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The safety and immunogenicity of an HIV-1 nef-expressing modified vaccinia virus Ankara (MVA) was investigated in 14 HIV-1-positive patients (CD4 >400/µl) on highly active antiretroviral therapy (HAART). Patients were vaccinated at weeks 0, 4 and 16, followed by interruption of HAART at week 18. MVA-nefwas well-tolerated except for local reactions, with only mild systemic side effects reported in a few patients. Vaccination with MVA-nefwas associated with recognition of new HIV-1 T-cell epitopes (cytotoxic T-lymphocyte epitopes in 9/14 patients, CD4 epitope/recombinant Nef protein in 2/14) and an increase in CD4+ and CD8+ T cells. All patients had been vaccinated against smallpox and a strong T-cell and antibody response to MVA was induced in all patients. After interruption of HAART, viral load rebounded in all patients, but after a median time of 36 (4–76) weeks in 9/14 patients, viraemia remained below the pre-HAART viral load and CD4 counts stayed above the pre-HAART levels. While six patients have remained off therapy for a median time of 64 (57–76) weeks, HAART was resumed in 8/14 patients after a median treatment interruption time of 15 (4–38) weeks. This study has demonstrated that MVA-nefis safe and immunogenic in HIV-1-infected subjects and has provided encouraging data on the potential of therapeutic vaccinations.

ABSTRACTMore effective vaccines against Mycobacterium tuberculosismay contribute to the control of this major human pathogen. DNA vaccines encoding single mycobacterial proteins stimulate antimycobacterial T-cell responses and induce partial protection against M. tuberculosisin animal models. The protective efficacy of these vaccines encoding a single antigen, however, has been less than that afforded by the current vaccine, Mycobacterium bovisbacillus Calmette-Guérin (BCG). The heterodimeric cytokine interleukin-12 (IL-12) potentiates the induction and maintenance of the type 1 helper T-cell response. We have developed a novel self-splicing vector based on the 2A protein of foot-and-mouth disease virus that permits the coordinate expression of both chains of IL-12 (p2AIL12). Coimmunization with this vector and DNA expressing M. tuberculosisantigen 85B or MPT64 enhanced the specific lymphocyte proliferative response and increased the frequency of specific gamma interferon-secreting T cells against the whole protein and a defined CD8+T-cell epitope on MPT64. Further, coimmunizing with p2AIL12 significantly increased the protective efficacy of DNA-85 in the lung against an aerosol challenge with M. tuberculosisto the level achieved with BCG. Therefore, codelivery of an IL-12-secreting plasmid may be a potent strategy for enhancing the protective efficacy of vaccines against M. tuberculosis.

More effective vaccines against Mycobacterium tuberculosis may contribute to the control of this major human pathogen. DNA vaccines encoding single mycobacterial proteins stimulate antimycobacterial T-cell responses and induce partial protection against M. tuberculosis in animal models. The protective efficacy of these vaccines encoding a single antigen, however, has been less than that afforded by the current vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG). The heterodimeric cytokine interleukin-12 (IL-12) potentiates the induction and maintenance of the type 1 helper T-cell response. We have developed a novel self-splicing vector based on the 2A protein of foot-and-mouth disease virus that permits the coordinate expression of both chains of IL-12 (p2AIL12). Coimmunization with this vector and DNA expressing M. tuberculosis antigen 85B or MPT64 enhanced the specific lymphocyte proliferative response and increased the frequency of specific gamma interferon-secreting T cells against the whole protein and a defined CD8(+) T-cell epitope on MPT64. Further, coimmunizing with p2AIL12 significantly increased the protective efficacy of DNA-85 in the lung against an aerosol challenge with M. tuberculosis to the level achieved with BCG. Therefore, codelivery of an IL-12-secreting plasmid may be a potent strategy for enhancing the protective efficacy of vaccines against M. tuberculosis.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 X 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked subunits (p40 and p35) encoded by separate genes. We used the apparent autocleavage property of a 2A peptide from the foot-and-mouth disease virus (FMDV) to express bovine (Bo) IL-12 as a self-processing polypeptide (p402Ap35). We demonstrate that 2A will mediate the cleavage of p402Ap35 into two separate subunits in a manner similar to that observed during the processing of the FMDV polypeptide. Furthermore, this 2A polypeptide encoded a functional heterodimer, which elicited activities associated with IL-12 in other species. We propose that this strategy of self-processing polypeptides may be used in many applications where the coordinated and stoichiometric expression of complex proteins is required.

The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin‐dependent uptake mechanisms, endocytic pathways involving clathrin‐coated pits, and the mannose receptor. Furthermore, co‐localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established. J. Leukoc. Biol.66: 50–58; 1999.

ABSTRACTA large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (ΔPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting ΔPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding ΔPLD. While all groups of sheep vaccinated with DNA encoding ΔPLD were afforded better protection against an experimental challenge withC. pseudotuberculosisthan those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.