BACKGROUNDTo observe the LMP2 specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad-LMP2 in rhesus.

METHODSThe rhesuses were immunized with Ad-LMP2 through intra muscular injection in three groups, high dosage (4.5 x 10(11) VP/kg), medium dosage (1.5 x 10(11) VP/kg) and low dosage (0.5 x 10(11) VP/kg) groups. They were totally immunized six times at intervals of 5 days. The specific cellular immune responses were tested during the 7th week by ELISPOT after immunization. And the titers of anti-LMP2 antibody were tested by EIA throughout the period of immunization.

RESULTSLMP2 induced specific cellular and humoral immune responses in all three dosage group. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response. Both the neutralizing antibody to adenovirus and anti-LMP2 antibody could be detected from 2 weeks after immunization. They would reach the peak during 3-4 weeks after immunization, then declined during the 7th week after immunization.

CONCLUSIONThe recombinant adenovirus LMP2 could induce specific cellular and humoral responses in rhesus after immunization.

Adenoviridae ; genetics ; Animals ; Antibodies, Viral ; blood ; Antibody Formation ; immunology ; Female ; Immunization ; methods ; Macaca mulatta ; Male ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

FMDV 2A peptide was introduced as a linker between GP5 and M protein of porcine reproduction and respiratory syndrome virus (PRRSV) to allow automatic self-cleavage the polyproteins. This strategy simultaneously displayed the neutralizing action of GP5 protein and cell-mediated immunity of M protein. We put them into the expression cassette of adenovirus vector. The results of RT-PCR, IFA and Western blotting showed that GP5 and M protein were not only expressed correctly, but also self-cleavaged and assemble heterodimers formation. To detect the advantages of rAd-GP5-2A-M, we also constructed some other recombinant adenoviruses (rAd-GP5, rAd-M and rAd-GP5-M) as control. After inoculated subcutaneously into BALB/c mice, the four recombinant adenoviruses can induce PRRSV-specific antibodies and cell-mediated immune response, but the level of humoral and cell-mediated immune response against PRRSV induced by rAd-GP5-2A-M is the strongest among the four recombinant adenoviruses. All of these suggested that it is possible to develop one multi-gene engineering vaccine utilizing FMDV 2A peptide, and also provided a novel strategy for developing other viral disease vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Swine ; Vaccines, Synthetic ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; metabolism ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; immunology

Animals ; Humans ; Influenza A virus ; immunology ; Influenza Vaccines ; immunology ; Protein Engineering ; Viral Matrix Proteins ; chemistry ; genetics ; immunology ; metabolism

OBJECTIVETo observe the specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys.

METHODSSixteen rhesuses were immunized with Ad5F35-LMP2 through intra-muscular injection in three groups: high dosage group (1.5 x 10(10) TCID(50)/rhesus), medium dosage group (1.5 x 10(9)TCID(50)/rhesus), low dosage group (1.5 x 10(8)TCID50/rhesus) and the last group was control (PBS 4 ml/rhesus). They were totally immunized three times at intervals of one month. The EBV-LMP2 specific cellular immune responses were tested during the 0, 4, 8, 12 weeks by Elispot after immunization respectively. And the titers of anti-LMP2 antibody were tested by EIA at the same time.

RESULTSEBV-LMP2 specific cellular and humoral immune responses which were induced by recombinant adenovirus Ad5F35-LMP2 can be found in all the three dosage groups. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response.

CONCLUSIONThe recombinant adenovirus Ad5F35-LMP2 could elicit LMP2 specific cellular and humoral immune responses in rhesus.

Adenoviruses, Human ; genetics ; Animals ; Cell Differentiation ; Herpesvirus 4, Human ; genetics ; Immunity, Cellular ; immunology ; Immunization ; methods ; Macaca mulatta ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology

In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines.
Adenoviridae ; genetics ; metabolism ; Animals ; Antibodies, Viral ; blood ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Immunization ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology ; Viral Matrix Proteins ; genetics ; immunology

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E + prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.
Animals ; Antibodies, Viral/blood ; Baculoviridae/genetics ; Blotting, Western ; Cloning, Molecular ; Encephalitis Virus, Japanese/genetics/*immunology ; Encephalitis, Japanese/*immunology/prevention&control ; Female ; Immunization ; Immunoglobulin Isotypes/blood ; Japanese Encephalitis Vaccines/*immunology/standards ; Mice ; Mice, Inbred ICR ; Microscopy, Fluorescence ; Plasmids ; Recombinant Proteins/genetics/immunology ; Viral Envelope Proteins/genetics/*immunology ; Viral Matrix Proteins/genetics/*immunology ; Viral Nonstructural Proteins/genetics/*immunology

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

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.
Antibodies, Viral/*blood/immunology ; Antigens, Viral/*genetics/immunology ; Cloning, Molecular ; Cytomegalovirus/genetics/*immunology ; Cytomegalovirus Infections/blood/*immunology/virology ; DNA, Complementary/genetics ; DNA, Viral/genetics ; Gene Library ; *Genes, Structural, Viral ; Human ; Recombinant Fusion Proteins/biosynthesis/immunology ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Viral Matrix Proteins/*genetics/immunology

We identified the critical amino-acid residues in antigen M derterminant (MAD) epitope of human cytomegalovirus protein M. On the basis of the peptide sequence of MAD, some conservative residues were mutated into the glycine residue. Then the gene fragment of mutants linked to amino terminal of Fc were cloned into the plasmid pET32-Fc and expressed by fusion with Fc. After purified by protein A affinity chromatography, the activity of mutants binding the goat polyclonal antibodies against human cytomegalovirus (HCMV) were detected by ELISA and Western blotting. Our results showed that when glutamine residue was mutated into glycine residue, the activity of MAD(Q --> G) binding the goat polyclonal antibodies against HCMV was reduced apparently. Other mutants did not have the same characteristics. The activity of MAD was closely related to the conformation of glutamine residue.
Amino Acids ; genetics ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Base Sequence ; Cytomegalovirus ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Goats ; Humans ; Immunoglobulin Fc Fragments ; genetics ; immunology ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Mutation ; Viral Matrix Proteins ; genetics ; immunology

The oncogenic Epstein-Barr virus (EBV) -encoded latent membrane protein 1 (LMP1) enables this virus's long-term survival within the cells of immune system. Mean while, LMP1 also plays a critical role for the transformation of resting B cells by EBV. It initiates the activation of signalling pathways, such as NF-kappaB, mitogen-activated protein kinase (MAPK), and JAK/STAT cascade by adaptor proteins including the tumor necrosis factor (TNF) receptor associated factors (TRAFs) and the TNF receptor associated death domain protein (TRADD). It increases the expression of adhesion molecules LFA-1, ICAM-1, and costimulatory molecule B7-1 of B cells, and regulates the antibody and cytokine secreted by B cells. LMP1 and CD40 have many common properties in signal transduction. Both of them co-localize in lipid rafts for signal transduction. Considering its close relationship with CD40, the research on LMP1 has become a hot spot in the immunology field.
Animals ; B-Lymphocytes ; immunology ; CD40 Antigens ; genetics ; physiology ; Gene Expression Regulation, Viral ; Herpesvirus 4, Human ; genetics ; metabolism ; physiology ; Humans ; Signal Transduction ; Viral Matrix Proteins ; genetics ; physiology