Maria Lina Tornesello

Human Immunodeficiency Virus Type 1 Subtype Distribution in the Worldwide Epidemic: Pathogenetic and Therapeutic Implications

Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS (10, 38, 105, 114). It is characterized by extensive and dynamic genetic diversity, generating variants falling into distinct molecular subtypes as well as recombinant forms; these forms display an uneven global distribution (55). This diversity has implications for our understanding of viral transmission, pathogenesis, and diagnosis and profoundly influences strategies for vaccine development. Here we review selected aspects of the genetic diversity of HIV-1, with particular emphasis on its pathogenetic and therapeutic implications.

Herpesvirus-like DNA sequences detected in endemic, classic, iatrogenic and epidemic Kaposi's sarcoma (KS) biopsies

The identification of Kaposis sarcoma (KS) clusters in subequatorial Africa (endemic KS, AKS) and the high frequency of KS in sexually transmitted AIDS (epidemic KS, EKS), have previously suggested a role for infectious agents in the etiopathogenesis of KS. The recent identification of herpesvirus (HHV)‐like DNA sequences in one case of EKS and their detection in >90% of all tested EKS, prompted us to determine the prevalence of these viral sequences in all types of KS, such as AKS, EKS, classic KS (CKS) and iatrogenic KS (IKS). The presence of herpesvirus (HHV)‐like DNA sequences has been examined in 61 KS skin tumors obtained from Greece, Italy, USA, Uganda and Kenya. All KS types (100%) were positive by polymerase chain reaction (PCR) and Southern‐blot analysis, while 5 out of 6 (83%) and 4 out of 7 (57%) uninvolved autologous skin biopsies from AKS and CKS patients, respectively, were positive for HHV‐like sequences. All samples from non‐KS patients were negative, i.e. 17 human biopsies from healthy individuals or patients affected by other pathologies, 5 human cell lines and 15 peripheral blood mononuclear cells (PBMC) from HIV‐positive subjects. These results suggest that HHV‐like sequences play a major role in the pathogenesis of this neoplasm.

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.

Baculovirus-Derived Human Immunodeficiency Virus Type 1 Virus-Like Particles Activate Dendritic Cells and Induce Ex Vivo T-Cell Responses

ABSTRACT We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model based on HIV-1 Pr55gag virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A (HIV-VLPAs). The HIV-VLPAs show the induction in BALB/c mice of systemic and mucosal neutralizing antibodies as well as cytotoxic T lymphocytes, by intraperitoneal as well as intranasal administration. In the present article, the effects of the baculovirus-expressed HIV-VLPs on human immature monocyte-derived dendritic cells (MDDCs) have been evaluated. The HIV-VLPs efficiently induce maturation and activation of MDDCs and are incorporated into MDDCs preferentially via an actin-dependent macropinocytosis and endocytosis. The HIV-VLP-activated MDDCs show enhanced Th1- and Th2-specific cytokine production, and the effects of HIV-VLPs on MDDCs are not mediated through Toll-like receptors 2 and 4 (TLR2 and -4) signaling. Finally, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4+ T cells in an ex vivo immunization assay. Our results on the interaction and processing of baculovirus HIV-VLPs by MDDCs give an insight into the mechanisms underlying the immune response induced by HIV-VLPAs in vivo.

Induction of systemic and mucosal cross-clade neutralizing antibodies in BALB/c mice immunized with human immunodeficiency virus type 1 clade A virus-like particles administered by different routes of inoculation.

ABSTRACT We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model, based on virus-like particles (VLPs) expressing gp120 from a Ugandan HIV-1 isolate of clade A (HIV-VLPAs), which shows the induction of neutralizing antibodies as well as cytotoxic T lymphocytes (CTL) in BALB/c mice by intraperitoneal (i.p.) administration. In the present study, immunization experiments based on a multiple-dose regimen have been performed with BALB/c mice to compare different routes of administration. i.p. and intranasal (i.n.), but not oral, administration induce systemic as well as mucosal (vaginal and intestinal) immunoglobulin G (IgG) and IgA responses. These immune sera exhibit >50% ex vivo neutralizing activity against both autologous and heterologous primary isolates. Furthermore, the administration of HIV-VLPAs by the i.n. immunization route induces a specific CTL activity, although at lower efficiency than the i.p. route. The HIV-VLPAs represent an efficient strategy to stimulate both arms of immunity; furthermore, the induction of specific humoral immunity at mucosal sites, which nowadays represent the main port of entry for HIV-1 infection, is of great interest. All these properties, and the possible cross-clade in vivo protection, could make these HIV-VLPAs a good candidate for a mono- and multicomponent worldwide preventive vaccine approach not restricted to high-priority regions, such as sub-Saharan countries.

High efficient production of Pr55gag virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A

The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.

Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with hepatitis B and hepatitis C virus infections

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Hepatocarcinogenesis is a multistep process mainly associated with persistent infection with hepatitis B (HBV) or C (HCV) viruses and always involving the accumulation of genetic alterations over decades of chronic liver disease. Mutations in TP53 and CTNNB1 genes are considered the cancer drivers for HCC development with variable frequencies depending on the etiology. Here we present a comprehensive review evaluating somatic mutations in TP53 and CTNNB1 genes in HBV- and HCV-related HCCs. Moreover, we report the mutational analysis of TP53 (exons 4-9) and CTNNB1 (exon 3) as well as PIK3CA (exon 9) genes in HCC from Southern Italy. The overall mutation frequency of TP53 and CTNNB1 was 33.3%, while hotspot variations in PIK3CA were completely absent. CTNNB1 mutations were significantly associated with young age (P=0.019) and moderately/poorly differentiated HCV-related HCC (P=0.015). The extended analysis of genetic alterations will help to identify molecular markers for liver cancer prevention, diagnosis and treatment of HBV and HCV-associated liver cancer.

Induction of neutralizing antibodies and cytotoxic T lymphocytes in Balb/c mice immunized with virus-like particles presenting a gp120 molecule from a HIV-1 isolate of clade A.

We have recently developed a candidate HIV-1 vaccine based on virus-like particles (VLPs) expressing a gp120 from an Ugandan HIV-1 isolate of the clade A (HIV-VLP(A)s). In vivo immunogenicity experiments were performed in Balb/c mice, with an immunization schedule based on a multiple-dose regimen of HIV-VLP(A)s without adjuvants, showing a significant induction of both humoral and cellular immunity. The Env-specific cellular response was investigated in vitro, scoring for both the proliferative response of T helper cells and the cytolytic activity of cytotoxic T lymphocytes (CTLs). Furthermore, immune sera showed >50% neutralization activity against both the autologous field isolate and the heterologous T cell adapted B-clade HIV-1(IIIB) viral strain. This is one of the first examples of HIV-1 vaccines based on antigens derived from the A clade, which represents >25% of all isolates identified world wide. In particular, the A clade is predominant in sub-Saharan countries, where 70% of the global HIV-1 infections occur, and where vaccination is the only rational strategy for an affordable prevention against HIV-1 infection.

Developments in virus-like particle-based vaccines for infectious diseases and cancer

Virus-like particles hold great promise for the development of effective and affordable vaccines. Indeed, virus-like particles are suitable for presentation and efficient delivery of linear as well as conformational antigens to antigen-presenting cells. This will ultimately result in optimal B-cell activation and cross-presentation with both MHC class I and II molecules to prime CD4+ T-helper as well as CD8+ cytotoxic T cells. This article provides an update on the development and use of virus-like particles as vaccine approaches for infectious diseases and cancer.

Update on Head and Neck Cancer: Current Knowledge on Epidemiology, Risk Factors, Molecular Features and Novel Therapies.

Tobacco use and alcohol consumption are the main risk factors associated with head and neck squamous cell carcinoma (SCC) development due to their cytotoxic and mutagenic effects on the exposed epithelia of the upper aerodigestive tract. Epstein-Barr virus (EBV) and high-risk human papillomaviruses (HPVs), both encoding viral oncoproteins able to interfere with cell cycle control, have been recognized as the etiological agents of nasopharynx carcinoma and a fraction of oropharyngeal carcinoma, respectively. Head and neck SCC is a deadly disease and despite innovative treatments represents a major challenge for patients. Recently, a number of genomic studies have highlighted the molecular heterogeneity of head and neck SCC based on methylation profiles, microRNA expression, mutated genes and new druggable pathways which may represent new targets for cancer-tailored therapies. To date, cetuximab is the only FDA-approved anti-epidermal growth factor receptor therapy for the treatment of head and neck SCC. In addition, a number of monoclonal antibodies targeting AKT, mTOR and PI3K pathways are under evaluation. Several therapeutic vaccines against HPV16 and EBV proteins are also under study. The purpose of this article is to review the epidemiology, pathogenesis and molecular features of head and neck SCC, with an emphasis on new therapies.