HIV-1 DNA vaccine efficacy is enhanced by coadministration with plasmid encoding IFN-alpha

Numerous strategies have been employed in an attempt to improve the immunogenicity and efficacy of nucleic acid vaccines. In the present study, the immunogenicity in the induction of humoral and cellular immune responses to HIV-1 DNA vaccine expressing a chimeric gene of gag and gp120 and the adjuvant effect of IFN-alpha on HIV-1 DNA vaccine were studied in a murine model. The DNA vaccine plasmid pVAX1-gag-gp120 and eukaryotic expression plasmid pVAX1-IFN were constructed by inserting the chimeric gene of gag and gp120 of HIV-1 and IFN-alpha into the downstream of CMV promoter of eukaryotic expression vector pVAX1, respectively. In vitro expression detected by RT-PCR and Western blotting showed that the genes of interest could be expressed in transfected HeLa cells. After BALB/c mice were immunized by three intramuscular inoculations of the HIV-1 DNA vaccine plasmids alone or in combination with IFN-alpha expression plasmids, the different levels of anti-HIV-1 humoral and cellular responses were measured comparable to the control groups immunized with pVAX1-IFN, parent plasmid pVAX1 or PBS. The percentage of CD3+CD4+ and CD3+CD8+ subgroups of spleen T lymphocytes and the specific cytotoxicity activities of splenic CTLs in the coinoculation group were significantly higher than those in the separate inoculation group, and an enhancement of antibody response was also observed in the coinoculation group compared with the separate inoculation group. Take together, coadministration of HIV-1 DNA vaccine plasmids and IFN-alpha expression plasmids can elicit stronger humoral and cellular immune responses in mice than HIV-1 DNA vaccine plasmids alone, and IFN-alpha can be an effective immunological adjuvant in DNA vaccination against HIV-1.     

Inhibition of intra- and extra-cellular Tat function and HIV expression by pertussis toxin B-oligomer

HIV-1-transactivating factor Tat contributes to virus replication and to the onset of AIDS-associated pathologies by targeting different infected and uninfected cell types. We previously demonstrated that the B-oligomer of pertussis toxin (PTX-B) inhibits HIV infection and replication in primary T cells and macrophages and Tat-dependent HIV-1 long terminal repeat (LTR) transactivation inT lymphoid Jurkat cells. Here we demonstrate that PTX-B inhibits Tat-dependent NF-kappaB activation and HIV-1 LTR-transactivation in non-permissive epithelial HL3T1 cells in a phosphatidylinositol 3'-kinase-dependent way. PTX-B exerts its inhibition both when Tat is produced endogenously in transfected cells and in cells incubated with the extracellular Tat protein. In this latter case, PTX-B does not interfere with extracellular Tat uptake by cells. PTX-B inhibited also interleukin-8 secretion and virus expression stimulated in chronically infected U1 promonocytic cells by intra- and/or extracellular Tat. The genetically modified holotoxin PT-9 K/129G retains the capacity to inhibit Tat transactivating activity and HIV replication in both HIV-permissive and non-permissive cells. Inconclusion, PTX-B acts as a "pleiotropic" inhibitor of Tat, and this may significantly contribute to the broad spectrum of anti-HIV-1 effects exerted by PTX-B in different cell types, and suggests PTX-B and its derivatives as prototypic for the development of anti-Tat drugs.     

Repression of tumor necrosis factor-β expression by the human immunodeficiency virus type-1 tat protein in central nervous system-derived glial cells

HIV-1 Tat is a potent transactivator that stimulates expression from the HIV-1 LTR, from certain cellular gene promoters and from several heterologous viral promoters. Previous reports show that HIV-1 Tat transactivates tumor necrosis factor-β (TNF-β) promoter-directed gene expression in lymphocytic and monocytic cell lines and further demonstrate that a ‘TAR-like structure' downstream of the TNF-β promoter is essential for Tat activity. The ability of Tat to activate TNF-β may have profound effects as TNF has been shown to be a potent activator of HIV-1 gene expression and an important immunomodulatory and growth regulatory factor. The studies presented herein demonstrate a novel finding where HIV-1 Tat specifically represses (>10-fold) TNF-β promoter-directed gene expression in central nervous system-derived glial cells. Amino acid residues 2 to 36 of HIV-1 Tat are required for TNF-β repression. Tat repression of TNF-β, a factor which upregulates HIV-1 gene expression, suggests a novel mechanism whereby HIV-1 is able to establish latent infection of glial cells that present no detectable virions and/or viral antigens.     

Viral and cellular gene expression in CD4+ human lymphoid cell lines infected by the simian immunodeficiency virus, SIV/Mne.

Our laboratory has undertaken an analysis of cellular and viral gene expression in CD4+ human lymphoid cell lines infected by the human and simian immunodeficiency viruses, HIV-1 and SIV/Mne, respectively. The purpose of the current study was to: (i) examine the effects of SIV/Mne infection on host macromolecular synthesis and compare the results to those in the HIV-1 system; and (ii) investigate the mechanisms responsible for the restriction of SIV/Mne infection in CD4 positive lymphoid cells which are readily infected by HIV-1. First we determined that SIV does not impose selective blocks on host macromolecular synthesis, unlike HIV-1, which induces both the selective inhibition of cellular protein synthesis and the degradation of cellular mRNAs (Agy, M., Wambach, M., Foy, K., and Katze, M. G., 1990, Virology 177, 251-258). No such selective reduction in cellular mRNA stability or protein synthesis was observed in cells infected by SIV/Mne. Additional differences between SIV and HIV-1 were observed using a panel of CD4+ human cell lines. While HIV-1-infected all cell lines. SIV/Mne efficiently infected only the MT-4, C8166, and 174 x CEM cell lines. Repeated efforts to infect CEM or Jurkat cells were unsuccessful as determined by PCR analysis of viral DNA. HUT 78 cells supported a limited infection detectable only by PCR analysis. These data suggest the block in viral replication in the nonsusceptible cell lines is at an early step. Interestingly, all the SIV susceptible cells were virally transformed, C8166 and MT-4 by HTLV-1, and 174 x CEM by Epstein-Barr virus. Furthermore FACS analysis revealed that all susceptible cells expressed two B cell associated markers, B7/BB1 and CD40. These observations taken together highlight differences between the HIV and SIV viruses, and suggest that for efficient replication, SIV/Mne may require an additional cell surface molecule, cofactors provided by transforming viruses, or a complex interplay between the two.     

A quantitative GFP-based bioassay for the detection of HIV-1 Tat transactivation inhibitors.

The Tat function of the human immunodeficiency virus (HIV) represents an important target for the development of new anti-HIV drugs. A rapid, sensitive and simple bioassay was developed for the detection of HIV transactivation inhibitors. A reporter plasmid based on the expression of the green fluorescent protein (GFP) under control of the HIV-1 long terminal repeat (LTR) was constructed. This reporter gene can be quantified by simply measuring the fluorescence irradiated by GFP-producing cells, without the need of extraction procedures or enzymatic assays. Cells, stably expressing HIV-1 Tat protein, were transfected with this plasmid and the inhibitory effect of anti-Tat drugs was assessed by measuring the inhibition of fluorescence. Using this assay system the anti-transactivation activity of several known compounds was confirmed. This is the first HIV transactivation assay using GFP reporter gene in microtiter plates. The assay can be used for the detection and quantification of HIV transactivation, and for the high throughput evaluation of anti-transactivation drugs in different cellular backgrounds.     

Exogenous human immunodeficiency virus type-1 Tat protein selectively stimulates a phosphatidylinositol-specific phospholipase C nuclear pathway in the Jurkat T cell line

We investigated the effect of extracellular Tat protein of human immunodeficiency virus-type 1 (HIV-1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV-1 p24 and cross-linked anti-CD3 monoclonal antibody (mAb) were added in culture for 1–60 min to Jurkat lymphoblastoid CD4⁺ T cells. The stimulation of T cell receptor by cross-linked anti-CD3 mAb resulted in a rapid increase of the phosphatidylinositol-specific phospholipase C (PI-PLC) activity in whole cell lysates. On the other hand, Tat protein, either alone or in combination with anti-CD3 mAb, showed little effect on the PI turnover of whole cell extracts. Tat, however, selectively stimulated a nuclear-specific PI-PLC with a peak of activity after 30 min from the addition in culture to Jurkat cells. Interestingly, this time corresponded to that required for the uptake and nuclear localization of recombinant Tat protein, as demonstrated by electron microscope immunocytochemistry experiments with anti-Tat mAb. Moreover, exogenous Tat reached the nucleus of Jurkat cells in a bioactive form, as shown in a HIV-1 long terminal repeat-chloramphenicol acetyl transferase transactivation assay. The specific increase of a nuclear PI-PLC activity was further demonstrated by the ability of Tat to stimulate PI turnover also when added directly to isolated nuclei. As a whole, these data demonstrate that Tat selectively stimulates a nuclear polyphosphoinositide hydrolysis, which appears to be independent of the cellular PI turnover. The relevance of these findings for a better understanding of the biological functions of extracellular Tat is discussed.     

Transfection optimization for primary human CD8+ cells

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.     

CD46 RNAi对CD59介导的T细胞信号转导的作用研究

目的构建携带针对CD46基因的pSUPER retro RNAi逆转录病毒载体,研究糖基磷脂酰肌醇(GPI)锚定蛋白CD59与CD46在介导T细胞信号转导中的相关性。方法将能转录产生靶向CD46小发夹RNA(shRNA)的寡核苷酸序列,克隆入逆转录病毒载体pSUPER retro,转化大肠杆菌JM109并转染Jurkat细胞。将Jurkat细胞分为未转染的Jurkat细胞组(Ⅰ组)、转染空质粒的Jurkat细胞组(Ⅱ组)、转染CD59干扰质粒的Jurkat细胞组(Ⅲ组)及转染CD46干扰质粒的Jurkat细胞组(Ⅳ组)。用RT-PCR、Western blot技术检测各组细胞中的CD59、CD46基因的表达水平。用噻唑蓝(MTT)比色法检测CD46与CD59联合作用对4组Jurkat细胞的增殖效应。结果重组载体经PCR及限制性内切酶酶切鉴定初步成功后送测序,结果表明序列正确,构建成功,稳定转染后,Ⅳ组细胞CD46分子的表达被成功抑制,Ⅲ组细胞CD59分子的表达被抑制。Ⅰ组和Ⅱ组细胞CD46与CD59单抗联合作用后,增殖能力明显高于Ⅲ组、Ⅳ组(P<0.05);但Ⅰ组和Ⅱ组,Ⅲ组和Ⅳ组之间无差异。结论 CD59可增强CD46对T细胞信号转导的效应。     

Entry of human immunodeficiency virus-1 into glial cells proceeds via an alternate, efficient pathway

Although the CD4 molecule is the cellular receptor for human immunodeficiency virus-1 (HIV-1) in cells of the lymphocyte/monocyte lineage, a number of investigators have also been able to infect cells, including several of central nervous system (CNS) origin, that do not express CD4 protein or mRNA. These infections are generally nonpermissive. To ascertain whether the nonpermissive nature of infection in glial cells is due to an inefficient entry pathway, we prepared a permanently transfected U373-MG cell line expressing the CD4 molecule and demonstrated that HIV-1 still replicates at a low level. Furthermore, a virus uptake assay indicated that HIV-1 enters glial cells effectively, even in the absence of CD4. These results demonstrate that HIV-1 entry is efficient and that the restrictive nature of the infection in glial cells is due to postentry mechanisms. In addition, these findings support the existence of an alternate, efficient, entry pathway in some glial cells.     

Activation of HIV-1 enhancer sequence by vaccinia virus

To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.     

Simultaneous Analysis of In Vivo CD8+ T Cell Cytotoxicity Against Multiple Epitopes using Multicolor Flow Cytometry

CD8+ T cells play a critical role in host defense against infections and tumors. Analysis of cytotoxic function of antigen-specific CD8+ T cells in animal models would be important in optimizing vaccine design against infections and tumors. In vivo cytotoxicity assays using fluorescent cellular dyes have been used as a popular alternative to traditionally used in vitro ⁵¹Cr-release assays. With the identification of multiple epitopes in various pathogen models, methods to simultaneously analyze cytotoxicity of CD8+ T cells to multiple epitopes in vivo would assist studies which aim to generate protective CD8+ T cell immunity to multiple epitopes. In this study, we evaluate the use of multiple fluorescent cellular dyes for the in vivo cytotoxicity assay. The use of 3 dyes allowed us to analyze the cytotoxicity of antigen-specific CD8+ T cell populations to multiple epitopes generated by virus infections, as well as their functional avidity, in vivo. Our studies extend the use of in vivo cytotoxicity assays to allow direct comparisons of cytotoxicity to various epitopes in the same animal and may also be applicable to assessment of in vitro cytotoxicity of human CD8+ T cells specific for multiple viral or tumor antigens in clinical settings.