In some cases these Abs have been suggested to have a protective role against some disease conditions (75,76), by beneficially influencing cytokine localization, half-life and activity (59,77,78). cytokines have a good safety profile in clinical studies. For example, GM-CSF is being used as an adjuvant in several Phase I/II trials against a variety of tumors (37,38), and IL-21 has been tested against metastatic melanoma and renal cancer (39C41). Here, we investigated whether trimeric HIV-1 Env proteins with GM-CSF or IL-21 cytokines incorporated into the V1V2 domain or fused to the C-terminus (Ct) would increase the induction of anti-Env Ab responses cells and isolated using an EndoFree Plasmid Giga kit (Qiagen, Venlo, the Netherlands). New Dihydroethidium Zealand White rabbits (6 or 12 per group) were immunized at weeks 0, 2, 4 and 8 with 125 g of endotoxin-free DNA via the abdominal dermis using gene gun technology (54). Blood samples were obtained at weeks 0, 2, 4, 6 and 8, with a final bleed at week 10. Outbred NMRI mice (4 or 5 5 per group) were immunized at weeks 0, 2, 4, and 6 with 20 g of DNA in the abdominal dermis using the same gene gun method. Blood samples were obtained from weeks 0, 2, 4, and 6, with a final bleed at week 6 or 8. All protocols dealing with animal manipulations were in accordance with guidelines published by FELASA (Federation of European Animal Science Association) Mouse monoclonal to CD54.CT12 reacts withCD54, the 90 kDa intercellular adhesion molecule-1 (ICAM-1). CD54 is expressed at high levels on activated endothelial cells and at moderate levels on activated T lymphocytes, activated B lymphocytes and monocytes. ATL, and some solid tumor cells, also express CD54 rather strongly. CD54 is inducible on epithelial, fibroblastic and endothelial cells and is enhanced by cytokines such as TNF, IL-1 and IFN-g. CD54 acts as a receptor for Rhinovirus or RBCs infected with malarial parasite. CD11a/CD18 or CD11b/CD18 bind to CD54, resulting in an immune reaction and subsequent inflammation and GV-SOLAS (German Society of Laboratory Animal Science) and were reviewed by the Harlan, MFD Diagnostics, or Davids Dihydroethidium Biotechnologie Animal Care Committees, as appropriate. RESULTS Generation and characterization of EnvrGM-CSF and EnvrIL-21 We have previously described the insertion of various cytokines into the V1V2 domain of Envwt (34C36). Here, we used the same approach with the cytokines GM-CSF and IL-21, using rabbit sequences, to generate the EnvrGM-CSF Dihydroethidium and EnvrIL-21 chimeric proteins for studies in rabbits (Fig. 1A). We note that during the course of these studies a better soluble mimic of the native Env spike was identified (BG505 SOSIP.664 gp140), but we did not use it here (45C47). Both chimeric EnvrGM-CSF and EnvrIL-21 proteins were expressed by transient transfection of 293T cells, EnvrGM-CSF expression Dihydroethidium was similar to Envwt whereas EnvrIL-21 was produced less efficiently (see Supplemental Fig. 1A) (36). The antigenic structures of EnvrGM-CSF and EnvrIL-21 were analyzed using an anti-trimer ELISA and were found to be similar to those of EnvhGM-CSF, EnvmGM-CSF, EnvhIL-21 and EnvmIL-21 (Supplemental Fig. 1B) (34C36). Open in a separate window FIGURE 1 Design and Dihydroethidium functionality of the Envwt, EnvrGM-CSF and EnvrIL-21 fusion proteins (A) Cartoon representation of Envwt, EnvrGM-CSF and EnvrIL-21. Ig production by human B cells stimulated with Envwt, EnvrIL-21 or rhIL-21 cellular responses). However, a disadvantage of this approach is that undesired autoimmune responses may be generated against the self-molecule when it is presented in a nonnatural context. Although the beneficial effects of including cytokines in vaccines, particularly DNA vaccines, are being studied extensively, whether autoimmunity arises is rarely investigated. Here, we studied, in both mice and rabbits, whether chimeric cytokine-Env molecules induced autoimmune responses to the cytokine moiety, and whether the location of the cytokine mattered. We found that substantial autoAb responses were raised against GM-CSF and IL-21, when the cytokine was either embedded in the body of the Env protein (i.e. within the V1V2 loop structure) or attached to the Ct (via a flexible linker sequence). We have not evaluated Ct fused cytokines in mice, but we would expect similar results. Whether induction of autoAbs accounts for the failure of the cytokines to boost the Ab responses to the Env remains to be determined. However, we note that EnvmGM-CSF did induce stronger anti-gp120 Ab and T cell responses in mice, indicating that the presence of anti-GM-CSF Abs does not necessarily abolish the adjuvant activity of the embedded cytokine (35). AutoAbs against cytokines may have multiple adverse effects. One example involves some formulations of recombinant human erythropoietin used to treat renal anemia. Several patients developed more severe anemia, which was attributed to the unwanted induction of NAbs against erythropoietin (58). AutoAbs can be induced when recombinant IFN-, IFN- and GM-CSF are repeatedly administered (59C61). In a clinical trial neutropenic cancer patients were treated with.
