Construction Of SFV-replicon Vaccine Based On PTD Fused With HA Of Influenza A (H5N1) Virus And Its Cellular Immune Response Induction

By analysing the antigenic peptides and homogeneity of the HA sequences(human isolates of the influenza A virus H5N1 subtype) and exploring a novel alphavirus replicon system of VP22 fused with HA,this study assesses whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented by fusing with VP22.Materials and methodsThe first step in developing an efficient gene vaccine against human-avian H5N1 influenza is to select a representative and highly immunogenic HA gene.A total of 232 reference sequences of the H5N1 human isolates during 1997-2008 retrieved from the influenza database(influenza virus resources) were used in this study.Phylogenetic trees and antigenicity analysis were constructed using neighbour-joining programs included in DNASTAR and DNAMAN software.The HA,VP22 and EGFP genes were amplified and cloned into the unique Xho I and Not I restriction sites of plasmid pSFV,resulting in the plasmid pSFV-HA,pSFV-VP22,pSFV-EGFP.To construct pSFV-VP22-HA,the VP22-HA fusion gene inserting five glycine-coding sequences at the fusion site was firstly produced,by SOE-PCR(i.e.,splicing by overlap extension PCR).This fusion gene was then subcloned into the unique BamHI and NotI restriction sites of the pSFV vector.The pSFV-HA,pSFV-VP22,pSFV-EGFP and pSFV-VP22-HA vectors were linearised and transcribed into the RNA transcripts in vitro.Then,VRP-HA,VRP-VP22,VRP-EGFP and VRP-VP22/HA were assembled in BHK-21 cells by using In vitro-transcribed RNAs of pSFV helper vectors and that of pSFV-HA,pSFV-VP22,pSFV-EGFP and pSFV-VP22-HA,respectively.EGFP expression was observed under a fluorescence microscope after the replicons infecting the BHK-21 cells,while RT-PCR and immunofluorescence staining were performed to detect HA,VP22 and VP22-HA expressions.The percentages of apoptotic and necrotic BHK-21 cells,which were infected by VRP-HA,VRP-VP22,VRP-EGFP and VRP-VP22/HA,were analysed by using a flow cytometry with annexin V apoptosis detection kitsSix- to eight-week-old BALB/c mice were immunised intramuscularly(i.m.; 200μl) at zero weeks and boosted at three weeks with VRP-HA and VRP-VP22/HA, respectively,and VRP-VP22,VRP-EGFP or normal saline(NS) was also used as controls.Lymphocytes were separated from the spleens of mice and detected for specific cell-mediated responses.IL-4 expression of CD4⁺ T cells and IFN-γexpression of CD8⁺ T cells were assayed by means of intracellular cytokine staining(ICCS) with flow cytometry.The percentage of cells expressing cytokines was analysed by using the ANOVA test with SPSS software,and all data are presented as mean±SD values.ResultsA phylogenetic analysis of the 232 reference sequences of the H5N1 human isolates during 1997-2008 revealed that the genetic relationships among the A/Anhui/1/2005 isolates were similar to most of the 1997-2008 H5N1 human isolates (i.e.,70%at the genetic distance of 7×10^-6 and 90%at the genetic distance of 7×10^-5). An antigenicity analysis revealed that the four isolates(A/Anhui/1/2005, A/Beijing/01/2003,A/China/2006 and A/human/China/GD02/2006) had 26 antigenic peptides,and A/Anhui/1/2005 isolates had frequent antigenic peptides.The pSFV-HA,pSFV-VP22,pSFV-EGFP and pSFV-VP22-HA vectors were confirmed by sequencing.The integrity and quantity of RNA transcripts of the pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA and pSFV helper vectors were determined via denaturing gel electrophoresis.Observation under a fluorescence microscope revealed EGFP expression in the infected BHK-21 cells,especially in the nuclei.HA,VP22 and VP22-HA expression in the infected BHK-21 cells was confirmed by RT-PCR and immunofluorescence staining.Furthermore,all the BHK-21 cells infected with any of the replicons showed high levels of apoptosis,but at significantly higher-than-normal levels-especially those of early apoptosis.Four groups of mice immunised with VRP-HA(10⁵ TU and 10⁶ TU) and VRP-VP22/HA(10⁵ TU and 10⁶ TU) presented high percentages of cells expressing IL-4 and IFN-γ,while the three control groups that received 10⁵ TU of VRP-VP22, VRP-EGFP or NS presented low percentages of expression.The ANOVA-SNK analysis revealed that the IL-4 expressions in three groups(10⁶ TU VRP-HA,and 10⁵ TU and 10⁶ TU VRP-VP22/HA) were significantly higher than those of the control groups(VRP-VP22,VRP-EGFP and NS)(p < 0.01).A dose titration effect for IL-4 expression,as well as for IFN-γexpression,was observed both in the VRP-HA-vaccinated mice and in the VRP-VP22/HA-vaccinated mice.ConclusionsOur results revealed that the HA gene of A/Anhui/1/2005 isolates presented with comparatively representative and high immunogenicity.VRP-HA,VRP-VP22,VRP-EGFP and VRP-VP22/HA expressing the target proteins had be successfully assembled.And,VRP-HA,VRP-VP22,VRP-EGFP and VRP-VP22/HA could induce infected cells apoptosis.Our results revealed that both VRP-VP22/HA and VRP-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could increase the immunogenicity of the HA antigens to which it is fused.