The virus-induced signaling adaptor molecule enhances DNA-raised immune protection against H5N1 influenza virus infection in mice
Introduction
The influenza A virus is able to cause severe morbidity and mortality to susceptible humans, making it one of the most deadly infectious pathogens. In 1997, a highly pathogenic avian H5N1 influenza virus was first reported to be transmitted from poultry to humans in Hong Kong, resulting in 18 infected people and six deaths [1]. During the period from 2003 to December 2010, 510 confirmed cases of human infection with H5N1 influenza viruses have been reported around the world, resulting in 303 deaths (http://www.who.int/csr/disease/avian_influenza). Although there have only been a few reports of direct human-to-human H5N1 transmission to date, H5N1 could potentially overcome this obstacle by reassortment with co-circulating human H1N1 or H3N2 influenza viruses [2], which may lead to the emergence of a pandemic influenza virus that could spread rapidly in the human population [3], [4].
Vaccination is currently the most effective method to reduce influenza virus transmission, as well as the associated socio-economic burden [5], [6]. The main forms of licensed human influenza virus vaccines are the traditional trivalent inactivated influenza vaccines, which are comprised of antigens from three circulating virus strains, currently including H1N1, H3N2 and influenza B. However, the difficulties associated with the egg-based production of traditional avian influenza vaccines, including the low yield of candidate vaccine viruses in chicken embryos, the necessity for biosafety level 3 containment facilities, and the poor immunogenicity of H5 hemagglutinin (HA) [7], have encouraged the development of new H5N1 vaccine strategies. As novel vaccine candidates, DNA vaccines have been proven to induce effective antibody response and long-term cell-mediated immunity in animal models [8], [9], [10], [11], [12], [13]. DNA vaccines are also stable, inexpensive and relatively simple to prepare. However, the magnitude of the immune response elicited by DNA vaccines in larger animals, including humans, has been disappointing [14]. Considerable effort has been made to improve the efficacy of DNA vaccines, including optimizing the plasmid delivery system [15], [16], improving antigen uptake and/or presentation [17], [18], combining DNA vaccines with different prime–boost vaccination strategies [19], and co-delivering cytokines to up-regulate the immune responses to the antigens [20], [21], [22], [23], [24], [25].
Viral infection results in induction of type I IFNs, including IFN-β and IFN-α family cytokines [26], [27], [28]. IFN-α/β play a key role in mediating both the induction of the innate immune response and the subsequent development of adaptive immunity to viruses [29], [30], [31]. At least two distinct mechanisms have been proposed for the production of IFN-α/β by virus-associated molecules [29], [30]. One mechanism is mediated by TLRs, such as TLR3, which recognizes viral dsRNA released by infected cells [32]. Viral dsRNA binds to TLR3 and triggers TRIF-mediated signaling pathways, leading to IRF-3 and NF-κB activation [33], [34], [35]. Another mechanism involves retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which function as cytoplasmic viral RNA sensors [36], [37]. It has been shown that RIG-I-mediated signaling is independent of TLR3 and TRIF [36]. In the RIG-I pathway, the virus-induced signaling adaptor molecule (VISA, also known as MAVS, IPS-1, or Cardif) plays an essential role in the activation of antiviral signaling [38], [39], [40], [41]. As an adaptor molecule in the RIG-I pathway, VISA is required for the activation of NF-κB and IRF-3 (interferon regulatory factor-3), and its activation therefore leads to the production of type I IFNs. VISA, which localizes to the mitochondria, triggers downstream signaling and is a critical link between mitochondria and innate antiviral immunity [41]. The overexpression of signaling molecules can be used to mimic infection by microbes, thereby eliciting the innate immune responses required for subsequent activation of adaptive immune responses against antigens. It has been found that incorporating the TLR adaptor molecule TRIF into a DNA vaccine triggers TLR signaling and thereby augments DNA-raised adaptive immune responses against the virus [42], [43], [44]. DNA vaccine immunogenicity is also enhanced by co-transfection of IRFs [45]. Adjuvanting a DNA vaccine with a TLR9 ligand together with Flt3 ligand results in enhanced cellular immunity against the corresponding virus [46]. In the present study, we used a mouse model to investigate the potential use of VISA as an adjuvant to augment DNA-raised immune responses against H5N1 and compared this with the TRIF genetic adjuvant.
Section snippets
Mice and viruses
Six-week-old female BALB/c mice were used for immunization and challenge experiments. All mice were maintained with free access to sterile food and water.
The avian H5N1 influenza virus isolate A/chicken/Hubei/489/2004(H5N1) (A/chicken/Hubei/489) and a reassortant influenza virus (A/Viet Nam/1194R) with HA and NA genes of human-isolated influenza virus (A/Viet Nam/1194/2004 (H5N1)) and the internal protein genes of A/Puerto Rico/8/1934 were used in this study. A/Viet Nam/1194R was kindly
VISA activated NF-κB and IFN-β promoters
To investigate whether VISA can be used as a potential genetic adjuvant for DNA vaccines, we tested its ability to activate the NF-κB and IFN-β promoters in Vero cells. The Toll-like receptor adaptor molecule TRIF, a known genetic adjuvant [42], [43] was used for comparison. The pNF-κB-Luc and pIFN-β-Luc reporter constructs were used to measure the activity of the NF-κB and IFN-β promoters, respectively. Our data demonstrate that the overexpression of VISA activated both the NF-κB and IFN-β
Discussion
H5N1 influenza virus continues to pose a public health threat to humans and animals. Consequently, there is an urgent need for effective vaccines against this emerging infectious disease. DNA vaccines hold considerable potential for the prevention and treatment of viral diseases, and studies to assess this strategy for protection against influenza virus have used both envelope and internal viral proteins [8], [12], [18], [48], [51], [57], [58], [59]. However, the early promise of DNA vaccines
Acknowledgments
This work was supported by the National High Technology Research and Development Program (2006AA02Z463) and the National Basic Research Program (2010CB911803) of China.
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These authors contributed equally to this work.