ResearchDevelopment of avian influenza virus H 5 DNA vaccine and MDP-1 gene of Mycobacterium bovis as genetic adjuvant

Background: Studies have shown that DNA vaccines can induce protective immunity, which demonstrated the high potential of DNA vaccines as an alternative to inactivated vaccines. Vaccines are frequently formulated with adjuvants to improve their release, delivery and presentation to the host immune system. Methods: The H5 gene of H5N1 virus (A/Ck/Malaysia/5858/04) was cloned separately into pcDNA3.1 + vector. The immunogenicity of the cloned H5 DNA vaccine was tested on SPF chickens using two different approaches. First approach was using H5 DNA vaccine (pcDNA3.1/H5) and the second was using H5 DNA vaccine in addition to the pcDNA3.1/MDP1 vaccine. Ten days old chickens inoculated three times with two weeks intervals. The spleen and muscle samples from chickens immunized with H5 (pcDNA3.1/H5) and H5 + MDP1 (pcDNA3.1/H5 + pcDNA3.1/MDP1) vaccines were collected after sacrificing the chickens and successfully expressed H5 and MDP1 RNA transcripts. The sera of immunized chickens were collected prior to first immunization and every week after immunization; and analyzed using enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. Results: Results of competitive ELISA showed successful antibody responses two weeks post immunization. The HI test showed an increased in antibody titers during the course of experiment in group immunized with H5 and H5 + MDP1 vaccines. The result showed that the constructed DNA vaccines were able to produce detectable antibody titer in which the group immunized with H5 + MDP1 vaccine produced higher antibody comparing to H5 vaccine alone. Conclusions: This study shows for the first time the usefulness of MDP1 as a genetic adjuvant for H5 DNA vaccine. Background Influenza virus can cause an acute, highly transmittable respiratory disease, which can result in high morbidity and mortality in both human and animals [1]. The 1997 Hong Kong outbreak of highly pathogenic avian influenza virus (HPAI)-H5N1 showed that avian influenza is a potential threat to human and is believed to be transmitted from infected birds [2]. The Hong Kong outbreak of avian influenza H5N1 was controlled by slaughtering 1.5 million chickens, which cost more than 245 million dollars in a single month. Therefore, antivirals and vaccines seem to be a more prospective solution to control the outbreaks of avian influenza virus [2]. Currently, whole virus inactivated vaccines containing HA as the main component, are the common vaccines to prevent avian influenza. However, these vaccines require large numbers of specific-pathogen-free embryonated chicken eggs and about 6 months to propagate the viruses [2]. On the other hand, this is not an ideal method to produce inactivated vaccine for highly pathogenic strains, as the embryos are killed shortly after propagation and require a high level of biosecurity to handle [3]. Commercial vaccines have been successful in producing protective immunity against infections by homologous virus but failed in preventing the outbreaks of heterologous virus and occasionally been reported as a possible cause of reemerging outbreaks [2]. The commercially available vac* Correspondence: aro@ibs.upm.edu.my 1 Institute of Bioscience, University Putra Malaysia, Serdang 43400, Selangor, Malaysia Full list of author information is available at the end of the article © 2010 Jalilian et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jalilian et al. Genetic Vaccines and Therapy 2010, 8:4 http://www.gvt-journal.com/content/8/1/4 Page 2 of 9 cines against H5N1 are inactivated whole virus vaccine and fowlpox virus vaccine expressing the H5 gene [4]. Moreover, various recombinant vaccines against avian influenza H5N1 virus which are able to induce different levels of protective immunity, such as DNA plasmidbased vaccine, baculovirus recombinant H5 vaccine, and reverse genetic H5 vaccine have been examined experimentally [5-7]. Concurrent studies have revealed that DNA vaccines encoding HA of influenza A virus can result in the development of protective immune response against influenza virus challenge in animals [8,9]. In most cases, two or three doses of naked plasmid DNA are required to induce immune response to the pathogen [10,11]. Nevertheless, other studies have shown that a single dose of DNA vaccine can trigger protective immunity, which demonstrated the high potential of DNA vaccines as an alternative to inactivated vaccines [12,13]. Recently, we have showed that the fusion of ESAT-6 of Mycobacterium tuberculosis to H5 DNA vaccine are able to improve the antibody titer of chickens against AIV showing the flexibility of modifying the efficacy of DNA vaccine [14]. Mycobacterial DNA binding protein 1 (MDP1) is a main cellular protein produced by Mycobacterium bovis. The protein has both nucleic acid binding activity and macro-molecular bio-synthesis inhibitory properties that play key role in modulating bacterial growth [15]. Prabhakar et al., in 1998, revealed that DNA binding proteins (orthologus with MDP1) may act as an immunodominant antigen which stimulates cellular and humoral responses presumably through TLR9 dependent pathway production of proinflammatory cytokines [16,17] and the induction of IFN-γ production [18,19]. Hence, MDP1 may play an important role as a potential adjuvant to boost the immunotherapeutic effects of DNA vaccines.