Annacarmen Petrizzo

Translating Tumor Antigens into Cancer Vaccines

ABSTRACT Vaccines represent a strategic successful tool used to prevent or contain diseases with high morbidity and/or mortality. However, while vaccines have proven to be effective in combating pathogenic microorganisms, based on the immune recognition of these foreign antigens, vaccines aimed at inducing effective antitumor activity are still unsatisfactory. Nevertheless, the effectiveness of the two licensed cancer-preventive vaccines targeting tumor-associated viral agents (anti-HBV [hepatitis B virus], to prevent HBV-associated hepatocellular carcinoma, and anti-HPV [human papillomavirus], to prevent HPV-associated cervical carcinoma), along with the recent FDA approval of sipuleucel-T (for the therapeutic treatment of prostate cancer), represents a significant advancement in the field of cancer vaccines and a boost for new studies in the field. Specific active immunotherapies based on anticancer vaccines represent, indeed, a field in continuous evolution and expansion. Significant improvements may result from the selection of the appropriate tumor-specific target antigen (to overcome the peripheral immune tolerance) and/or the development of immunization strategies effective at inducing a protective immune response. This review aims to describe the vast spectrum of tumor antigens and strategies to develop cancer vaccines.

Challenges in cancer vaccine development for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver malignancy, representing the third and fifth leading cause of death from cancer worldwide in men and women, respectively. The main risk factor for the development of HCC is the hepatitis B and C virus (HBV and HCV) infection; non-viral causes (e.g., alcoholism and aflatoxin) are additional risk factors. HCC prognosis is generally poor because of the low effectiveness of available treatments and the overall 5-year survival rate is approximately 5-6%. In this framework, immunotherapeutic interventions, including cancer vaccines, may represent a novel and effective therapeutic tool. However, only few immunotherapy trials for HCC have been conducted so far with contrasting results, suggesting that improvements in several aspects of the immunotherapy approaches need to be implemented. In particular, identification of novel specific tumor antigens and evaluation of most advanced combinatorial strategies could result in unprecedented clinical outcomes with great beneficial effect for HCC patients. The state of the art in immunotherapy strategies for HCC and future perspectives are reported in the present review.

Antigen-specific vaccines for cancer treatment

Vaccines targeting pathogens are generally effective and protective because based on foreign non-self antigens which are extremely potent in eliciting an immune response. On the contrary, efficacy of therapeutic cancer vaccines is still disappointing. One of the major reasons for such poor outcome, among others, is the difficulty of identifying tumor-specific target antigens which should be unique to the tumors or, at least, overexpressed on the tumors as compared to normal cells. Indeed, this is the only option to overcome the peripheral immune tolerance and elicit a non toxic immune response. New and more potent strategies are now available to identify specific tumor-associated antigens for development of cancer vaccine approaches aiming at eliciting targeted anti-tumor cellular responses. In the last years this aspect has been addressed and many therapeutic vaccination strategies based on either whole tumor cells or specific antigens have been and are being currently evaluated in clinical trials. This review summarizes the current state of cancer vaccines, mainly focusing on antigen-specific approaches.

Combinatorial immunotherapy strategies for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver malignancy. The prognosis for HCC patients greatly varies according to the stage at diagnosis. Overall it is poor, with a 5-year survival rate of approximately 5-6%. Immunotherapeutic interventions represent a novel and effective therapeutic tool. However, only few immunotherapy trials for HCC have been conducted so far with contrasting results, suggesting that significant improvements are needed. Indeed, the liver is characterized by a strong intrinsic immune suppressive microenvironment which needs to be counterbalanced with immune stimulatory approaches. Therefore, the implementation of combinatorial protocols combining immune stimulatory strategies with specific immunotherapy approaches could result in a dramatic improvement of efficacy and clinical outcome in HCC patients. The present review aims at describing the state of the art in immunotherapy strategies for HCC and future perspectives.

Immune signatures in human PBMCs of idiotypic vaccine for HCV-related lymphoproliferative disorders

Hepatitis C virus (HCV) is one of the major risk factors for chronic hepatitis, which may progress to cirrhosis and hepatocellular carcinoma, as well as for type II mixed cryoglobulinemia (MC), which may further evolve into an overt B-cell non-Hodgkins lymphoma (NHL).It has been previously shown that B-cell receptor (BCR) repertoire, expressed by clonal B-cells involved in type II MC as well as in HCV-associated NHL, is constrained to a limited number of variable heavy (VH)- and light (VL)-chain genes. Among these, the VK3-20 light chain idiotype has been selected as a possible target for passive as well as active immunization strategy.In the present study, we describe the results of a multiparametric analysis of the innate and early adaptive immune response after ex vivo stimulation of human immune cells with the VK3-20 protein. This objective has been pursued by implementing high-throughput technologies such as multiparameter flow cytometry and multiplex analysis of cytokines and chemokines.

Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy

ObjectiveThe continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population.MethodsThe distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995–2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses.Results18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification.ConclusionThe Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.

Functional characterization of biodegradable nanoparticles as antigen delivery system

BackgroundPeptide based vaccines may suffer from limited stability and inefficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development.ResultsFlow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14+ monocytes and mouse Hepa 1–6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4+ T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8+ T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery.ConclusionsOur study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines.

Innate immunity and hepatitis C virus infection: a microarray's view

Hepatitis C virus (HCV) induces a chronic infection in more than two-thirds of HCV infected subjects. The inefficient innate and adaptive immune responses have been shown to play a major pathogenetic role in the development and persistence of HCV chronic infection. Several aspects of the interactions between the virus and the host immune system have been clarified and, in particular, mechanisms have been identified which underlie the ability of HCV to seize and subvert innate as well as adaptive immune responses. The present review summarizes recent findings on the interaction between HCV infection and innate immune response whose final effect is the downstream inefficient development of antigen-specific adaptive immunity, thereby contributing to virus persistence.

Immunological effects of a novel RNA-based adjuvant in liver cancer patients

Evaluation of biological effects of adjuvants on immune cells has been assessed in a limited number of studies. Moreover, no data are available on samples derived from cancer patients who may have a severe immune impairment. The effects of a novel RNA-based adjuvant (RNAdjuvant® developed by CureVac) were assessed in an ex vivo setting on PBMCs obtained from 8 healthy volunteers and 17 HCC patients, using a multiparametric approach to analyze network dynamics of early immune responses. Evaluation of CD80, CD86 and HLA-DR expression, cytokine production as well as gene expression was performed. Moreover, the downstream effect on CD4+ T cell phenotyping was evaluated. Treatment with RNAdjuvant® showed comparable effects on PBMCs of both HCC and healthy subjects. In particular, CD80, CD86 and HLA-DR expression was found up-regulated in circulating dendritic cells, which promoted a CD4+ T cell differentiation toward an effector phenotype. A mixed Th1/Th2 cytokine pattern was induced, although a more predominant production of TNFα and IFNγ was observed in HCC patients versus healthy controls. The cytokine profile was further confirmed by gene transcriptional analysis, which showed up-regulation of several genes involved in innate and adaptive immune-related pathways. The present study is the first demonstration that HCC patients and healthy subjects are equally responsive to an adjuvant. This may suggest that the same vaccine formulation including the RNAdjuvant® might have similar potency in healthy subjects and cancer patients.

Cellular prognostic markers in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the five big killers worldwide and is frequently associated with chronic hepatitis B and C virus (HBV and HCV) infections. Tumor microenvironment consists of a complex network of cells and factors that plays a key role in the tumor progression and prognosis. This is true also for HCC. Several studies have shown strikingly strong correlation between HCC clinical prognosis and intratumoral infiltration of cells affecting tumor growth, invasion, angiogenesis and metastasis. None of such cells is yet validated for routine diagnostic and prognostic assessment. The present review aims at providing a state-of-the-art of such studies.