Paola Rizza

Chimeric Plant Virus Particles as Immunogens for Inducing Murine and Human Immune Responses against Human Immunodeficiency Virus Type 1

ABSTRACT The high-yield expression of a neutralizing epitope from human immunodeficiency virus type 1 (HIV-1) on the surface of a plant virus and its immunogenicity are presented. The highly conserved ELDKWA epitope from glycoprotein (gp) 41 was expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. The resulting chimeric virus particles (CVPs), purified and used to immunize mice intraperitoneally or intranasally, were able to elicit high levels of HIV-1-specific immunoglobulin G (IgG) and IgA antibodies. Furthermore, the human immune response to CVPs was studied with severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). hu-PBL-SCID mice immunized with CVP-pulsed autologous dendritic cells were able to mount a specific human primary antibody response against the gp41-derived epitope. Notably, sera from both normal and hu-PBL-SCID mice showed an anti-HIV-1-neutralizing activity. Thus, PVX-based CVPs carrying neutralizing epitopes can offer novel perspectives for the development of effective vaccines against HIV and, more generally, for the design of new vaccination strategies in humans.

Recent advances on the immunomodulatory effects of IFN-α: Implications for cancer immunotherapy and autoimmunity

Interferons alpha (IFNs-α) are pleiotropic cytokines belonging to the type I IFN family, originally described for their antiviral activity. These cytokines exhibit a long record of clinical use in patients with some types of cancer and viral diseases. Notably, certain autoimmune disorders have been postulated to be mediated by endogenous IFN-α and are often observed in some IFN-treated patients. IFN-α can induce multiple biological effects, including induction/promotion of apoptosis and inhibition of cell growth. In addition, these cytokines promote the differentiation and activity of host immune cells. Early studies in mouse tumor models showed the importance of host immune mechanisms in the generation of a long-lasting antitumor response after injection of the animals with either IFN or tumor cells genetically modified for IFN-α production. Several studies have shown that IFN-α can induce the rapid differentiation of monocytes into highly activated dendritic cells (DCs). Of note, these DCs (IFN-DCs) are particularly effective in taking up complex antigens and inducing T- and B-cell immunity. The ensemble of these results suggests that IFN-DCs can play a role in the generation of antitumor T-cell immunity, pointing out that these cells could be successfully used in strategies of cancer immunotherapy. Likewise, IFN-α-DC interactions could also play a role in the pathogenesis of some autoimmune disorders, often associated with IFN-α treatment. All this reveals the complexity of the IFN-α-DC interactions under normal and pathological conditions and stimulates further studies for identifying optimal modalities in either using these cytokines or controlling their production/action in patients.

Constitutive expression of specific interferon isotypes in peripheral blood leukocytes from normal individuals and in promonocytic U937 cells

Constitutive expression of IFN‐α5 and IFN‐β was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase–polymerase chain reaction and direct sequencing of the amplified product. The activated form of the interferon‐induced transcription factor complex ISGF3 was also dectected in nuclear extracts from uninduced cells. Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene, indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU/mL. This endogenous IFN was also shown to play a role in mamtaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells. These results suggest that IFN‐α5 and IFN‐β are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis.

Cytokines as natural adjuvants for vaccines: where are we now?

Abstract The International Meeting on Cytokines as Natural Adjuvants: Perspectives for Vaccine Development was held at the Istituto Superiore di Sanita, Rome, Italy from 22–24 April 2002.

Recombinant interferon-α2b improves immune response to hepatitis B vaccination in haemodialysis patients: Results of a randomised clinical trial

The use of adjuvants capable of improving the deficient immune response to hepatitis B virus (HBV) vaccine in haemodialysis patients is highly needed. Among potential adjuvants, type I interferons deserve a special attention in view of their known effects promoting cellular and humoral immune responses. The aim of the present trial was to evaluate the effects of recombinant interferon-alpha2b (IFN) administered as an adjuvant of HBV vaccine in unvaccinated haemodialysis patients. A significant and early enhancing effect on the antibody response was observed in patients receiving IFN. In addition, a predominance of IgG1 anti-HBs along with a transient normalization of circulating Th1 lymphocytes was only found in patients receiving IFN who achieved an early seroprotection. However, 6 months after the last vaccine dose, no significant differences were observed in the seroprotection rate achieved in patients vaccinated with IFN compared to that in patients receiving HBV vaccine alone. Mild to moderate fever, asthenia, and arthromyalgia were the most common reactions that occurred in vaccinees given IFN. In conclusion, addition of IFN to HBV vaccine, under the conditions used in this trial, is safe and achieves an earlier and higher seroprotection rate improving Th1-dependent immune response in haemodialysis patients.

Translational research on advanced therapies

Fostering translational research of advanced therapies has become a major priority of both scientific community and national governments. Advanced therapy medicinal products (ATMP) are a new medicinal product category comprising gene therapy and cell-based medicinal products as well as tissue engineered medicinal products. ATMP development opens novel avenues for therapeutic approaches in numerous diseases, including cancer and neurodegenerative and cardiovascular diseases. However, there are important bottlenecks for their development due to the complexity of the regulatory framework, the high costs and the needs for good manufacturing practice (GMP) facilities and new end-points for clinical experimentation. Thus, a strategic cooperation between different stakeholders (academia, industry and experts in regulatory issues) is strongly needed. Recently, a great importance has been given to research infrastructures dedicated to foster translational medicine of advanced therapies. Some ongoing European initiatives in this field are presented and their potential impact is discussed.

IFN-α as a vaccine adjuvant: recent insights into the mechanisms and perspectives for its clinical use

The IFN-α family are pleiotropic cytokines with the longest record of clinical use. Over the last decade, new biological effects of IFN-α on immune cells, including dendritic cells, have been described, supporting the concept that these cytokines can act as effective vaccine adjuvants. Recently, an important advance in our understanding of the mechanisms of interferon adjuvant activity has been achieved. Some clinical studies have been performed to assess the adjuvant activity in individuals immunized with preventive vaccines, showing variable results depending on interferon/vaccine formulation and vaccinated subjects. In spite of many data in animal models, little information is available on the possible advantage of utilizing IFN-α as an adjuvant for cancer vaccines in humans. Further clinical trials specifically designed to explore vaccine adjuvant activity are needed in order to define the best conditions for using IFN-α or IFN-α-conditioned dendritic cells for the development of therapeutic vaccines.

The SCID mouse reaction to human peripheral blood mononuclear leukocyte engraftment. Neutrophil recruitment induced expression of a wide spectrum of murine cytokines and mouse leukopoiesis, including thymic differentiation.

In this study, we describe the kinetics of host immune reactions occurring in mice with severe combined immunodeficiency (SCID) at different times after the intraperitoneal injection of human peripheral blood mononuclear leukocytes (huPBL). At 24 hr, a massive neutrophil recruitment and an induced expression of a wide spectrum of murine cytokine mRNA (i.e., interleukin [IL]-10, IL-4, IL-6, IL-10, IL-12, tumor necrosis factor [TNF]-α and interferon [IFN]-γ) occurred in the huPBL-SCID mouse peritoneal cavity. By using ELISAs specific for mouse cytokines, large amounts of IL-1-α, TNF-α, IL-6, and IFN-γ were detected in the peritoneal washings of huPBL-SCID mice 1 day after intraperitoneal injection. IL-6 and IFN-γ production persisted for up to 2 weeks after PBL transplantation. Medullary and extramedullary expansion of the SCID mouse hematopoietic cells also occurred in the chimeras as early as 1 week after injection, together with a marked thymic differentiation (murine CD4+/CD8+ cells) at 10–12 weeks after transplantation. On the whole, these results indicate that, after huPBL injection, SCID mice mount a complex multistage immune response. These host reactions should be taken into consideration for any accurate interpretation of results obtained using the huPBL-SCD3 model. The control of responses (by means of specific antibodies to murine cytokines and to granulocytes or through the use of anti-inflammatory drugs) may be helpful in improving the engraftment of huPBL in SCID mice and in furthering our knowledge of the T and B cell-independent natural immune reactions.

Role of type I interferon in inducing a protective immune response: Perspectives for clinical applications

Type I IFNs (IFN-I) are antiviral cytokines endowed with many biological effects, including antitumor activity. Over the last 15 years, an ensemble of studies has revealed that these cytokines play a crucial role in the induction of a protective antitumor immune response. Early in vivo studies in mouse models have been instrumental for understanding the IFN-I-induced host-mediated mechanisms. IFN-α is currently recognized as a powerful inducer of the differentiation/activation of dendritic cells (DCs) and today IFN-α-conditioned DCs represent promising DC candidates for the development of therapeutic cancer vaccines. Moreover, data from pilot clinical trials support the concept of using IFN-α as an enhancer of the response of patients to cancer vaccines. Notably, endogenous IFN-I production does also play a critical role in the antitumor response to some chemotherapeutic agents. Thus, we can now envisage new strategies of clinical use of IFN-α, based on the injection of IFN-conditioned cells as well as the usage of these cytokines as cancer vaccine adjuvants, alone or in combination with other treatments (including epigenetic drugs) to induce an immunogenic cell death and a long lasting antitumor response.

U937-SCID mouse xenografts: a new model for acute in vivo HIV-1 infection suitable to test antiviral strategies

In this study we attempted to develop a new xenochimeric model for HIV infection in SCID mice, characterized by an easy engraftment of target cells, high levels of viremia and long-lasting HIV-1 infection. SCID mice were injected subcutaneously with uninfected human U937 cells and cell-free HIV-1 (IIIB strain) or HIV-1-infected human peripheral blood lymphocytes (PBL). Mice were evaluated for tumor growth, viral infection at the tumor level (DNA-polymerase chain reaction (PCR), RNA-PCR) and immunostaining for the p55/p18 HIV protein) and p24 antigenemia or serum HIV-1 RNA copies. Pretreatment of mice with antibodies to either mouse-IFN alpha/beta or granulocytes resulted in a tumor take and levels of p24 antigenemia higher than in control mice. In mice treated with these antibody preparations, there was a long-lasting HIV infection with the presence of high levels of circulating infectious virus (serum p24 values up to 4000 pg/ml and serum RNA copies up to 5 x 10(7)/ml over 3 months, with the majority of the cells expressing HIV-antigens at the tumor site). Intraperitoneal treatment of SCID mice with AZT (480 mg/kg per day) resulted in a complete inhibition of both p24 and RNA HIV-1 copies in the serum, together with a marked reduction in the number of infected cells and the levels of virus expression at the tumor site. We conclude that some specific features of this model (i.e. easy establishment, high reproducibility, well defined kinetics of virus infection, massive and long persistent viremia) underline the special advantages of its use for testing new antiviral therapies.