To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.
Cancer Vaccines ; biosynthesis ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Female ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Papillomavirus Vaccines ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Uterine Cervical Neoplasms ; prevention & control ; Vaccines, DNA ; biosynthesis

In 1990, it was reported that the naked DNA encoding an antigen (so-called DNA vaccine) transduced directly into the muscle is able to induce immune responses just like antigen inoculation. Since then, a number of DNA vaccines against different diseases have been developed and shown to induce different levels of specific humoral and/or cell-mediated immunity. Efforts have been made to develop effective DNA vaccines against classical swine fever (CSF). This review covered the following aspects in the development and application of CSF DNA vaccines: construction and evaluation, application of adjuvants, combination with other vaccines and the existing problems and solutions.
Adjuvants, Immunologic ; pharmacology ; Animals ; Classical Swine Fever ; prevention & control ; Swine ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), has been epidemic or endemic in many countries, and causes great economical losses to pig industry worldwide. Attenuated vaccines (such as C-strain) have played an important role in the control of CSF. Recently some new phenomena appear, such as atypical and persistent infections of CSF, immunization failure and so on. Meanwhile, eradication programs have been implemented in many countries, restricting the widespread applications of attenuated vaccines. Thus, currently the priority is to strengthen the research in pathogenesis and transmission mechanisms, as well as to develop marker vaccines. Recently, the applications of reverse genetics technology open up a new way for research of structure and function of CSFV proteins and development of novel vaccines against CSF. This review focuses on the progress of applications of reverse genetics in the functional analysis and marker vaccine development of CSFV, and also discusses the problems confronted now and prospective aspects in the study of CSFV.
Classical swine fever virus ; genetics ; Cloning, Molecular ; Genetics, Microbial ; methods ; RNA, Viral ; genetics ; Recombination, Genetic ; Vaccines, Synthetic ; biosynthesis ; immunology ; Viral Vaccines ; biosynthesis ; genetics

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

Six recombinant plasmids co-expressing the wild-type GP5 gene or the codon-optimized GP5 gene (containing pan-DR epitope) of porcine reproductive and respiratory syndrome virus (PRRSV) and the E2 gene of classical swine fever virus (CSFV) or the E2 fused with the UL49 of pseudorabies virus (PrV) were constructed based on the suicidal DNA vaccine pSFV1CS-E2 described previously. Expression of GP5 and E2 was confirmed by indirect immunofluorescence assay. The immunogenicity of six plasmids was evaluated in BALB/c mouse model. For the six plasmids, low-level of E2 and GP5 protein specific antibodies could be detected in the sera of the immunized mice. Specific lymphoproliferative responses to the PRRSV or CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. Antigen specific IFN-gamma and L-4 secretion was detected in the splenocytes of some immunized mice by cytokine ELSIA. Fusion with the PrV UL49 in the suicidal vaccines induced significantly higher lymphoproliferative responses and cytokine secretion. Taken together, the suicidal DNA vaccines co-expressing GP5 and E2 could induce PRRSV and CSFV specific humoral and cell-mediated immune responses.
Animals ; Antibodies, Viral ; blood ; Antibody Formation ; Cytokines ; blood ; Female ; Immunity, Cellular ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

OBJECTIVETo analyze the influence of optimal codon usage on the expression levels and immunogenicity of DNA vaccines, encoding the human papillomavirus type 6b (HPV 6b) E7 gene.

METHODSThe full length E7 gene of HPV 6b was modified to substitute human preferred codon for rarely used codon, and three mutations were introduced into the pRB binding site of HPV 6b E7 to eliminate its transformation potential. The codon optimized and mutated E7 gene (hu-mE7) were cloned into the Kpn I and EcoR I site of the pcDNA3 mammalian expression vector, the in vitro expression of the hu-mE7 gene and the immunogenicity of hu-mE7 DNA vaccine were compared with the wt-E7gene.

RESULTSThe in vitro expression of pcDNA3-hu-mE7 was much higher than the classical wt-E7 plasmid in monkey COS-1 cell line. Mice immunized intramuscularly with the pcDNA3-hu-mE7 showed that the codon modified E7 gene induced a stronger IFN-gamma ratios than the wt-E7 gene.

CONCLUSIONSThese results suggest that the optimized codon usage contributes to the enhancement of gene expression and immunogenicity of HPV 6b E7 gene.

Animals ; Cell Line ; Codon ; genetics ; Female ; Gene Expression ; Genes, Viral ; genetics ; Genetic Vectors ; Mice ; Papillomaviridae ; genetics ; Papillomavirus Vaccines ; Transfection ; Vaccines, DNA ; immunology ; Viral Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

OBJECTIVETo construct and characterize recombinant adenoviruses containing glycoprotein (GP) gene from rabies virus CVS-N2C strain.

METHODSTo obtain the recombinant adenovirus by pAdEasy system, identify recombinant virus with cDNA sequencing, Northern blot, Dot blot, Western blot and challenge-protection experiment of mice.

RESULTSRecombinant adenovirus showed typical adenovirus morphological characteristics; the viral genome was stable; GP specific mRNA and presence of glycoprotein were determined in rAdGPcvs and rAdGPcvs' infected cells. The glycoprotein produced by recombinant adenovirus had a molecular mass of 66,000, which was similar to that of natural glycoprotein. In the group of rAdGPcvs immunized mice, 87.5%-100% of mice survived from a 35.8LD50/38.0LD50 lethal rabies intracerebral challenge. Finally 73.3%-83.3% of the mice that had received eAdGPcvs survived, and all the Ad5 immunized mice succumbed to rabies.

CONCLUSIONRecombinant adenovirus rAdGPcvs and rAdGPcvs' hold great potential to be developed as recombinant rabies vaccines, and at the same time, it is actually the first study that on high neuropathogenicity rabies CVS-N2C glycoprotein based adenoviral recombinants.

Adenovirus E3 Proteins ; biosynthesis ; genetics ; immunology ; Adenoviruses, Human ; genetics ; Animals ; Antibodies, Viral ; biosynthesis ; genetics ; immunology ; Antigens, Viral ; Base Sequence ; Genetic Vectors ; Glycoproteins ; biosynthesis ; genetics ; immunology ; Humans ; Mice ; Molecular Sequence Data ; Nucleoproteins ; biosynthesis ; genetics ; immunology ; Rabies ; immunology ; prevention & control ; Rabies Vaccines ; biosynthesis ; genetics ; immunology ; Rabies virus ; genetics ; immunology ; Vaccines, Synthetic ; biosynthesis ; genetics ; immunology ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology ; Virus Replication

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

The avirulent QU strain of fowl adenovirus, a member of duck adenovirus type 1, could be a potential vector in recombinant vaccine development. To identify a non-essential region for replication of QU virus, a 3.4 kb fragment near the E4 region of QU virus genome was amplified by PCR to construct a plasmid pADGFP, in which ORF1, ORF8 and ORF9 was replaced with a system expressing enhanced green fluorescence protein. Further, a recombinant virus rQUGFP was constructed by homologous recombination after pADGFP and QU virus were co-transfected into chick embryo fibroblast. The one step growth curve of the rQUGFP was found to be identical with that of parent QU virus and the TCID50 titers of different generation recombinants maintained stable. These findings suggest that the region including ORF1, ORF8 and ORF9 of QU virus genome is dispensable for virus replication, and the foreign gene inserted into virus genome can be efficiently and stably expressed. The work lays the foundation for further studies of developing this virus as a vector of recombinant vaccine.
Adenovirus E4 Proteins ; genetics ; immunology ; Animals ; Fowl adenovirus A ; classification ; genetics ; Genes, Viral ; genetics ; Genetic Vectors ; genetics ; Open Reading Frames ; genetics ; Recombination, Genetic ; Transfection ; Vaccines, Synthetic ; biosynthesis ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; genetics ; immunology ; Virus Replication

To construct a recombinant vaccinia virus RVJ1175M2 expressing influenza A3 virus (H3N2) M2 gene, full length gene encoding influenza virus (H3N2) M2 protein was amplified with PCR and cloned into plasmid pJSC1175 which was used for homologous recombination with vaccinia virus Tiantan strain. Along with this, a recombinant vaccinia virus RVJ1175M2 containing the M2 gene was subsequently constructed. It was identified by PCR that the gene of M2 protein was inserted into the TK locus of vaccinia virus Tiantan strain correctly and M2 protein was expressed by recombinant vaccinia virus RVJ1175M2 effectively. Two electrophoretic bands of M2 protein expressed by the infected HeLa cells, one of 15kD and the other of 13kD in accordance with related documents, was deteced by Western-blot. M2 protein distributing on the surface of the infected cells was demonstrated by immunofluorescence and flow cytometry. The results suggested that recombinant vaccinia virus RVJ1175M2 could express M2 protein effectively, this laid a foundation for comparative research on the immune effect of universal vaccine of influenza virus with different kinds of vaccine expressing M2 protein.
HeLa Cells ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza Vaccines ; immunology ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Vaccines, Synthetic ; immunology ; Vaccinia virus ; genetics ; Viral Matrix Proteins ; genetics