DNA vaccine constructs against enterovirus 71 elicit immune response in mice

Genetic Vaccines and Therapy BioMed Central Research Open Access DNA vaccine constructs against enterovirus 71 elicit immune response in mice Wong Siew Tung1, Sazaly Abu Bakar2, Zamberi Sekawi3 and Rozita Rosli*1 Address: 1Dept. of Human Growth and Development, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 2Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya Medical Center, 50603 Kuala Lumpur, Malaysia and 3Dept of Clinical Laboratory Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Email: Wong Siew Tung - ; Sazaly Abu Bakar - ; Zamberi Sekawi - ; Rozita Rosli* - * Corresponding author Published: 19 April 2007 Genetic Vaccines and Therapy 2007, 5:6 doi:10.1186/1479-0556-5-6 Received: 5 September 2006 Accepted: 19 April 2007 This article is available from: http://www.gvt-journal.com/content/5/1/6 © 2007 Tung 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. Abstract Background: Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo. Methods: The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71. Results: The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71. Conclusion: The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine. Page 1 of 13 (page number not for citation purposes) Genetic Vaccines and Therapy 2007, 5:6 1. Background Enterovirus 71 (EV71) belongs to the genus of enteroviruses from the family Picornaviridae. It possesses a single stranded RNA genome of approximately 7500 nucleotides of positive polarity, which is encapsulated in a capsid containing 60 copies of each of the four structural proteins, VP1 through VP4 [1]. The antigenic diversity among the enteroviruses is caused by variations within capsid proteins VP1 to VP3, but neutralization epitopes are most densely clustered on VP1 [2]. Enterovirus 71, along with coxsackievirus A16 (CA16), is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. EV71 is thought to spread by contact with fecal contaminated materials. Infection by the virus is often asymptomatic or may manifest as mild self-limiting illness which is often characterized by the presence of characteristic lesions on the palms, soles and oral mucosa. EV71 and CA16 are genetically closely related. However, unlike CA16 that is more limited in its pathogenicity to HFMD, EV71 is also associated with severe complications involving the central nervous system (CNS) such as aseptic meningitis, encephalitis and poliomyelitis-like paralysis [3,4]. Since the first report of EV71 infection which occurred in California in 1969 [5], world-wide reports of outbreaks have followed. The neurovirulence of EV71 first came to attention in 1975 in Bulgaria when 44 people died of a polio-like disease [6]. Epidemics of EV71 causing CNS diseases subsequently occurred in New York, Australia, Europe and Asia [7-10]. An unusual epidemic of HFMD complicated by fatal myocarditis and pulmonary edema occurred in Malaysia in 1997, and EV71 had been implicated as the etiology of the outbreak [11]. Thirty-one children in Sarawak, Malaysia and four children in Peninsular Malaysia succumbed to the infection within hours of admission to the hospitals [12]. The largest EV71 epidemic reported to date occurred from April to December of 1998 in Taiwan in which a variety of clinical manifestations were observed. These included HFMD, encephalitis, meningitis, herpangina and poliomyelitits-like paralysis. In this outbreak, more than 90,000 children infected with HFMD were reported. Among these patients, more than 320 children were hospitalized with suspected meningitis, encephalitis, or acute flaccid paralysis, and at least 55 died, suggesting neurovirulence of the pathogen [13]. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. Thus, the only current means to prevent EV71 infection is through avoidance of contact between infected and susceptible individuals [14]. Since no effective antiviral agents are available, the need for an effective EV71 vaccine is urgent http://www.gvt-journal.com/content/5/1/6 to immunize the public before an outbreak occurs. A formalin-inactivated EV71 vaccine was developed in response to the Bulgarian epidemic in 1975 [6,15] but was not used to control the epidemic and has not been used since. Thus, no data on the efficacy of the Bulgarian vaccine is available. Recently, several candidates of EV71 vaccines using different approaches are being investigated. These include formalin-inactivated whole virus vaccine, DNA vaccine and recombinant protein vaccine. These vaccine constructs remain promising vaccine strategies that require further refinement, thus further study and development are required [16-18]. In this study, a DNA vac (...truncated)