Inhibition of tumor growth by cancer vaccine combined with metronomic chemotherapy and anti-PD-1 in a pre-clinical setting.

Tumor microenvironment (TME) is characterized by multiple immune suppressive mechanisms able to suppress anti-tumor effector cell immunity. Combinatorial strategies, including vaccine and immunomodulatory drugs, need to be developed for improved immunotherapy efficacy. A novel combinatorial approach was assessed in C57BL/6 mice injected with mouse melanoma B16F10 cells. A multi-peptide vaccine (PEPT) was combined with a low dose metronomic chemotherapy (MCT) and an anti-PD-1 checkpoint inhibitor (CI). Statistical analysis were performed with the unpaired two-sided Student's t-test and ANOVA. Animals treated with the multi-peptide vaccine combined with MCT or CI showed remarkable delay in tumor growth and prolonged survival as compared to control groups. The multi-pronged combination including PEPT+MCT+CI was able to prolong survival in all mice and inhibit tumor growth in 66.6% of mice. All animals which did not show tumor growth were re-challenged with the same melanoma cells and one of them showed complete tumor growth inhibition. The anti-tumor effect was associated with strong T cell immune response to vaccine mutated peptides and significant reduction of regulatory T cells. The combination of a vaccine with MCT and CI was highly efficient in potentiating the vaccine's anti-tumor effects. The approach is highly promising to be moved into clinical trial.

Use of adjuvants for immunotherapy.

Cancer vaccines are designed to stimulate the body's immune system to kill tumor cells. To improve their immunogenicity, vaccine antigens must be combined with adjuvants which are able to stimulate the innate immunity and potentiate the adaptive immune response. In the last years a new generation of adjuvants mimicking the natural microbial ligands have been developed. In particular, several TLR ligands have been extensively explored as vaccine adjuvants and many preclinical and clinical studies have been conducted. However, the road to approval of such adjuvants for clinical use is still to go.

A novel multi-drug metronomic chemotherapy significantly delays tumor growth in mice.

BACKGROUND: The tumor immunosuppressive microenvironment represents a major obstacle to an effective tumor-specific cellular immune response. METHODS: In the present study, the counterbalance effect of a novel metronomic chemotherapy protocol on such an immunosuppressive microenvironment was evaluated in a mouse model upon sub-cutaneous ectopic implantation of B16 melanoma cells. The chemotherapy consisted of a novel multi-drug cocktail including taxanes and alkylating agents, administered in a daily metronomic fashion. The newly designed strategy was shown to be safe, well tolerated and significantly efficacious. RESULTS: Treated animals showed a remarkable delay in tumor growth and prolonged survival as compared to control group. Such an effect was directly correlated with CD4(+) T cell reduction and CD8(+) T cell increase. Furthermore, a significant reduction in the percentage of both CD25(+)FoxP3(+) and CD25(+)CD127(low) regulatory T cell population was found both in the spleens and in the tumor lesions. Finally, the metronomic chemotherapy induced an intrinsic CD8(+) T cell response specific to B16 naturally expressed Trp2 TAA. CONCLUSION: The novel multi-drug daily metronomic chemotherapy evaluated in the present study was very effective in counterbalancing the immunosuppressive tumor microenvironment. Consequently, the intrinsic anti-tumor T cell immunity could exert its function, targeting specific TAA and significantly containing tumor growth. Overall, the results show that this represents a promising adjuvant approach to significantly enhance efficacy of intrinsic or vaccine-elicited tumor-specific cellular immunity.

Systems vaccinology for cancer vaccine development.

Results of therapeutic vaccines for established chronic infections or cancers are still unsatisfactory. The only therapeutic cancer vaccine approved for clinical use is the sipuleucel-T, for the treatment of metastatic prostate cancer, which induces a limited 4-month improvement in the overall survival of vaccinated patients compared to controls. This represents a remarkable advancement in the cancer immunotherapy field, although the clinical outcome of cancer vaccines needs to be substantially improved. To this aim, a multipronged strategy is required, including the evaluation of mechanisms underlying the effective elicitation of immune responses by cancer vaccines. The recent development of new technologies and computational tools allows the comprehensive and quantitative analysis of the interactions between all of the components of innate and adaptive immunity over time. Here we review the potentiality of systems biology in providing novel insights in the mechanisms of action of vaccines to improve their design and effectiveness.

Development of a stable insect cell line constitutively expressing rotavirus VP2.

An insect High-Five cell line was generated constitutively and stably expressing the VP2 protein of rotavirus RF strain leading to the formation of core-like particles. The integration of the VP2 gene was confirmed by PCR, Real-time PCR and Southern blot, and the protein expression was confirmed by Western blot and immunofluorescence microscopy. Integrity and self assembly of VP2 as core-like particles was demonstrated by electron microscopy. The High-Five cell system stably expressing rotavirus core-like particles can be applied to the study of viral protein structure and functions; it may be useful for vaccine development, gene therapy and drug delivery.

Molecular characterization analysis of the outer protein layer (VP7) from human rotavirus A genotype G1 isolate identified in Iran: implications for vaccine development.

The full open reading frame of the outer protein layer VP7 from an isolate of human rotavirus identified in 2010 in an Iranian child admitted to hospital with gastroenteritis was amplified from a clinical stool specimen and subjected to molecular characterization. Genetic and phylogenetic analyses indicated that the analyzed gene falls into the G1 genotype forming a sub-cluster with sequences recently identified in Iran and geographically distant countries. Such results were confirmed by protein sequence alignment, showing a highly conserved "G1-like?? amino acid sequence pattern within the known three main immunodominant regions. These results are extremely relevant in a perspective of vaccine development. Indeed, the present study confirms that the A group G1 genotype is the most prevalent Rotavirus circulating in Iran and supports the development of G1 genotype-based rotavirus vaccine for this country.

Immunogenomics approaches for vaccine evaluation.

Vaccines represent a potent tool to prevent or contain diseases with high morbidity or mortality. However, despite their widespread use, there is still a limited understanding of the mechanisms underlying the effective elicitation of protective immune responses by vaccines. The integrated co-operation between cells and molecules of innate and adaptive immune systems is under intense study by several groups and constantly updated. The recent development of new technologies and computational tools permits the comprehensive and quantitative analysis of the interactions between all of the components of immunity over time. This study reviews recent progress in exploiting an immunogenomics approach, within the systems biology strategy, to study and evaluate vaccine strategies for infectious and neoplastic diseases. The final goal of this approach is 2-fold, looking for novel and unpredictable mechanisms as well as identifying common immune signatures, relevant for predicting immune responsiveness to improve the design of vaccine strategies. Such approach, indeed, would enable the switch from 'empirical' to 'knowledge-based' vaccinology, leading to a patient-tailored treatment.

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-Hodgkin's 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.

Gene profiling, biomarkers and pathways characterizing HCV-related hepatocellular carcinoma.

BACKGROUND: Hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The molecular mechanisms of HCV-induced hepatocarcinogenesis are not yet fully elucidated. Besides indirect effects as tissue inflammation and regeneration, a more direct oncogenic activity of HCV can be postulated leading to an altered expression of cellular genes by early HCV viral proteins. In the present study, a comparison of gene expression patterns has been performed by microarray analysis on liver biopsies from HCV-positive HCC patients and HCV-negative controls. METHODS: Gene expression profiling of liver tissues has been performed using a high-density microarray containing 36'000 oligos, representing 90% of the human genes. Samples were obtained from 14 patients affected by HCV-related HCC and 7 HCV-negative non-liver-cancer patients, enrolled at INT in Naples. Transcriptional profiles identified in liver biopsies from HCC nodules and paired non-adjacent non-HCC liver tissue of the same HCV-positive patients were compared to those from HCV-negative controls by the Cluster program. The pathway analysis was performed using the BRB-Array- Tools based on the "Ingenuity System Database". Significance threshold of t-test was set at 0.001. RESULTS: Significant differences were found between the expression patterns of several genes falling into different metabolic and inflammation/immunity pathways in HCV-related HCC tissues as well as the non-HCC counterpart compared to normal liver tissues. Only few genes were found differentially expressed between HCV-related HCC tissues and paired non-HCC counterpart. CONCLUSION: In this study, informative data on the global gene expression pattern of HCV-related HCC and non-HCC counterpart, as well as on their difference with the one observed in normal liver tissues have been obtained. These results may lead to the identification of specific biomarkers relevant to develop tools for detection, diagnosis, and classification of HCV-related HCC.

Evolution of the HIV-1 V3 region in the Italian epidemic.

The epidemic of Human Immunodeficiency Virus type 1 infection in Italy is mostly ascribed to the B subtype, which represents the prevalent subtype in Western Countries. The virus isolates of the B subtype, moreover, show an increasing nucleotide heterogeneity over time, indicating a continuous intra-subtype dynamic evolution, typical of long-lasting epidemics. In recent years, however, the progressive decrease in the transmission rate among the historically defined risk groups (i.e. homosexuals and IDUs) and the parallel increase in heterosexual transmission are slowly introducing variants of non-B subtype into the Italian HIV-1 epidemic. This appears to be strictly linked to the growing number of immigrants from non-Western Countries, where non-B clades and CRFs are prevalent, and consequent inter-racial blending. The distribution of these novel genetic forms needs to be evaluated by continuous molecular monitoring nationwide to verify whether they will overcome the pre-existing B-clade epidemic, which could have significant implications for diagnosis, treatment and vaccine development. Here we review the genetic evolution of HIV-1 spreading within the Italian epidemic.