was to investigate whether administration of DNA coding for cytokines such as interleukin 12 (IL-12) and granulocyte–macrophage colony-stimulating factor (GM-CSF) could increase the protective immune response induced by vaccination with DNA coding for viral antigens. Study design: For the influenza A virus or LACV model, C57BL/6 or interferon-α/β receptor (IFNAR-1)-deficient mice, respectively, were vaccinated once or twice with 100 μg of DNA encoding viral antigens. At the same time plasmid DNAs (100 μg) coding either for mouse GM-CSF or mouse IL-12 were administered. The mice were subsequently challenged with a lethal dose of influenza A virus or LACV and monitored for clinical symptoms (weight loss) and survival. Results: To achieve a high degree of protection (70% survival) two injections of DNA encoding the influenza A virus surface protein hemagglutinin (HA) were required. Intriguingly, administration of DNA coding for IL-12 alone also led to a pronounced protective effect against virus challenge. Co-administration of DNAs encoding IL-12 and HA significantly increased the protective immunity against influenza A virus, while IL-12 expression did not improve protection upon vaccination with DNA coding for the internal nucleocapsid protein N of LACV. Co-injection of DNA coding for mouse GM-CSF and HA also showed an adjuvant effect. Conclusions: The data clearly indicate that co-administration of DNA encoding cytokines such as IL-12 and GM-CSF with DNA coding for viral antigens has adjuvant effects on the protective immune response against different viral pathogens.

Introduction

Direct injection of plasmid DNAs encoding neo-antigens to raise specific immune responses against infectious agents or tumors represents a novel approach to vaccination (for reviews see Hassett and Whitton, 1996, McDonnell and Askari, 1996, Donnelly et al., 1997, Moelling, 1997). DNA based vaccines have been reported to elicit protective immunity against various viral agents, including influenza A virus (Ulmer et al., 1993, Donnelly et al., 1995), human immunodeficiency virus 1 (HIV-1) (Wang et al., 1993, Boyer et al., 1997), rabies virus (Xiang et al., 1994, Xiang et al., 1995), measles virus (Cardoso et al., 1996, Yang et al., 1997), Ebola virus (Xu et al., 1998), Japanese encephalitis virus (Lin et al., 1998) and several members of the herpes virus family (Manickan et al., 1995, McClements et al., 1996, Kuklin et al., 1997). DNA vaccination involves the injection of DNA into tissues that support the in vivo expression of DNA-encoded antigens and their presentation to the host immune system, thereby eliciting humoral as well as cellular responses leading to the production of antibodies and/or cytotoxic T cells (Donnelly et al., 1997). The mode of application of the DNA and the nature of the expressed antigen are factors determining the induction of either a T-helper 1 (Th1) or T-helper 2 (Th2) type immune response (Feltquate et al., 1997). The balance of the Th1 versus the Th2 response can also be influenced by combined application of DNA vaccines with plasmid DNAs encoding cytokines such as granulocyte–macrophage colony-stimulating factor (GM-CSF) or interleukin 12 (IL-12), or co-stimulatory molecules such as B7.2 (Xiang and Ertl, 1995, Iwasaki et al., 1997, Tsuji et al., 1997). The protective quality of the humoral and cellular immune response is dependent on the type of cytokines secreted by activated CD4+ Th cells. Antigen specific Th1 cells secrete IL-2 and interferon-γ (IFN-γ), activate macrophages and stimulate production of antibodies of the IgG2a isotype (reviewed in Mosmann and Coffman, 1989, Abbas et al., 1996). Th2 cells produce mainly IL-4, IL-5 and IL-10, leading to activation of eosinophils and the production of IgE and IgG1 antibodies (Fig. 4). There is growing evidence that induction of a Th1 type response plays a decisive role in the generation of protective immunity against various pathogens such as Leishmania major (Heinzel et al., 1993) and HIV-1 (Kim et al., 1997). A critical factor for promoting the development of Th1 cells is the cytokine IL-12. It is a heterodimeric cytokine produced by B lymphocytes, macrophages and dendritic cells and is characterized by its ability to stimulate cytotoxic activity and IFN-γ production by T cells and natural killer (NK) cells (Kobayashi et al., 1989, Chan et al., 1991, Trinchieri, 1994). GM-CSF represents another cytokine which enhances the immune response against pathogens or tumors. GM-CSF, a hematopoietic growth factor that stimulates proliferation of neutrophils, monocytes and eosinophils, has a recognized activity as a molecular adjuvant to enhance immunity (Metcalf, 1985, Xiang and Ertl, 1995).

Using knock-out mice with specific defects in the immune system, we have recently shown that the protective immunity against influenza A virus by DNA vaccination is primarily mediated by a humoral response directed against the viral surface antigen hemagglutinin (HA) (Pavlovic et al., 1999a). Moreover, taking advantage of mice deficient for a functional interferon-α/β receptor (IFNAR-1) as animal model, we have assessed the feasibility of DNA vaccination against La Crosse virus (LACV), which represents the major arboviral cause of pediatric encephalitis in the USA. Again, protection was observed following vaccination with DNA coding for the viral surface proteins G1 and G2 which was mediated by neutralizing antibodies (Pavlovic et al., 1999b). However, effective protection against both viruses still requires multiple injections of DNA coding for viral antigens. In the present study we therefore evaluated whether the protective immune response against influenza A virus and LACV with DNA encoding viral antigens could be enhanced by co-administration of IL-12 or GM-CSF expressing vectors.

Section snippets

Mice and viruses

C57BL/6 mice and 129Sv mice deficient for the α/β-subunit of IFNAR-1 (Muller et al., 1994) were bred in our animal facility. Influenza A/PR/8/34 virus was grown in embryonated eggs yielding a titer of 4.6×108 50% tissue culture infectious dose (TCID50) per ml. The original strain of La Crosse Virus (LACV) (Thompson et al., 1972) was grown on BHK-21 cells yielding a titer of 2.3×108 TCID50 per ml.

Plasmids

The plasmid pV1J–HA carries the cDNA of the HA segment of influenza A/PR/8/34; its expression is

Results

A plasmid DNA encoding the hemagglutinin (HA) gene of influenza A has been shown to protect mice against challenge with influenza A virus (Montgomery et al., 1993). Protection can be determined by intranasally inoculation of the vaccinated mice with influenza A virus. The vaccination protocol can be varied in many parameters such as time of application, DNA concentration, injection route, etc. Furthermore, combinations with DNA encoding cytokines can be envisaged as an improvement of the

Discussion

In this study we show that the protective immune response in mice against influenza A virus induced by injection of DNA encoding the viral surface antigen HA can be enhanced by co-administration of DNAs encoding IL-12 or GM-CSF. For LACV the adjuvant effects of IL-12 or GM-CSF are less pronounced. Using DNAs coding for viral surface proteins or internal viral nucleocapsid proteins of influenza A virus and LACV we have recently shown that the protective immunity induced by DNA vaccination

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