Cristina Bergamaschi

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor α (IL-15Rα) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Rα, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Rα expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Rα also increased the number of CD44high memory cells with effector phenotype (CD44highCD62L-). Thus, mutual stabilization of IL-15 and IL-15Rα leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor α either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Rα in the same cell and suggest that the IL-15Rα is part of the active IL-15 cytokine rather than part of the receptor.

Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum

IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα. We purified human IL-15/IL-15Rα complexes from overproducing human cell lines and developed an ELISA specifically measuring the heterodimeric form of IL-15. Analysis of sera from melanoma patients after lymphodepletion revealed the presence of circulating IL-15/IL-15Rα complexes in amounts similar to the total IL-15 quantified by a commercial IL-15 ELISA that detects both the single-chain and the heterodimeric forms of the cytokine. Therefore, in lymphodepleted cancer patients, the serum IL-15 is exclusively present in its heterodimeric form. Analysis of the form of IL-15 present in either normal or lymphodepleted mice agrees with the human data. These results have important implications for development of assays and materials for clinical applications of IL-15.

Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia. Furthermore, after vaccination with SIV expression plasmids the recall responses to the SIV antigens were very high, indicating that DNA is a strong boost in the presence of antiretroviral treatment in SIV-infected animals. There was substantial animal-to-animal variability in DNA expression, revealed by plasma measurements of IL-15 produced by co-injected IL-15 DNA. IL-15 expression levels correlated with peak immune responses. Electroporation led to an expansion of antigen-specific CD4+ and CD8+ T cells of both central and effector memory phenotype. These results indicate that improved gene delivery and expression by electroporation dramatically increases immunogenicity of DNA vaccines. Electroporation is thus an important method to improve the effectiveness of DNA vaccination.

Differential Involvement of Vascular Endothelial Growth Factor in the Survival of Hypoxic Colon Cancer Cells

The recent approval of bevacizumab (Avastin), a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with chemotherapy for the treatment of patients with metastatic colorectal cancer, has provided proof of principle of the efficacy of antiangiogenic strategies for cancer therapy. The activity of bevacizumab is primarily attributed to its ability to inhibit endothelial cell survival. Whether anti-VEGF strategies may also have a direct effect on cancer cell survival is poorly understood. We show that serum-starved colon cancer cells differentially respond to autocrine production of VEGF with the induction of hypoxia inducible factor-1 alpha (HIF-1 alpha) and survival under hypoxic conditions. Inhibition of VEGF or VEGF receptor 2 (VEGFR2)/KDR, but not VEGFR1/Flt-1, was sufficient to abrogate VEGF-mediated induction of HIF-1 alpha and survival in sensitive HCT116, but not in resistant HT29, colon cancer cells. These results provide evidence that a VEGF/KDR/HIF-1 alpha autocrine loop differentially mediates survival of hypoxic colon cancer cells, and they suggest that colon cancer cells may be intrinsically sensitive or resistant to anti-VEGF strategies, which may determine the therapeutic efficacy of bevacizumab.

DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge

Optimized plasmid DNAs encoding the majority of SIVmac239 proteins and delivered by electroporation (EP) elicited strong immune responses in rhesus macaques. Vaccination decreased viremia in both the acute and chronic phases of infection after challenge with pathogenic SIVmac251. Two groups of macaques were vaccinated with DNA plasmids producing different antigen forms, “native” and “modified,” inducing distinct immune responses. Both groups showed significantly lower viremia during the acute phase of infection, whereas the group immunized with the native antigens showed better protection during the chronic phase (1.7 log decrease in virus load, P = 0.009). Both groups developed strong cellular and humoral responses against the DNA vaccine antigens, which included Gag, Pol, Env, Nef, and Tat. Vaccination induced both central memory and effector memory T cells that were maintained at the day of challenge, suggesting the potential for rapid mobilization upon virus challenge. The group receiving the native antigens developed higher and more durable anti-Env antibodies, including neutralizing antibodies at the day of challenge. These results demonstrate that DNA vaccination in the absence of any heterologous boost can provide protection from high viremia comparable to any other vaccine modalities tested in this macaque model.

Long-lasting humoral and cellular immune responses and mucosal dissemination after intramuscular DNA immunization.

Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat and Vif), long-lasting (up to 90 weeks post third vaccination) and were boosted with each subsequent immunization, even after an extended 90-week rest period. The SIV-specific cellular immune responses were consistently more abundant in bronchoalveolar lavage (BAL) than in blood, and were characterized as predominantly effector memory CD4(+) and CD8(+) T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.

The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit IMPLICATIONS FOR IMPROVED IL-12 CYTOKINE PRODUCTION

Background: The biosynthesis of IL-12p70 depends on the intracellular interaction of its p35 and p40 subunits. Results: The p40 subunit stabilizes p35 and promotes its secretion. Conclusion: Understanding the regulatory steps of IL-12 biosynthesis led to the generation of optimized IL-12 plasmids. Significance: Availability of expression-optimized IL-12 DNA plasmids is important for practical applications as DNA vaccine adjuvants and in cancer immunotherapy. IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ∼1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

Characterization and favorable in vivo properties of heterodimeric soluble IL-15/IL-15Rα cytokine compared to IL-15 monomer

Background: IL-15 acts in vivo in association with IL-15Rα, as a heterodimeric cytokine. Results: Authentically processed and glycosylated IL-15·sIL-15Rα heterodimer was purified from human cells and characterized by sequencing and functional studies. Conclusion: IL-15 heterodimer shows favorable in vivo properties compared with monomeric IL-15. Significance: Characterization and availability of the IL-15 heterodimer is important for functional studies and clinical applications. Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.

Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques

Optimized DNA vectors were constructed comprising the proteome of SIV including the structural, enzymatic, regulatory, and accessory proteins. In addition to native antigens as produced by the virus, fusion proteins and modified antigens with altered secretion, cellular localization and stability characteristics were generated. The DNA vectors were tested for expression upon transfection in human cells. In addition, the vectors were tested either alone or in combinations in mice and macaques, which provided an opportunity to compare immune responses in two animal models. DNA only immunization using intramuscular injection in the absence or presence of in vivo electroporation did not alter the phenotype of the induced T cell responses in mice. Although several fusion proteins induced immune responses to all the components of a polyprotein, we noted fusion proteins that abrogated immune response to some of the components. Since the expression levels of such fusion proteins were not affected, these data suggest that the immune recognition of certain components was altered by the fusion. Testing different DNA vectors in mice and macaques revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine.

Secretion and Biological Activity of Short Signal Peptide IL-15 Is Chaperoned by IL-15 Receptor Alpha In Vivo

The two known isoforms of IL-15 contain either a long signal peptide (LSP) or a short signal peptide (SSP), and are produced by alternatively spliced transcripts. It has been proposed that SSP IL-15 remains exclusively intracellular, and its function is unclear. In this study, we show that, similar to LSP IL-15, the SSP IL-15 is stabilized and secreted efficiently upon coexpression of IL-15Rα. Coinjection of SSP IL-15- and IL-15Rα-expressing plasmids into mice resulted in increased plasma levels of bioactive heterodimeric IL-15 and mobilization and expansion of NK and T cells. Therefore, SSP IL-15 is secreted and bioactive when produced as a heterodimer with IL-15Rα in the same cell. The apparent t1/2 of this heterodimer is lower compared with LSP IL-15/IL-15Rα, due to different intracellular processing. Coexpression of both LSP IL-15 and SSP IL-15 in the presence of IL-15Rα results in lower levels of bioactive IL-15, indicating that LSP and SSP IL-15 compete for the binding to IL-15Rα when expressed in the same cell. Because the SSP IL-15 interaction to IL-15Rα leads to a complex with lower apparent stability, SSP IL-15 functions as competitive inhibitor of LSP IL-15. The data suggest that usage of alternative splicing is an additional level of control of IL-15 activity. Expression of both SSP and LSP forms of IL-15 appears to be conserved in many mammals, suggesting that SSP may be important for expressing a form of IL-15 with lower magnitude or duration of biological effects.