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Construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18.

Selection of potent cytokine adjuvants is important for the development of Staphylococcus aureus DNA vaccines. Several potential cytokines have been proven to induce enhanced immune responses in animal models and clinical tests. There is still no reported use of IL18 as an adjuvant to design DNA vaccines against S. aureus. In this study, we cloned the main fibronectin binding protein gene (a fragment from clumping factor A, ClfA(221-550)) of S. aureus and bovine interleukin 18 (bIL18). Then recombinant plasmids were constructed based on the eukaryotic expression vector pVAX1 with or without bIL18. Indirect immunofluorescence assays in transfected HeLa cells indicated that the recombinant DNAs (rDNAs) could be expressed correctly and had antigenicity. BALB/c mice were used as experimental models to examine the immunogenicity of rDNAs in vivo. The ClfA(221-550) rDNA provoked antibody production. The bIL18 rDNA induced production of the Th1 type cytokines IL2 and IFNgamma, and ClfA(221-550) and bIL18 synergistically stimulated T-lymphocyte proliferation. The data demonstrated that bIL18 is a potent adjuvant that could be used to enhance cellular immunity.

1662 related Products with: Construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18.

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A hollow-fiber bioreactor for expanding HIV-1 in human lymphocytes used in preparing an inactivated vaccine candidate.

An inactivated HIV vaccine intended to elicit broadly neutralizing antibodies is designed to use a pool of population-prevalent HIV-1 from plasma (PHIV), isolated before evolution of antibody-mediated genetic mutations. A suitable cell substrate (CS) for isolating such PHIV is peripheral blood mononuclear cells (PBMC) after stimulating with phytohemagglutinin (PHA) and interleukin-2 (IL-2). Feasibility of employing a hollow-fiber bioreactor under optimized conditions was investigated for large-scale expansion and efficient recovery of concentrated PHIV. Each CS batch was infected in vitro with a prototype PHIV, the infected cells were introduced into the bioreactor for 7-10 days in co-culture, and the cell-free supernatants were assayed for p24 antigen as an index of HIV synthesis. PBMC versus CD8-depleted (CD8D) CS, 20kDa versus 5kDa molecular weight cut-off (MWCO) bioreactor cartridges, 7- versus 10-day culture periods, and varying concentrations of IL-2, fetal bovine serum (FBS) and glucose content in the medium were functionally evaluated for p24 yield. PBMC cultures in 20kDa MWCO cartridges with 15% FBS, 80IU/mL IL-2 and 2.0g/L glucose produced the highest p24 yield; however, CD8D-CS, 20-30% FBS and 80 IU/mL IL-2 within 5kDa cartridges and 2.0 g/L glucose in the circulating medium was more cost-effective for synthesis of virion p24.

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Analysis of recombinant and modified proteins by capillary zone electrophoresis coupled with electrospray ionization tandem mass spectrometry.

A method for rapid characterization of recombinant and modified proteins with known sequences is described. The analytical system consists of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization ion trap tandem mass spectrometer via a sheath-flow interface. Following the procedure consists of proteolytic fragmentation, CZE peptide separation, tandem mass spectrometry (MS-MS) analysis of separated peptides, sequence database search and monitoring of the specific peptides, C 125 S mutated interleukin 2 (S-125-IL2) and bovine beta-casein were characterized as a model of recombinant protein and naturally modified protein, respectively. A tryptic peptide mixture derived from the synthetic salmon calcitonin (s-CT) was also analyzed to test the performance of the system. Although a conventional sheath-flow interface with much higher flow-rate compared to the microspray interface and nanospray interface was used, the proteins were identified at the low picomole level.

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Delivery of the Theileria parva p67 antigen to cattle using recombinant vaccinia virus: IL-2 enhances protection.

To evaluate vaccinia virus as a delivery system for recombinant antigen in cattle, calves were immunized with a recombinant vaccinia virus (rVV) expressing the sporozoite surface antigen (p67) of Theileria parva (V-67) combined with those expressing bovine IL-4 (V-IL4) or IL-2 (V-IL2). The anti-p67 antibody levels detected in calves inoculated with the combination of V-67 and V-IL4 were higher than those produced by animals injected with V-67 alone or V-67 and V-IL2. On challenge with cryopreserved sporozoites, 5 of 7 animals receiving V-67 combined with V-IL2 were protected, while those receiving V-67 in conjunction with V-IL4 behaved like unimmunized control calves. Vaccination with a recombinant virus expressing a chimaeric p67(p583)/IL2 product gave rise to a lower level of protection, whereas V-IL2 provided no immunity. The results of this study demonstrate the potential of rVV as a delivery system for use in vaccination of cattle against Theileria parva infection.

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Oral tolerization to adenoviral antigens permits long-term gene expression using recombinant adenoviral vectors.

Recombinant adenoviruses (Ads) efficiently transfer foreign genes into hepatocytes in vivo, but the duration of transgene expression is limited by the host immune response which precludes gene expression upon readministration of the virus. To test if this immune response can be abrogated by oral tolerization, we instilled protein extracts of a recombinant adenovirus type-5 via gastroduodenostomy tubes into bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient jaundiced Gunn rats. Control rats received BSA. Subsequent intravenous injection 5 x 10(9) pfu of a recombinant adenovirus-expressing human BUGT1 (Ad-hBUGT1) resulted in hepatic expression of human BUGT1 (hBUGT1) with reduction of serum bilirubin levels by 70%. After 2 mo serum bilirubin increased gradually. In orally tolerized rats, but not in controls, a second dose of the virus on day 98 markedly reduced serum bilirubin again. In the tolerized rats, the development of antiadenoviral neutralizing antibodies and cytotoxic lymphocytes were markedly inhibited, and transplantation of their splenocytes into naive Gunn rats adoptively transferred the tolerance, indicating a role for regulatory cells. Lymphocytes from the tolerized rats hyperexpressed TGFbeta1, IL2, and IL4 upon exposure to viral antigens, whereas IFNgamma expression became undetectable. Thus, oral tolerization with adenoviral antigens permits long-term gene expression by repeated injections of recombinant adenoviruses.

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Recombinant human interleukin 2 induces proliferation and immunoglobulin secretion by bovine B-cells: tissue differences and preferential enhancement of immunoglobulin A.

Recombinant human interleukin 2 (rhIL2) was found to have activity on bovine B-cells in vitro, inducing both their proliferation and differentiation into immunoglobulin secreting cells. Only low and medium density (activated) B-cells were responsive; high density (resting) B-cells did not respond to rhIL2. Proliferative responses by unfractionated and purified B-cell populations were similar for all tissues tested. In contrast, immunoglobulin secretion was stimulated to a greater degree in prescapular lymph node (PSLN) and bronchial lymph node (BLN) cells than in mesenteric lymph node (MLN) cells and splenic lymphocytes on culture with IL2. All isotypes were stimulated equally in the case of PSLN, BLN, and splenic lymphocytes. However, culture of unfractionated MLN lymphocytes with IL2 resulted in enhanced secretion of IgA relative to the other isotypes. Removal of T-cells and accessory cells from the MLN lymphocyte preparation resulted in this effect being lost.

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Differential in situ expansion and gene expression of CD4+ and CD8+ tumor-infiltrating lymphocytes following adoptive immunotherapy in a murine tumor model system.

In previous reports, we demonstrated that adoptively transferred T cells homed to the tumor site (among other sites) and that amplification of immune responses occurred in situ leading to the generation of cytotoxic CD8+ tumor-infiltrating lymphocytes (TIL) and macrophages. The present report extends these findings and shows that following adoptive immunotherapy (AIT) of mice bearing the immunogenic transplanted methylcholanthrene-induced rhabdomyosarcoma (MCA/76-9) there was a differential expansion of CD4+ and CD8+ TIL, the numbers peaking on days 6 and 8, respectively. At this time, CD8+ TIL accounted for the majority of Thy-1+ cells. Northern analyses of RNA extracted from positively selected (by panning) Thy-1+, CD8+ and CD4+ TIL isolated 8 days after AIT indicated the following: in five separate experiments, CD4+ cells expressed three- to sixfold more interleukin (IL)2 mRNA and six- to eightfold more IL6 mRNA than CD8+ cells, while CD8+ TIL expressed three- to sixfold more IL2 receptor (IL2R) mRNA and four- to sixfold more interferon-gamma mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL2 over a 24-h period, whereas both IL6 and interferon-gamma activities were demonstrable. The level of IL2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL2 and only a low response by CD4+ cells suggesting that most of the CD4+ TIL were in the resting stage. This was confirmed when it was shown that the incubation of panned CD4+ TIL with IL2 supplemented with irradiated spleen cells and "spent" 76-9 tumor culture supernatant (as a source of antigen) induced expansion of TIL resulting in a population consisting of greater than 90% CD4+ TIL. The overall data suggest that the relatively deactivated state of the CD4+ TIL at this particular time reflects the status of the rejection process in terms of the absence or low concentration of stimulating tumor-associated antigen.

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Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.

Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.

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Bovine interleukin 2: cloning, high level expression, and purification.

We utilized a human IL2 probe to isolate bovine IL2 sequences from a lymph node cDNA library. Bovine IL2 was subsequently expressed in both bacteria and yeast. Using a rapid, two-step purification scheme, we have been able to isolate over 20 mg/l of homogenous bovine rIL2 secreted from the yeast. The availability of sizable quantities of bovine rIL2 should make it possible to ascertain potential therapeutic or prophylactic utility of this lymphokine in cattle.

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