Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes.

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes.

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins.

Strategies to inhibit the spread of HIV infection consist of a number of specific molecular approaches. Since viral production is dependent upon Tat-mediated transactivation of the HIV promoter through the Tat activating region (TAR), tat antisense RNA, anti-tat ribozymes, TAR decoys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant proteins, including a newly generated form Tat delta 58, for the ability to inhibit Tat-mediated transactivation and HIV production. In addition, we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (Tat delta 53) and the Drosophila Engrailed (Eng) transcription repressor domain to test the hypothesis that transcriptional repression can be targeted to the HIV promoter. This fusion mutant was also examined for its capacity to block both Tat-mediated transactivation and HIV replication. We show that three mutants Tat delta 53. Tat delta 58 and Tat delta 53/Eng result in a transdominant phenotype inhibiting wild-type Tat-mediated transactivation, and that the inhibiting potential is increased by the presence of the entire basic domain or the fusion of a repressor domain. However, only the transdominant mutants Tat delta 58 and Tat delta 53/Eng significantly inhibit HIV-1 replication after infection of transfected T cell lines. These results demonstrate the potent inhibiting activity of Tat mutants on HIV replication, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.

High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.

Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV.

Broadly neutralizing, MN-like PND-directed antibodies in Rwandan children with long-term HIV1 infection.

Sera from 11 perinatally HIV1-infected Rwandan children with prolonged survival were tested in vitro for the presence of neutralizing antibodies against different HIV1 strains. These 11 sera from long survivor (LS) children were compared with 16 sera from Rwandan children with AIDS. Sera from HIV1-infected children exhibited the greatest neutralizing activity against HIV1MN cell-free infection. They also inhibited HIV1RII and HIV1LAI cell-free infection with lower titres. Higher neutralization titres were observed in sera from LS compared to the AIDS group, with a significant difference for HIV1MN and HIV1LAI strains. Sera from LS children also inhibited syncytium formation induced by HIV1MN-infected cells with higher titres than AIDS children. Sera from the HIV1-infected children showed reactivity to the HIV1MN V3 peptide, as well as to both the US/European and the African consensus V3 peptides. Higher reactivity was observed in sera from LS than from AIDS children, and the difference was significant toward the African consensus peptide. The LS children also had significantly higher V3MN IgG avidity than the AIDS children. These data support the notion that the humoral response to the V3 domain, associated with a broadly neutralizing activity, may be an important factor in the prolonged survival of these children. The specificity against HIV1MN also suggests that an antigenically MN-related strain may be prevalent in Rwanda, and that an MN-related principal neutralizing domain sequence could be an important determinant for candidate vaccines in this part of Africa.

Cell surface phenotypic changes induced in H9 T cells chronically infected with HTLV type I or HIV type 1 or coinfected with the two viruses.

To investigate whether HTLV-I infection, HIV-I infection, or HIV-I infection of HTLV-I-infected cells affect the expression of cellular surface molecules, an HTLV-I-infected T cell line derived from the H9 T cell line was established (H36). H9 cells uninfected or infected with HTLV-I were then infected with HIV-1. We have compared the density of different surface markers on these three infected H9 T cell lines. These markers consist of T cell-specific antigens (CD2, CD3, CD4, and CD8), activated T cell antigens (CD25 and CD71), major histocompatibility complex (MHC) antigens (class I and II), and adhesion molecules (LFA-1 and ICAM-1). The experiments reported in this article show that chronic HTLV-I infection, HIV-1 infection, and HIV-1 infection of HTLV-I-infected T cells modulate the expression of several immunologically important cell surface antigens. The nature and the extent of T lymphoid cell phenotypic modulation depend on the infecting virus. Furthermore, HTLV-I and HIV-1 interact with each other in the phenotypic modulation of coinfected cells.

Primary in vitro immunization with multimeric synthetic peptides of HIV-1 envelope glycoproteins: generation of neutralizing human monoclonal antibodies.

Peripheral blood lymphocytes from healthy HIV-1 seronegative donors were immunized in vitro with the following synthetic peptides: (i) an octameric poly-L-lysine conjugated peptide of the HIV-1MN V3 loop and (ii) a resin bound synthetic peptide aa642-665 of HIV-1 gp41. Lymphoblastoid cell lines (LCL) were obtained by immortalization with Epstein-Barr virus (EBV). We produced four LCL secreting human monoclonal antibodies (HuMoAbs) of the IgM isotype: three were directed against the V3 domain (FC10, FC81 and CF41) and one against aa642-665 (CA45C). Two of these HuMoAbs (FC81 and CA45C) reacted to viral surface antigen on HIV-1-infected cells. All the HuMoAbs inhibited 40-53% of cell fusion induced by HIV-1-infected H9 cells at 5 micrograms/ml. They also neutralized, at lower concentrations, cell-free infection with HIV-1MN, HIV-1IIIB and four primary clinical HIV-1 isolates. No enhancing activity of the HuMoAbs in the presence of complement was observed. The results presented here show the feasibility of generating neutralizing human monoclonal antibodies against HIV-1 by primary in vitro immunization with selected synthetic peptides of HIV-1 envelope glycoproteins. This approach has provided tools for further studies of synergistic neutralization assays, and generated potential immunoglobulin candidates for passive immunotherapy.

Human and murine monoclonal antibodies directed against a conserved sequence from gp41 (aa583-599) of human immunodeficiency virus type 1.

Human spleen cells from an HIV-seropositive donor were immunized in vitro with the aa583-599 peptide conjugated to an heptalysyl core. This sequence was derived from the putatively HIV-immunosuppressive region of HIV1 gp41. The same conjugated peptide was used to immunize mice. One human and one mouse IgM monoclonal antibody (mAb) directed against the aa583-599 peptide were obtained. The two mAb had distinct patterns of reactivity against a panel of 42 peptides with modified sequences. Neither of the mAb inhibited the immunosuppressive effect of aa583-599 octopus-lys-conjugated peptide on anti-CD3 Ab-induced lymphoproliferation. In addition, both mAb did not neutralize cell-free virus transmission or enhance HIV infection. However, HmAb inhibited formation of syncytia between HIV1-infected (but not HIV2-infected cells) and non-infected target cells at concentrations above 20 micrograms/ml, whereas MmAb did not have any effect. The degree of conservation of the aa583-599 region makes HmAb a candidate for use as a group-specific reagent in future HIV1 passive immunotherapy protocols.