Construction and identification of HSV-1 vector vaccine carrying HIV-1 antigen / 生物工程学报

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.

IL15 DNA adjuvant enhances cellular and humoral immune responses induced by DNA and adenoviral vectors encoding HIV-1 subtype B gp160 gene / 病毒学报

To enhance the immunogenicity of DNA and adenoviral vector vaccines expressing HIV-1 subtype B gp160, human interleukin 15 (hIL15) DNA adjuvant (pVR-hIL15) was constructed. BALB/c mice received DNA prime/protein boost immunization with pVR-HIVgp160/Ad5-HIVgp160 alone or combined with pVR-hIL15. Cellular and humoral immune responses were evaluated by IFN-gamma enzyme-linked immunosorbent spot assay and enzyme-linked immunosorbent assay, respectively. Compared with those immunized with vaccines alone, the mice immunized with vaccines combined with pVR-hIL15 had significantly increased specific cellular response and antibody titer (P < 0.05). It suggests that the IL15 DNA adjuvant can enhance the immune responses induced by prime-boost regimen using DNA and adenoviral vector encoding HIV-1 subtype B gp160.

Immunogenicity of plasmid DNA and adenoviral vectors encoding HIV-1 subtype B env gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct DNA and recombinant adenovirus vector vaccines containing an env gene from the prevalent subtype B strain in China and try to use them for therapeutic and prophylactic vaccines.</p><p><b>METHODS</b>The candidate plasmid DNA vaccine pVR-gp160 and recombinant adenovirus vaccine rAdV-gp160 were constructed separately. BALB/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gp120-specific cellular responses and antibody levels were detected by ELISPOT and ELISA respectively.</p><p><b>RESULTS</b>DNA vaccine alone and combined vaccines in a DNA prime/rAdV-gp160 boost vaccination regimen induced high level of Gp120-specific cellular responses. While low level of Gp120-specific antibodies were elicited in all groups.</p><p><b>CONCLUSION</b>DNA and rAdV vaccines could efficiently express Gp160 protein and activate specific cellular responses.</p>

Expression, purification, and characterization of an anti-human RBC ScFv-HIV gp160 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct and express anti-human RBC and HIVgp160 fusion protein for rapid detection of antibody to HIV.</p><p><b>METHODS</b>The gene of the anti human RBC ScFv and HIV antigen were constructed together into expression vector. The fusion protein was expressed in E. coli.</p><p><b>RESULTS</b>The fusion protein was proved to be able to bind both anti-RBC and HIVgp160. It could cause agglutination of human RBC when HIVgp160 was present.</p><p><b>CONCLUSION</b>The fusion protein has the potential in rapid detection of HIV.</p>

Activation of mitogen activated protein kinases via complement receptor type 2 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein-Barr virus. The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen-activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD4+ cell deletion in AIDS.</p><p><b>METHODS</b>HOS cells (HOS-CR2) and HOS-CD4 cells (HOS-CD4CR2) stably expressing CR2 were established and then identified by FACS and Western blotting. Activation and blocking tests of MAPKs were assessed by Western blot. Cell proliferation was determined using Cell Titer 96((R)) Aqueous One Solution Reagent.</p><p><b>RESULTS</b>FACS results showed that the positive rates of HOS-CR2 and HOS-CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS-CR2 and HOS-CD4CR2 cells were high. Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS-CR2, HOS-CD4, and HOS-CD4CR2 cells. The activation of MAPKs in HOS-CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical. The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time-dependent manner; the activation of MAPKs was blocked by anti-CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI-3K), respectively. In HOS-CD4 cells, MAPKs were activated by HIV-gp160. In HOS-CD4CR2 cells, MAPK activation was induced by HIV-gp160, 10% NHS, and HIV-gp160 + 10% NHS; phosphorylation of p38MAPK was dramatically induced by HIV-gp160 + NHS, and lasted for 1 hour. The cell proliferation results showed that HIV-gp160 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells (P < 0.01) and that NHS enhanced the effect of HIV-gp160 (P < 0.01).</p><p><b>CONCLUSIONS</b>The activation of MAPKs is independently mediated by CR2 and that anti-CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively. The results of the signal transduction and cell proliferation assays of HOS-CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD4+ cell proliferation.</p>

Immunization with HIV-1 subtype B gp160-DNA induces specific as well as cross reactive immune responses in mice.

BACKGROUND & OBJECTIVES: HIV-1 gp160 is an important structural protein for the virus cell interaction and virus entry. Therefore, it is regarded as the most important target for HIV-1 vaccine development. In this study we investigated the use of HIV-1 gp160-DNA construct in eliciting specific and cross reactive cell mediated immune response in mice. METHODS: DNA segment encoding env, tat and rev genes of HIV-1 subtype B (strain BRU-2) was amplified and cloned into mammalian expression vector pCI to generate plasmid pCIBRU-TRE. Mice were injected intramuscularly four times at biweekly intervals with 100 micrograms/dose of pCIBRU-TRE in normal saline, and subsequently analysed for anti HIV-envelope (env) immune responses. RESULTS: A low antibody level was detected as determined by ELISA after 4 doses. Subsequent inoculations failed to increase the antibody titres significantly. Spleen cells from the immunized mice were used for the detection of cellular immune response by lymphocyte proliferation assays (LPA), in vitro production of cytokines and cytotoxic T lymphocyte (CTL) assays. T cell response which was seen from the second week onwards, persisted even at the end of 24 wk following the last dose. Similar levels of T cell proliferation were observed on stimulation with either homologous or heterologous peptides. Cytokine studies showed a Th1 type of response. A cross clade MHC class I restricted CTL response was observed against target cells stimulated with either homologous or heterologous HIV antigens. INTERPRETATION & CONCLUSION: This study demonstrated that DNA encoding full length HIV-1 env glycoprotein gp160 induces specific as well as cross reactive cell mediated immune responses in mice. However, the induction of antibody response was poor.