DNA vaccine constructs against enterovirus 71 elicit immune response in mice

Abstract

BackgroundEnterovirus 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.MethodsThe 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.ResultsThe 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.ConclusionThe promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine.

Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity

“…However, there is no effective therapy for vaccineassociated paralytic poliomyelitis, which is caused by virulent revertants of vaccine strains at a rate of one case per 520 000 doses associated with the first dose of the vaccine (Nkowane et al, 1987). For EV71, various vaccine candidates and therapies are being developed, but no vaccine has been established (Chen et al, 2006;Chiu et al, 2006;Liu et al, 2005Liu et al, , 2007Shih et al, 2004;Tan & Cardosa, 2007;Tung et al, 2007;Wu et al, 2007;Yu et al, 2000). The results obtained in this study would be useful for the identification of novel targets to develop antienterovirus drugs that could serve as therapeutic and/or prophylactic agents against acute neurological diseases caused by enterovirus infection.…”

Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime

“…However, there is no effective therapeutic means for vaccine-associated paralytic poliomyelitis caused by virulent revertants of vaccine strains, which occurred at a rate of one case per 520 000 vaccinations with the first dose of the live-attenuated vaccine (Nkowane et al, 1987). For EV71, various vaccine candidates and therapeutic means are being developed, but no vaccine has been established (Arita et al, 2005a(Arita et al, , 2007Chen et al, 2006;Chiu et al, 2006;Liu et al, 2005Liu et al, , 2007 Shih et al, 2004;Tan & Cardosa, 2007;Tung et al, 2007;Wu et al, 2007; Yu et al, 2000).…”

“…However, there is no effective therapeutic means for vaccine-associated paralytic poliomyelitis caused by virulent revertants of vaccine strains, which occurred at a rate of one case per 520 000 vaccinations with the first dose of the live-attenuated vaccine (Nkowane et al, 1987). For EV71, various vaccine candidates and therapeutic means are being developed, but no vaccine has been established (Arita et al, 2005a(Arita et al, , 2007Chen et al, 2006;Chiu et al, 2006;Liu et al, 2005Liu et al, , 2007 Shih et al, 2004;Tan & Cardosa, 2007;Tung et al, 2007;Wu et al, 2007; Yu et al, 2000).To date, several anti-enterovirus compounds that target cellular factors and inhibit various stages of virus replication have been identified. Brefeldin A blocks membrane traffic between the cis-and trans-Golgi compartments by targeting a cellular guanine nucleotide- exchange factor, GBF1, and inhibits the replication of PV, but not encephalomyocarditis virus (EMCV) (Belov et al, 2008;Cuconati et al, 1998;Irurzun et al, 1992;Maynell et al, 1992).…”

Condition and Prospects of Development of Vaccines for Specific Prevention of Enterovirus (Nonpolio) Infection

“…Tung et al [22] разработали ДНК-вакцину против ЭВ 71 типа путем включения гена, отве-чающего за синтез белка VP1 вируса в эукари-отический вектор. В ответ на введение мышам ДНК-вакцины уровень IgG против VP1 антигена у них повышался, при бустерной иммунизации -понижался.…”