Correction: Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh, IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0059604.].

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro.

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2Apro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID50) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT-PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-ß did not differ significantly in any group. These results suggest that siRNAs targeting the 2Apro region of the EV71 genome exerted antiviral effects in vitro.

Enhancing specific-antibody production to the ragB vaccine with GITRL that expand Tfh, IFN-γ(+) T cells and attenuates Porphyromonas gingivalis infection in mice.

The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ(+) T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ(+) T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ(+) T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ(+) T cells and antibody production to P. gingivalis.

[Construction of eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) and its immunogenic analysis].

AIM: To construct eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and mouse glucocorticoid-induced tumor necrosis factor receptor ligand (mGITRL) and to analyze its immunogenicity in vivo. METHODS: The ragB gene was obtained from pMD18-T-ragB, and then cloned into the eukaryotic expression vector pIRES and pIRES-mGITRL, respectively. The eukaryotic expression vectors: pIRES-ragB and pIRES-ragB-mGITRL were identified by double enzyme digestion and DNA sequencing, then transfected into COS7 cells by Lipofectamine(TM);2000. The expressions of ragB or mGITRL in COS7 cells were detected by Western blotting. The mice were immunized with the recombinant pIRES-ragB-mGITRL plasmid. The serum antibody level was determined by ELISA. RESULTS: pIRES-ragB and pIRES-ragB-mGITRL plasmids were successfully constructed. Western blotting showed that the targeted gene was over-expressed in COS7 cells and skeletal muscle cells, respectively. The high titers of antibodies against RagB were detected in mouse serum. CONCLUSION: The construction of pIRES-ragB-mGITRL co-expression vector provides the experimental basis for Porphyromonas gingivalis vaccine research, prevention and treatment of periodontitis.