NIH Format Biographical sketch: Franco M. Buonaguro, M.D. Director of Molecular Biology and Viral Oncology Unit at Natl Cancer Inst, Naples - ITALY Dr Franco M. Buonaguro, born in 1952 in Sant'Agata dei Goti (Benevento - Italy), has been interested since his years at the Medical School on cell differentiation and transformation with several agents, such as chemical, physical and biological agents. Since 1983 his activity has been focused on the role of viral agents in human cancers, in particular on the role of DNA viruses such us Herpesvirus (HCMV, HHV-8, EBV) and Papillomavirus (HPV) in the etiopathogenesis of Kaposi’s sarcoma (KS) and in genital cancers, respectively. Several studies have been performed also on HIV/AIDS and more recently an HIV Vaccine Program has been established. HIV – AIDS studies. In collaboration with the Uganda Virus Research Center, since 1986, he has conducted AIDS research within the frame of the Uganda AIDS Program and the World Health Organization (Geneva), supported also by the MCD-2 project of the ICSC-World Laboratory (Lausanne), which in 1995 financed the establishment of the East African AIDS Research Center. The following results have been obtained: Biomolecular and phylogenetic analysis on Ugandan isolates. The bio-molecular analysis performed in two sequential studies on the hypervariable region V3 of the envelope gp120 indicates that HIV-1 isolates identified in Northern and Southern Uganda cluster into the A and D clades with a prevalence of the clade A (Buonaguro L. et al J. Virol., 1995 and in preparation). Although the divergence among these samples is constantly increasing (>20%), the V3 loop region, which is one of the HIV-1 major antigenic epitopes, shows a high stability that suggests a possibility of a wide immune protection with a discrete number of epitopes. These studies are performed within the frame of the UNAIDS Network for the Isolation and Characterization of HIV-1 Isolates from different regions of the World, particularly from regions with highest incidence/prevalence of HIV-1 infections. Biomolecular and phylogenetic characterization of HIV-1 isolates in Italy. The surveillance of HIV high-risk groups allowed the identification of HIV B-clade members as the prevalent strains in the Italian infected i.v. drug users, which are characterized by a slow viral growth and low infectivity (Buonaguro L. et al. AIDS, 1994). On going is the enrollment of a cohort of subjects seroconverted in the last year in order to evaluate the biomolecular evolution of HIV-1 strains involved in the epidemic of our region. Furthermore in 1999 we have started a collaboration with Prof. A. Lazzarin, Head of the Division of Infectious Diseases, and Dr L. Lopalco, Head of the Immunobiology Laboratory, of the San Raffaele Hospital in Milan, to analyze the HIV-1 strains in the HIV-1 recent seroconverted individuals, who attend such structure. The viral isolates identified in the first 10 recruited subjects cluster into the B clade, indicating that also in Lombardia, the Italian region with the highest incidence of HIV-1 infection, the B clade is still prevalent, although the transmission route has been shifting from the needle sharing to heterosexual contacts. Development of an anti-HIV-1 vaccine based on the production of Virus-like particles (VLPs) in a Baculovirus expression system. In collaboration with Prof. H. Wolf, Director of the Microbiology Institute of the University of Regensburg, Germany, a vector expressing HIV-1 gag and env genes has been engineered for the presentation of the gp120 on the p55 gag precursor (Pr55gag)-based VLPs. The gp120 used for these VLPs has been characterized in our laboratory from one Ugandan isolate of the clade A, and shows a high degree of stability in the hypervariable region V3 (Buonaguro L. et al. AIDS Res. Hum. Retroviruses, 1998). These HIV-VLPs show a strong in vivo immunogenicity in Balb/c mice with induction of both humoral and cellular arms of immune response, without adjuvants (Buonaguro L. et al., 2001). This represents, in our knowledge, one of the first vaccines designed to be targeted versus non-B HIV-1 strains, which are overwhelmingly prevalent in the world epidemic. Dr Buonaguro is Director of the Molecular Biology and Viral Oncology Unit at the Natl Cancer Institute “Fond Pascale “ Napoli-Italy. He graduated cum laude in 1977 at the “Federico II” Medical School in Naples - Italy, where achieved the specialty in Endocrinology in 1982 and the Specialty in Microbiology and Virology in 1992. After a postdoctoral fellowship at the Department of Cell Biology, Argonne National Laboratory, Argonne, IL, USA (1979-81), a WHO Fellowship and research associate position at the Tumor Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA (1983-86), he became an assistant member of the Experimental Oncology F – Viral Oncology at Natl Cancer Institute in Naples. He was promoted associate member in 1991, and became full member in 2001. Since 2008 he is Director of Molecular Biology and Viral Oncology Unit, as well as of the AIDS Ref Center, at the Natl Cancer Inst "Fond Pascale", Naples - ITALY Dr Buonaguro dedicated a lot of energy for Developing-Countries’ needs, in particular he has been involved in a large International Programs on AIDS Research in Uganda, one of the Countries most heavily hit by the epidemic, developing and coordinating the ICSC World Lab East Africa AIDS Research Center at the Uganda Virus Research Institute (UVRI) - Entebbe, of which he has been co-Director since 1995 and Director since April 2001. He is member of the World Federation of Scientists since 1997, has served as WHO virology consultant in 1996 and 2003. Member of WHO/ UNAIDS Network for HIV isolation and Characterization since 1996; Member WHO HPV DNA International Collaborative Study Group since 2002. With respect to editorial activities Dr Buonaguro is Editor in chief of Infectious Agents and Cancer a 2006-established online journal published by BioMed Central at www.infectagentscancer.com; Managing Editor of Frontiers in Bioscience; Editorial Board Member of Chinese Journal of Medicine [http://www.cjmed.net/]; Guest editor of Plant-derived vaccines [http://www.future-science-group.com/m/244].
Abstract: Kaposi's sarcoma (KS) is a rare cancer in Iran and there is no epidemiological and molecular information about HHV-8 variants circulating among the Iranian population. In this study HHV-8 sequences have been analyzed in 43 cutaneous KS biopsies from Iranian patients mainly affected by classic KS. DNA samples were subjected to PCR amplification of HHV-8 ORF26, T0.7 and K1 followed by direct nucleotide sequencing and phylogenetic analysis. The analysis of ORF26 showed that 30 (69.8%) and 13 (30.2%) samples belonged to subtypes A/C and K, respectively. In general, the clustering of HHV-8 T0.7 variants paralleled that of ORF26. Genotyping of K1 sequences showed that the majority of samples (39 out of 41) fall into the large C clade with only 2 belonging to the A clade. In conclusion, HHV-8 variants identified among classic Iranian KS are largely related to Eurasian genotypes previously identified in KS from Mediterranean, Middle East, and East Asian regions.
Abstract: To evaluate differences and changes in quality of life (QoL), lifestyle behavior and employment experience of young in comparison to midlife adults in response to early stage gynecologic cancer diagnoses.
Abstract: ABSTRACT: BACKGROUND: Recent emerging evidences identify Human Papillomavirus (HPV) related Head and Neck squamous cell carcinomas (HN-SCCs) as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC); 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR); and 3 viral integration into the host by in situ hybridization method (ISH). This triple method has been applied to HN-SCC originated from oral cavity (OSCC) and oropharynx (OPSCC), the two anatomical sites in which high risk (HR) HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP) was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. RESULTS: All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC) but lower specificity level (74% in OSCC and 93% in OPSCC). Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC (kappa = 0.38) and a moderate agreement in OSCC (kappa = 0.44). Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive) increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE) samples (100% in OSCC and 78.5% in OPSCC), but reduced the sensitivity (33% in OSCC and 60% in OPSCC). The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. CONCLUSIONS: Although HR-HPVs detection is of utmost importance in clinical settings for the Head and Neck Cancer patients, there is no consensus on which to consider the 'golden standard' among the numerous detection methods available either as single test or combinations. Until recently, quantitative E6 RNA PCR has been considered the 'golden standard' since it was demonstrated to have very high accuracy level and very high statistical significance associated with prognostic parameters. In contrast, quantitative E6 DNA PCR has proven to have very high level of accuracy but lesser prognostic association with clinical outcome than the HPV E6 oncoprotein RNA PCR. However, although it is theoretically possible to perform quantitative PCR detection methods also on FFPE samples, they reach the maximum of accuracy on fresh frozen tissue. Furthermore, worldwide diagnostic laboratories have not all the same ability to analyze simultaneously both FFPE and fresh tissues with these quantitative molecular detection methods. Therefore, in the current clinical practice a p16-IHC test is considered as sufficient for HPV diagnostic in accordance with the recently published Head and Neck Cancer international guidelines. Although p16-IHC may serve as a good prognostic indicator, our study clearly demonstrated that it is not satisfactory when used exclusively as the only HPV detecting method. Adding ISH, although known as less sensitive than PCR-based detection methods, has the advantage to preserve the morphological context of HPV-DNA signals in FFPE samples and, thus increase the overall specificity of p16/Consensus PCR combination tests.
Abstract: ABSTRACT: BACKGROUND: Virus-Like Particles (VLPs) represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN 1826 or poly(I:C) adjuvants, has been evaluated in an animal model. RESULTS: Adjuvanted VLPs elicited a higher titer of total specific IgG compared to VLPs alone. Furthermore, while VLPs alone induced a balanced TH2 pattern, VLPs formulated with either adjuvant elicited a TH1-biased IgG subclasses (IgG2a and IgG3), with poly(I:C) more potent than CpG ODN1826. CONCLUSIONS: The results confirmed that adjuvants efficiently improve antigen immunogenicity and represent a suitable strategy to skew the adaptive immune response toward the differentiation of the desired T helper subset, also using VLPs as antigen.
Abstract: Elicitation of a potent and broadly neutralizing antibody response is the main goal of an effective preventive HIV-1 vaccine. It has been shown by us and others that the expression of Env glycoproteins on the surface of particulate structures, such as Virus-Like Particles (VLPs), could be a more efficient strategy to deliver conformational epitopes to the immune system. To this aim, VLPs expressing native HIV Env gp140 or gp41 glycoproteins have been produced in insect cells using a baculovirus expression system and characterized for appropriate protein expression. VLP-bound HIV gp140 glycoprotein showed the appropriate expression and trimeric conformation. Immunogenicity studies have been performed in BALB/C mice by intra-peritoneal administration and sera from immunized mice have been tested in ELISA assays, for their reactivity with HIV specific antigens, as well as in ex vivo neutralization assay. Sera from immunized animals showed a high reactivity with individual HIV proteins expressed in VLPs. Results of TZM-bl based neutralization assay show that combined sera from animals independently immunized with gp140- or full-length-gp41-expressing VLPs have an additive/synergistic effect in the neutralization activity of HIV pseudoviruses. In conclusion, novel VLPs expressing different HIV Env glycoproteins with native trimeric conformation have been generated, showing the induction of effective antibody response with neutralization activity in TZM-bl neutralization assay. These results confirm the effectiveness of VLPs as presentation and delivery system for conformational proteins and show the improved neutralization activity upon the combination of anti-sera elicited by different HIV envelope antigens, suggesting the possibility of broadening the spectrum of viral epitopes targeted by immune response.
Abstract: We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efficient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specific systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag-VLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins. The persistence of HIV coding genes has been verified in clonal resistant insect cells, the protein expression and conformation has been verified by Western blot analysis. The resulting HIV-VLPs have been visualized by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. This represents, to our knowledge, the first example of stable double transfected insect cell line for the constitutive production of enveloped HIV-Gag VLPs presenting trimeric HIV-gp140 on their surface.
Abstract: Dendritic cells (DCs) represent the bridging cell compartment between a variety of nonself antigens (i.e., microbial, cancer and vaccine antigens) and adaptive immunity, orchestrating the quality and potency of downstream immune responses. Because of the central role of DCs in the generation and regulation of immunity, the modulation of DC function in order to shape immune responses is gaining momentum. In this respect, recent advances in understanding DC biology, as well as the required molecular signals for induction of T-cell immunity, have spurred many experimental strategies to use DCs for therapeutic immunological approaches for infections and cancer. However, when DCs lose control over such 'protective' responses - by alterations in their number, phenotype and/or function - undesired effects leading to allergy and autoimmune clinical manifestations may occur. Novel therapeutic approaches have been designed and currently evaluated in order to address DCs and silence these immunopathological processes. In this article we present recent concepts of DC biology and some medical implications in view of therapeutic opportunities.
Abstract: 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.
Abstract: The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 presents conserved binding sites for binding to the primary virus receptor CD4 as well as the major HIV chemokine coreceptors, CCR5 and CXCR4. Concerted efforts are underway to understand the specific interactions between gp120 and coreceptors as well as their contribution to the subsequent membrane fusion process. The present review summarizes the current knowledge on this biological aspect, which represents one of the key and essential points of the HIV-host cell interplay and HIV life cycle. The relevance of conformational HIV-1 Envelope proteins presented on Virus-like Particles for appropriate assessment of this molecular interaction, is also discussed.
Abstract: Immunotherapies, including vaccines, represent a potent tool to prevent or contain disease with high morbidity or mortality such as infections and cancer. However, despite their widespread use, we still have a limited understanding of the mechanisms underlying the induction of protective immune responses.Immunity is made of a multifaceted set of integrated responses involving a dynamic interaction of thousands of molecules; among those is a growing appreciation for the role the innate immunity (i.e. pathogen recognition receptors - PRRs) plays in determining the nature and duration (immune memory) of adaptive T and B cell immunity. The complex network of interactions between immune manipulation of the host (immunotherapy) on one side and innate and adaptive responses on the other might be fully understood only employing the global level of investigation provided by systems biology. In this framework, the advancement of high-throughput technologies, together with the extensive identification of new genes, proteins and other biomolecules in the "omics" era, facilitate large-scale biological measurements. Moreover, recent development of new computational tools enables the comprehensive and quantitative analysis of the interactions between all of the components of immunity over time. Here, we review recent progress in using systems biology to study and evaluate immunotherapy and vaccine strategies for infectious and neoplastic diseases. Multi-parametric data provide novel and often unsuspected mechanistic insights while enabling the identification of common immune signatures relevant to human investigation such as the prediction of immune responsiveness that could lead to the improvement of the design of future immunotherapy trials. Thus, the paradigm switch from "empirical" to "knowledge-based" conduct of medicine and immunotherapy in particular, leading to patient-tailored treatment.
Abstract: Surveillance of human papillomavirus (HPV) prevalence and genotype distribution in migrant women from middle and low-income countries to developed countries is limited. The aim of this study was to analyze the spectrum of HPV genotypes and prevalence of cervical abnormalities in women emigrated mainly from Eastern Europe and West Africa and living in Southern Italy. The study included 233 migrant and 98 Italian-born women who self-referred to two gynecological outpatient clinics in the Campania region. Cervical specimens were subjected to cytological examination and viral testing by broad spectrum PCR. The prevalence rates of HPV infection were 57.9% and 94.1% among migrant and 19.4% and 88.5% among Italian women with normal and abnormal cytology respectively. HPV infection was detected in 56.1% of Southern and Eastern European, 62.5% of Central and South American, 55.5% of West African, and 73.3% of Southern Asian women with normal cervix. Among the 140 HPV-positive migrants, a total of 28 mucosal HPV genotypes were identified of which 11 types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58), epidemiological classified as carcinogenic to humans (group 1), accounted for 73.4% of all infections. As expected, HPV16 was the most common viral type in all groups with frequency rates ranging from 12.5% in African to 30.1% in Eastern and Southern European women. In conclusion, the estimated prevalence of HPV infection among migrant women is very high, probably reflecting either lifestyle or high incidence of HPV in their country of origin. The implementation of vaccination strategies and cervical cancer surveillance are critical for women in this risk group.
Abstract: Adjuvants are becoming the key players of vaccine formulations to enhance the immunogenicity of subunit (peptides, proteins, virus-like particles (VLPs)) and DNA vaccines, as well as to reach the current new goals of preventing and/or treating chronic infectious diseases and cancers. Induction of humoral response, in particular neutralizing antibodies able to inhibit the binding of pathogens to their cellular receptors, remains a major goal of vaccines targeted to prevent acute lytic infections; induction/modulation of cellular immunity is, however, critical to fight latently/chronically infected cells as well as cancer cells. The new adjuvants, included in vaccine preparations, are currently able to modify the presentation of epitopes to the immune system with a specific T(H)1 versus T(H)2 polarization efficacy. A paradigm of the relevance of these new adjuvants is the immunological result obtained with the inclusion of monophosphoryl lipid A in the formulation of L1-based human papillomavirus (HPV)-naked VLPs. In the May issue of this journal, Garcon and colleagues describe the highly enhanced humoral and memory B cellular immunity of the AS04-adjuvanted HPV vaccine, which results in a long-lasting and broad spectrum immunity.
Abstract: A single-nucleotide polymorphism in the MDM2 promoter (SNP309; rs2279744) causes elevated transcription of this major negative regulator of p53 in several cancer types. We investigated MDM2 SNP309 and CDKN1A (p21/Waf1/Cip1) codon 31 (rs1801270) polymorphisms in 86 cases of cutaneous Kaposi's sarcoma (KS) from African and Caucasian patients, and 210 healthy controls. A significant increase of the MDM2 SNP309 T/G genotype was observed among classic KS cases (odds ratio 2.38, 95% confidence interval 1.0-5.5). Frequencies of CDKN1A codon 31 genotypes were not significantly different between cases and controls. The results suggest that the MDM2 SNP309 G allele may act as a susceptibility gene for the development of classic KS in Caucasian patients.
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.
Abstract: Human papillomavirus (HPV) genotypes have been extensively studied in uterine cervix squamous cell carcinoma and HPV16 variants have been found to be associated with increased cancer risk, but few reports have been published on genotype distribution and HPV16 variant prevalence in adenocarcinoma tumors. The objective of this study was to analyze viral genotypes and HPV16 intratypic variants in cervical adenocarcinoma and squamous cell carcinoma of Italian women.
Abstract: The African Society of Human Genetics (AfSHG), founded in 2003 with its inaugural meeting in Accra, Ghana,1 has the stated missions of (1) disseminating information about human genetics research in Africa, (2) establishing a mentorship network providing educational resources, including the development of appropriate technology transfer, (3) providing advocacy for human genetic research in Africa, and (4) encouraging collaborative research. Despite its young age, the AfSHG has developed a strong cadre of active researchers, both within and outside of Africa, with more than 400 members (from 16 countries across Africa as well as 8 other countries), and has held six successful meetings, five in Africa and one in the United States.
Abstract: 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.
Abstract: We have previously developed HIV-1 Pr55gag-based virus-like particles (HIV-VLPs) as presentation and delivery model using a transient Baculovirus expression system. Here we describe the establishment and characterization of stably transfected insect cell line for the constitutive and reproducible production of HIV-VLPs. The persistence of HIV gag coding gene has been verified in clonal resistant insect cells and the protein expression has been confirmed by Western blot analysis. The resulting HIV-VLPs have been evaluated by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. Our results demonstrate that this strategy is highly efficient for constitutive expression of conformational enveloped VLPs which can be employed as presentation and delivery system for pathogen as well as tumor-associated antigens. This represents, to our knowledge, the first example of stably transfected insect cell line for the constitutive production of VLPs.
Abstract: Human herpesvirus-8 (HHV-8) variants have been found heterogeneously distributed among human populations living in diverse geographic regions, but their differential pathogenicity in Kaposi's sarcoma development remains controversial. In the present study, HHV-8 variant distribution has been analyzed in classic, iatrogenic, endemic as well as epidemic Kaposi's sarcoma (KS) during pre-AIDS and AIDS period (1971-2008) in countries with different KS incidence rate. DNA samples from cutaneous KS lesions of 68 patients living in Africa (n=23, Cameroon, Kenya and Uganda), Europe (n=34, Greece and Italy) and North America (n=11) have been subjected to PCR amplification of HHV-8 ORF 26, T0.7, K1 and K14.1/15, followed by direct nucleotide sequencing and phylogenetic analysis. Among the 23 African samples, the majority of HHV-8 ORF 26 variants clustered with the subtype R (n=12) and B (n=5). Conversely, the viral sequences obtained from 45 European and North European tumors belonged mainly to subtype A/C (n=36). In general, HHV-8 and K1 variant clustering paralleled that of ORF 26 and T0.7. Genotyping of the K14.1/15 loci revealed a large predominance of P subtype in all tumors. In conclusion, comparison of the HHV-8 sequences from classic or endemic versus AIDS-associated KS showed a strong linkage of the HHV-8 variants with specific populations, which has not changed during AIDS epidemic.
Abstract: The introduction of highly active antiretroviral therapy has drastically changed HIV infection from an acute, very deadly, to a chronic, long-lasting, mild disease. However, this requires continuous care management, which is difficult to implement worldwide, especially in developing countries. Sky-rocketing costs of HIV-positive subjects and the limited success of preventive recommendations mean that a vaccine is urgently needed, which could be the only effective strategy for the real control of the AIDS pandemic. To be effective, vaccination will need to be accessible, affordable and directed against multiple antigens. Plant-based vaccines, which are easy to produce and administer, and require no cold chain for their heat stability are, in principle, suited to such a strategy. More recently, it has been shown that even highly immunogenic, enveloped plant-based vaccines can be produced at a competitive and more efficient rate than conventional strategies. The high variability of HIV epitopes and the need to stimulate both humoral neutralizing antibodies and cellular immunity suggest the importance of using the plant system: it offers a wide range of possible strategies, from single-epitope to multicomponent vaccines, modulators of the immune response (adjuvants) and preventive molecules (microbicides), either alone or in association with plant-derived monoclonal antibodies, besides the potential use of the latter as therapeutic agents. Furthermore, plant-based anti-HIV strategies can be administered not only parenterally but also by the more convenient and safer oral route, which is a more suitable approach for possible mass vaccination.
Abstract: Particulate structures hold great promise for the development of effective and affordable recombinant prophylactic as well as therapeutic vaccines. Different types of particulate structures, including virus-like particles (VLPs) and virosomes, have been developed depending on the nature of the viral pathogen to be targeted and the type of immune response (humoral vs cellular) to be elicited. Particulate structures allow the insertion or fusion of foreign antigenic sequences, resulting in chimeric particles delivering foreign antigens on their surface. Similarly, they are used as carriers for foreign antigens, including non-protein antigens, via chemical conjugation. Particulate structures, indeed, represent a very efficient system for delivering antigens to antigen presenting cells (APC) which, in turn, trigger and amplify the adaptive immune response. The present review will address the biological and immunological properties of particulate structures, in particular VLPs, as platform for vaccine development.
Abstract: The aim of this multicentric study was to identify human papillomavirus (HPV) type distribution in invasive cervical cancer and high-grade cervical intraepithelial neoplasia 2/3 (CIN2/3) in Italy.
Abstract: Renal allograft recipients have a well-documented increased incidence of human papillomavirus (HPV)-related malignancies and preventive strategies should be specifically implemented. While in females the use of the Papanicolau test and HPV detection assay are used currently as a screening test for cervical cancer, no diagnostic procedures have been implemented to monitor HPV infection in males. The aim of this study was to test for HPV infection and to determine the spectrum of viral genotypes in urine samples of men with renal transplants. The study included 88 patients who underwent kidney transplantation between 1999 and 2005. HPV sequences were detected by nested PCR, using the broad-spectrum consensus-primer pairs MY09/MY11 and the new MGP system, and characterized by nucleotide sequence analysis. Overall, 43 (48.9%) samples were found positive for HPV sequences and the most common genotypes were HPV 16 (53.5%) and HPV 54 (9.3%) followed by HPV 6, 53, 56, 58, 66, 11, 12, 20, 45, 62, and 71, in descending order of prevalence. The majority of HPV 16 isolates were classified as European and only one as African-1 variant on the basis of nucleotide signature present within the MGP L1 region. The high prevalence of HPV 16 among renal allograft recipients suggests that an HPV-16-based preventive or therapeutic vaccine may be effective for prevention or treatment of HPV-related neoplasia in this group of immune compromised patients.
Abstract: We describe a fatal case of myopericarditis presenting with cardiac tamponade in a previously healthy 11-year-old child. Pandemic H1N1 2009 influenza A virus sequences were identified in throat and myocardial tissues and pericardial fluid, suggesting damage of myocardial cells directly caused by the virus.
Abstract: The global transcriptional profile of peripheral blood mononuclear cells (PBMCs) stimulated with HIV candidate vaccine (virus-like particles, VLPs) has been evaluated in HIV-infected patients with low/high viral load compared to healthy volunteers. Baseline activation of chemokine production was observed in PBMC from HIV-infected patients and innate immune stimulation with HIV-VLPs was not blunted. The immune profile among HIV-infected patients was found to be qualitatively similar but quantitatively extremely variable. This diversity was independent of viral load and it might be dependent on individual immunogenetic traits or concurrent immunological status. This ex vivo screening strategy represents an efficient tool for guiding modifications/optimizations of vaccination strategies and understanding failures in individuals enrolled in clinical trials.
Abstract: We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) 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, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, 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. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.
Abstract: We have recently shown that 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-VLP(A)s), induce maturation and activation of antigen-presenting cells (APCs) in fresh peripheral blood mononuclear cells (PBMCs) from seronegative as well as seropositive, with either low or high viremia, HIV-1 subjects. A Th2 polarization has been observed in both HIV seropositive groups, which is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern. Here we show that the production of the known immune-suppressive IL-10 is induced in both HIV-seropositive groups at a significantly lower level by HIV-VLPs compared to LPS. These levels, however, appear to still negatively interfere with the innate as well as adaptive Th1-polarized response observed in HIV-seropositive groups. These results indicate that vaccines and novel adjuvants (i.e., TLR agonists, such as LPS) must be evaluated not only for their immunogenicity but also for their potential immune-suppressive effects. In this perspective, fresh ex vivo PBMCs can be of high value for screening the responses as well as eventual failures of vaccinees enrolled in clinical trials.
Abstract: 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.
Abstract: OBJECTIVES: We aimed to determine the incidence of women's breast cancer in Italy without using statistical approximations. METHODS: We analyzed the national hospitalizations database at the Ministry of Health to calculate the number of major surgeries in Italian women (mastectomies and quadrantectomies) due to breast cancer between 2000 and 2005, overall and by age groups (<44, 45-64, 65-74 and >or= 75 years old). RESULTS: Over the six years examined, an overall number of 100,745 mastectomies and 168,147 quadrantectomies were performed. A total of 41,608 major surgeries due to breast cancer were performed in the year 2000 and this number rose to 47,200 in 2005, with a 13.4% increase over six years. CONCLUSION: by analyzing the hospitalizations database concerning major breast surgery, incidence of breast cancer in Italy was found to be 26.5% higher than the official estimations which have been computed using statistical models (namely 47,200 vs. 37,300 cases in year 2005).
Abstract: Complex antigen structures currently represent the most-studied approach for prophylactic as well as therapeutic vaccines. Different types of complex vaccines, including virus-like particles and virosomes, have been developed depending on the nature of the viral pathogen they are trying to replicate (enveloped vs naked) or the modality to express antigenic epitopes (i.e., the binding of envelope protein on liposomic structures). The complex structure of these vaccines provides them with some adjuvanted properties, not uniformly present for all virus-like particle types. The further inclusion of specific adjuvants in vaccine preparations can modify the presentation modality of such particles to the immune system with a specific Th1 versus Th2 polarization efficacy. A paradigm of the relevance of these new adjuvants are the immunological results obtained with the inclusion of monophosphoryl lipid A adjuvant in the formulation of L1-based human papillomavirus-naked virus-like particles to reduce a Th1 cellular immunity impairment, peculiar for alum-derived adjuvants, along with the induction of highly enhanced humoral and memory B-cellular immunity.
Abstract: OBJECTIVES: Several studies have examined the association of codon 72 polymorphism of the TP53 gene, encoding either arginine or proline, in several tumor types but none have investigated its role in Kaposi's sarcoma (KS) development. METHODS: In this prevalent case-control study, 67 cutaneous lesions of classic, iatrogenic, endemic as well as epidemic KS from African (n = 22) and Caucasian (n = 45) patients, and blood samples from 150 healthy controls (n = 57 African, n = 93 Caucasian) have been analyzed for arginine and proline allele distribution. RESULTS: Among African cases the proline homozygous, heterozygous and arginine homozygous genotype frequencies were 50.0, 31.8 and 18.2%, respectively, and among controls 54.4, 40.3, and 5.3%, respectively (p = 0.1872). Conversely, among Caucasian cases genotype distributions were 6.7, 55.6, and 37.8%, and among controls 7.5, 34.4, and 58.1%, respectively (p = 0.0567). No significant differences in arginine and proline allele distribution were observed when the cases were stratified by HIV status/tumor type. CONCLUSIONS: The results obtained in this study suggest that p53 polymorphism at codon 72 does not represent a risk factor for the development of all forms of KS, either among African or among Caucasian populations.
Abstract: Epidemiologic evidence points to a connection between viral infections by the human papillomavirus (HPV) and a subgroup of squamous cell carcinomas of the oropharynx. Still controversial is the association of HPV infection with oesophageal neoplasia.
Abstract: Virtually all cases of cervical cancer are caused by persistent infections with a restricted set of human papillomaviruses (HPV). Some HPV types, like HPV16 and HPV18, are clear and powerful carcinogens. However, the categorization of the most weakly carcinogenic HPV types is extremely challenging. The decisions are important for screening test and vaccine development. This article describes for open discussion an approach recently taken by a World Health Organization International Agency for Research on Cancer (IARC) Monographs Working Group to re-assess the carcinogenicity of different HPV types.
Abstract: Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.
Abstract: Cancer has periodically been proposed as transmissible disease in animals and humans, and specific pathogens have long been searched for. Several biomolecular studies, fundamental in understanding cancer pathogenesis, have identified mechanisms directly/indirectly involved in pathogens-related cancer, including 1) oncogene transduction, with introduction of exogenous oncogenic genes; 2) activation of endogenous oncogenes, comprising those from endogenous retroviruses; 3) inactivation of constitutive suppressor genes, with enhanced susceptibility to exogenous oncogenic agents. Further pathogens' indirect role is associated to cancer promotion through inflammation and angiogenesis. The global burden of cancer associated with infectious agents approaches 20% of all malignancies. Most of the common "infectious" cancers occur in developing countries and their "attributable risk" (i.e. the proportion of cancers that would not occur if the agent were removed) is considerable. Although the cancer role of often ubiquitous pathogens, and the molecular mechanisms involved in the infrequent progression of chronic infections to cancers are still often unknown, the identity of the agents and efforts to mitigate their effects can lead to effective cancer prevention and substantive public health benefit.
Abstract: The association of the p53 polymorphism at codon 72 and susceptibility to develop human papillomavirus (HPV)-related cancer has been investigated in several studies with controversial results. In this study, 78 penile squamous cell carcinoma biopsies (n=17 from Uganda, n=61 from Italy) and blood samples from 150 healthy controls (n=57 from Uganda, n=93 from Italy) have been analyzed for the arginine and proline allele distribution. Among Ugandan cases the heterozygous, proline homozygous and arginine homozygous genotype frequency was 41.2%, 52.9% and 5.9%, respectively, and among controls was 40.3%, 54.4%, and 5.3%, respectively (P=0.9917). Conversely, among Italian cases genotype distribution was 42.6%, 4.9%, and 52.5%, and among controls was 34.4%, 7.5%, and 58.1%, respectively (P=0.5343). No significant differences in arginine and proline allele distribution were observed when the cases were stratified by HPV status. Therefore, no evidence of association between homozygosity for p53 arginine and HPV-related or HPV-unrelated penile squamous cell carcinoma was observed neither among Ugandan nor among Italian populations.
Abstract: Human papillomaviruses (HPVs) are the cause of cervical intraepithelial neoplasia and invasive carcinomas of the uterine cervix. The distribution of specific HPV genotypes varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies. The aim of this study was to determine the spectrum of HPV genotypes and HPV-16 variants among women with cervical lesions living in Ecuador. A total of 71 cases have been analyzed, including 32 chronic cervicitis, 29 cervical intraepithelial neoplasia grade 1, and 10 cervical intraepithelial neoplasia grade 2-3. HPV sequences were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction and characterized by nucleotide sequence analysis. Overall, 31 (43.7%) cases were HPV positive with prevalence rates of 37.5%, 44.8%, and 60% in patients with chronic cervicitis, cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 2-3, respectively. Among the positive cases, the most common genotypes were HPV 16 (64.5%) and HPV 81 (29%) followed by HPV 31, 53, 56, and 58, in descending order of prevalence. Seventeen (85%) HPV-16 isolates were classified as European and three (15%) as African-1 variant on the basis of nucleotide signature present within the MY09/MY11 L1 sequence. The results suggest that HPV 16 has a very high prevalence among women with cervical lesions in Ecuador; therefore, an effective HPV-16 based vaccine should prevent the development of cervical cancer in a large proportion of Ecuadorian women.
Abstract: Human immunodeficiency virus (HIV)-positive women have high rates of cervical squamous intraepithelial lesions (SIL) and concurrent human papillomavirus (HPV) infections with a variety of genotypes whose oncogenic risk is poorly documented. The prevalence and persistence of HPV genotypes and HPV16 variants were analysed in 112 HIV-positive and 115 HIV-negative Italian women. HIV-positive women were more likely than HIV-negative women to be infected by HPV at the initial examination (39.3 vs 13.9 %, P<0.001) and to have a higher period prevalence of HPV infection over a 3-year follow-up (43.8 % vs 17.4 %, P<0.001), regardless of CD4+ cell counts and anti-retroviral therapy. 'High-risk' and 'probable high-risk' HPVs (types 16, 18, 31, 33, 35, 45, 52, 58 and 66), among the 20 different viral genotypes identified, were predominant in HIV-positive (33.9 %) compared with HIV-negative (13.9 %) women. Among HIV-infected women, with normal cytology as well as with SIL of any grade, the most common genotypes were HPV16 followed by HPV81, -58, -72, -33 and -62. HPV16 isolates from 18 HIV-positive and eight HIV-negative women were classified into variant lineages based on sequencing analysis of E6 and E7 genes and the long control region. Whilst the HPV16 G350 European variant was prevalent in both HIV-positive (10.7 %) and -negative women (3.5 %), HPV16 African 2 variant was only detected in HIV-positive women (3.6 %), suggesting different sexual mixing behaviours. The increased prevalence of uncommon viral genotypes and HPV16 variants in HIV-positive Italian women underscores the need to target a wide range of HPV types in cervical screening of high-risk women.
Abstract: Baculovirus-expressed HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs) induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines.
Abstract: The causative role of human papillomaviruses (HPV) and HPV16 variants has been extensively studied in uterine cervix dysplastic lesions and invasive carcinoma; few such studies, however, have been performed in penile tumors. We have investigated HPV genotype and HPV16 variant distribution on 41 penile cancer biopsies from Italian patients. Cases were extracted from the respective pathology departments databases of National Cancer Institutes in Naples and Milan. HPV sequences were detected by PCR and characterized by direct sequence analysis. Among the 19 HPV-positive cases (46.3%) 2 viral genotypes were identified (HPV16 and 18) with HPV16 accounting for 94.7% (18 out of 19) of the infections. Sequence analysis of E6, E7 genes and long control region (LCR) of 18 HPV16 isolates allowed the identification of European (E-G-350) and non-European (AA and Af-1) variants in 44.4% and in 55.6% of the samples, respectively. The AA variant alone represented 44.4% of all HPV16 infections, a significantly higher frequency of that observed in cervical carcinoma from Italian women (Tornesello et al., J Med Virol 2004;74:117-26). Our results suggest that HPV16 has a very high prevalence among penile cancer patients in Italy and the increased frequency of HPV16 non-European classes, particularly the AA, suggests that they are more oncogenic than European variants in penile tissue.
Abstract: Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production. In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native viral (L1(v) gene) or a synthetic sequence optimized for expression in plant plastids (L1(pt) gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up to 1.5% total leaf proteins only when vectors included the 5'-UTR and a short N-terminal coding segment (Downstream Box) of a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes and by electron microscopy.
Abstract: The 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.
Abstract: The Human Immunodeficiency Virus type 1 (HIV-1) is classified into genetic groups, subtypes and sub-subtypes which show a specific geographic distribution pattern. The HIV-1 epidemic in Italy, as in most of the Western Countries, has traditionally affected the Intra-venous drug user (IDU) and Homosexual (Homo) risk groups and has been sustained by the genetic B subtype. In the last years, however, the HIV-1 transmission rate among heterosexuals has dramatically increased, becoming the prevalent transmission route. In fact, while the traditional risk groups have high levels of knowledge and avoid high-risk practices, the heterosexuals do not sufficiently perceive the risk of HIV-1 infection. This misperception, linked to the growing number of immigrants from non-Western Countries, where non-B clades and circulating recombinant forms (CRFs) are prevalent, is progressively introducing HIV-1 variants of non-B subtype in the Italian epidemic. This is in agreement with reports from other Western European Countries.In this context, the Italian HIV-1 epidemic is still characterized by low subtype heterogeneity and represents a paradigmatic example of the European situation. The continuous molecular evolution of the B subtype HIV-1 isolates, characteristic of a long-lasting epidemic, together with the introduction of new subtypes as well as recombinant forms may have significant implications for diagnostic, treatment, and vaccine development. The study and monitoring of the genetic evolution of the HIV-1 represent, therefore, an essential strategy for controlling the local as well as global HIV-1 epidemic and for developing efficient preventive and therapeutic strategies.
Abstract: 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.
Abstract: The immune response to HIV-1 virus-like particles (VLPs), presenting a clade A Ugandan gp120, has been evaluated in a mouse model by intra-nasal (i.n.) administration by a VLP+VLP homologous or a DNA+VLP heterologous prime-boost immunization protocol, including a HIV-1 DNA gp160/rev plasmid. Furthermore, the effect of the Eurocine lipid-based mucosal L3 adjuvant on the VLP immunogenicity has been assessed as well. The designed heterologous protocol is able to increase the env-specific humoral and cellular immune response, compared to the homologous protocol, which is to some extent increased by the administration of L3-adjuvanted VLP boosting dose. The anti-gag response is statistically increased in both homologous and heterologous protocols, particularly when the VLP boosting dose is adjuvanted. Immune sera from immunized animals exhibit >50% ex vivo neutralizing activity against heterologous A and B-clade viral isolates. An envelope B-cell epitope mapping shows an enhanced response against V3 epitopes all across the C2-V5 region in the heterologous prime-boost immunization strategy. The induction of humoral immunity at mucosal sites, which represents the main port of entry for the HIV-1 infection, is extremely relevant. In this framework, the DNA-VLP heterologous prime-boost protocol appears a promising preventive vaccine approach which can significantly benefit from specific mucosal adjuvants, as the Eurocine L3.
Abstract: BACKGROUND: The distribution of human papillomaviruses (HPVs) varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies. Little information, however, exists regarding HPV genotypes distribution in immigrant women from countries at high incidence for cervical cancer. The aim of this study was to determine the spectrum of HPVs and their variants among HIV-positive and HIV-negative women immigrants in South Italy mainly from West Africa and with a history of prostitution. RESULTS: Cervical cytological samples have been collected from 14 HIV-positive and 31 HIV-negative immigrants (38 out of 45 were born in Nigeria), attending a gynecological outpatient clinic in the Campania region. Human papillomaviruses were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction and characterized by nucleotide sequence analysis. Altogether, 42.2% (19/45) of samples were HPV positive with detection rates of 57.1% (8/14) in HIV-positive and 35.5% (11/31) in HIV-negative women. Among the twelve different viral genotypes identified, HPV33, 58, 70 and 81 were the prevalent genotypes with a frequency of 6.7% each, followed by HPV16, 35, 42, 54, 31, 52, 56 and 67, in descending order of prevalence. Sequence homology studies performed on the L1 amplified fragments of HPV16, 52 and 58 isolates allowed the identification of nucleotide changes distinctive of non-European variants. CONCLUSION: The overall HPV prevalence (42.2%) was high in this immigrant women group with the most common viral types other than HPV16 and 18, against which current vaccine strategies have been developed. The distribution of HPV genotypes and their variants in high-risk immigrants reflects that of their original countries. The surveillance of risk groups that may act as viral reservoirs of uncommon genotypes within different countries are necessary to determine the severity of HPV infection with the different viral types and to monitor a possible shift of prevalent strains following vaccination.
Abstract: HIV-1 C2-V3 subgenomic regions of the env gene from Iranian seropositive injecting drug users (IDUs) living in Mashhad have been analyzed to evaluate molecular and phylogenetic relationships with IDUs living in Tehran and identify possible common founder virus isolates. The results show that the viral sequences of the Iranian IDUs are strongly related and form a single cluster within the A subtype related to African Ugandan/Kenyan sub-Saharan isolates. Pairwise nucleotide alignment shows higher average divergence values within the Mashhad group than the Tehran group. Furthermore, the Mashhad sequences show much less conserved amino acid residues in the V3 loop than the Tehran sequences. These data represent the first macro-analysis of the HIV-1 molecular evolution in the Iran and Middle East epidemics and may be extremely relevant to guide the development and implementation of diagnostic as well as preventive/therapeutic approaches in this region.
Abstract: Genetic and phylogenetic information on the HIV-1 epidemic in Middle-East Countries, and in particular in Iran, are extremely limited. By March 2004, the Iranian Ministry of Health officially reported a cumulative number of 6'532 HIV positive individuals and 214 AIDS cases in the Iranian HIV-1 epidemic. The intra-venous drug users (IDUs) represent the group at highest risk for HIV-1 infection in Iran, accounting for almost 63% of all HIV-infected population. In this regards, a molecular phylogenetic study has been performed on a sentinel cohort of HIV-1 seropositive IDUs enrolled at the end of 2005 at the University of Mashhad, the largest city North East of Tehran. The study has been performed on both gag and env subgenomic regions amplified by Polymerase Chain Reaction (PCR) from peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. The results reported here show that the HIV-1 subtype A is circulating in this IDUs sentinel cohort. Moreover, the single phylogenetic cluster as well as the intra-group low nucleotide divergence is indicative of a recent outbreak. Unexpectedly, the Iranian samples appear to be phylogenetically derived from African Sub-Saharan subtype A viruses, raising stirring speculations on HIV-1 introduction into the IDUs epidemic in Mashhad. This sentinel study could represent the starting point for a wider molecular survey of the HIV-1 epidemics in Iran to evaluate in detail the distribution of genetic subtypes and possible natural drug-resistant variants, which are extremely helpful information to design diagnostic and therapeutic strategies.
Abstract: Mucosal, cutaneous and Epidermodysplasia verruciformis (EV)-related human papillomaviruses (HPVs) were searched by broad-spectrum PCR in 86 conjunctival neoplasia biopsies and 63 conjunctival non-neoplastic control tissue from Ugandan subjects. Seven different EV-related HPV types, including a putative new HPV, and two mucosal HPVs were detected in 25% (14 out of 56) of HIV-positive, in 10% (three out of 30) of HIV-negative conjunctival neoplasia samples, and rarely (0-1.6%) in control subjects. The absence of high-risk HPVs and the low detection frequency of EV-related HPV types in more advanced tumour stages (10%) raise doubts about their role in conjunctival carcinomas.
Abstract: The aim of the present investigation was to define the spectrum of mucosotropic human papillomaviruses among 414 Italian women with normal cervices (n = 183), low- and high-grade cervical squamous intraepithelial lesions (n = 101 and 65, respectively), and invasive squamous cervical carcinomas (n = 65). Human papillomaviruses were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction using three amplification methods and were characterized by nucleotide sequence analysis. The prevalence rates of HPV infections was 19.7%, 63.4%, 80%, and 81.5% in patients with normal cervices, low-grade, and high-grade squamous intraepithelial lesions, and cervical carcinomas, respectively. Among the 205 HPV-positive patients, a total of 31 mucosal HPV genotypes were identified of which 16 types, epidemiological classified as high-risk viruses (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 66, 68, 73, and 82), have been found in 16.9%, 50.1%, 69.2%, and 78.5% of normal cervix, low-, and high-grade cervical squamous intraepithelial lesions, and cervical carcinoma groups, respectively. As expected, the HPV16 was the most represented viral type in all groups examined with frequency rates ranging from 8.7% in normal subjects to 58.5% in invasive carcinoma patients. Ten epidemiologically defined low-risk HPV types (HPV6, 11, 42, 54, 61, 70, 71, 72, 81, 83) were detected in 2.7%, 7.9%, and 6.1% of normal cervix, low-, and high-grade cervical squamous intraepithelial lesions, respectively, and in none of invasive carcinomas. Furthermore, five unknown risk viruses were detected in 3% of low-grade cervical squamous intraepithelial lesions (HPV30, 32, 67), in 3.1% of high-grade cervical squamous intraepithelial lesions (HPV62, 90), and in 1.5% of cervical carcinomas (HPV62). Larger epidemiological screening studies, with PCR amplification and followed by either hybridization-based procedures against sequence targets of all known HPV types or sequence analysis studies, are needed in order to assess the epidemiological risk of less represented HPV types, to identify unknown viruses, and to monitor the future eventual spread of unusual viral types related to vaccination programs and/or population mobility.
Abstract: We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model based on HIV-1 Pr55(gag) 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-VLP(A)s). The HIV-VLP(A)s 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-VLP(A)s in vivo.
Abstract: A molecular and phylogenetic characterization on env and gag subgenomic regions has been performed in our laboratory on HIV-1 variants identified in seropositive individuals residing in Italy, infected in the 1999-2001 period, and five non-B-subtype HIV-1 isolates have been described. To confirm the phylogenetic characterization and to determine the genomic organization of three non-B HIV-1 isolates (A, G, and CRF02- AG), the complete gag, pol, and gp120 ORFs (approx. 6900 bp) have been sequenced for each of them. The phylogenetic tree analyses performed on the whole sequence or on individual genes suggested, for the A and G isolates, the identification of divergent strains that do not cluster into any of the known subsubtypes. This has been further validated by pairwise distance analysis. On the contrary, the phylogenetic classification of the CRF02-AG isolate has been confirmed and an overall typical pattern of intragenomic breakpoints has been observed by a Simplot analysis. These results confirm the constant HIV-1 molecular evolution and indicate the relevance of a continuous molecular monitoring of HIV-1 isolates for the development of appropriate vaccine candidates.
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-VLP(A)s), 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-VLP(A)s by the i.n. immunization route induces a specific CTL activity, although at lower efficiency than the i.p. route. The HIV-VLP(A)s 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-VLP(A)s a good candidate for a mono- and multicomponent worldwide preventive vaccine approach not restricted to high-priority regions, such as sub-Saharan countries.
Abstract: BACKGROUND: The most common TP53 gene polymorphism, which alters the amino acid sequence of the oncosuppressor p53 protein, is located at the codon 72, resulting in either Pro72 or Arg72 p53 variant. Several studies have associated this polymorphism with different types of cancer. We have analyzed the distribution and the role of TP53 Arg72 and Pro72 alleles in conjunctival neoplasia. METHOD: The study included 41 invasive conjunctival squamous cell carcinoma (ICSCC), 33 conjunctival intraepithelial neoplasia of grade 3 (CIN3), 33 of moderate grade (CIN1 and CIN2), and 115 controls from Uganda, a sub-Saharan country with the highest incidence rate of conjunctival neoplasia in the World, particularly in the era of AIDS. The TP53 Arg/Arg codon 72 genotype was detected in 21.9% of ICSCC and in 18.2% of CIN3 but only in 6% of CIN1-2 and in 5.2% of controls (P<0.05). Results: These data show an increased risk of ICSCC (odds ratio (OR)=6.2, 95% confidence interval (CI): 1.6-24.6) and CIN3 (OR=4.1, 95% CI: 1.0-18.0) associated with TP53 Arg homozygosity, not observed in CIN1-2 lesions (OR=0.8, 95% CI: 0.1-5.1). Moreover, the frequency of the Arg homozygosity was similar in HIV-positive and HIV-negative groups. We conclude that TP53 Arg/Arg codon 72 genotype is a relevant risk factor for invasive squamous cell carcinoma of the conjunctiva and for CIN3 in the Ugandan population. DISCUSSION: The absence of statistically significant difference in the distribution of TP53 Arg72 or Pro72 encoding alleles between HIV-positive and -negative subjects, affected by conjunctival neoplasia, suggests that HIV infection and/or the associated immunodeficiency represent further independent risk factors for ICSCC.
Abstract: We have recently developed a candidate 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 an Ugandan HIV-1 isolate of the clade A (HIV-VLPAs).The HIV-VLPAs induce in Balb/c mice systemic and mucosal neutralizing Antibodies as well as cytotoxic T lymphocytes, by intra-peritoneal as well as intra-nasal administration. Moreover, we have recently shown that the baculovirus-expressed HIV-VLPs induce maturation and activation of monocyte-derived dendritic cells (MDDCs) which, in turn, produce Th1- and Th2-specific cytokines and stimulate in vitro a primary and secondary response in autologous CD4+ T cells.In the present manuscript, the effects of the baculovirus-expressed HIV-VLPAs on the genomic transcriptional profile of MDDCs obtained from normal healthy donors have been evaluated. The HIV-VLPA stimulation, compared to both PBS and LPS treatment, modulate the expression of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation.The results of gene profiling analysis here presented are highly informative on the global pattern of gene expression alteration underlying the activation of MDDCs by HIV-VLPAs at the early stages of the immune response and may be extremely helpful for the identification of exclusive activation markers.
Abstract: The geographical distribution of human immunodeficiency virus type 1 (HIV-1) subtypes show, with the exception of some African Countries, a specific pattern. However, the significant phenomenon of migration to Western Countries, coupled to inter-ethnic blending, may result in a constant introduction and spread of novel subtypes and/or recombinant forms in previously homogeneous HIV-1 epidemics. The need to identify and trace these events prompted the development of a rapid and specific bio-molecular tool for the HIV-1 screening, based on the well-established Heteroduplex Mobility Assay (HMA). This modified version of HMA (rHMA) has been designed to detect, by a short electrophoretic analysis, HIV-1 isolates remarkably divergent form the local predominant clade, for subsequent more accurate genetic and phylogenetic analyses. The method has been validated for both C2-V5 region of env gene and the p24-p7 region of the gag gene, by proof-of-concept experiments performed on a panel of reference standards representing the globally most prevalent HIV-1 subtypes, and applied to screen Italian and Ugandan field isolates. The rHMA experimental conditions identified in this study have been shown to be specific and reliable for both sub-genomic regions of each subtype used. In the context of nationwide monitoring programs, the rHMA may represent a powerful tool for the HIV-1 molecular surveillance in both developed and developing countries, particularly those characterized by mono/dual-clade HIV-1 epidemic, which is relevant for epidemiological studies and for the development of preventive and therapeutic strategies.
Abstract: In research on control of the HIV/AIDS epidemic there are many ethical issues to be considered. The problem of personal autonomy versus the interest of society to prevent the spread of the disease in various settings makes it difficult to follow the regulations of the Declaration of Helsinki in all respects. This is particularly clear in the evaluation of trials aimed at preventing mother-to-child transmission of HIV. The interest of the child does not always conform to the policy of avoiding stigmatization of the mother. Programmes for the implementation of antiretroviral therapy and vaccine trials may differ in countries with different mean incomes of the inhabitants, and are also influenced by local patterns. For this reason, the Declaration of Helsinki should be changed in such a way that it conforms with the ways in which it may be possible to combat such a disastrous epidemic as that caused by HIV.
Abstract: Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.
Abstract: OBJECTIVE: The increasing prevalence of HIV-1 transmission through heterosexual contacts and the growing number of immigrants from non-Western countries, where non-B subtypes and recombinant forms are prevalent, suggest the possible emergence in Italy of a new epidemic wave of HIV-1 non-B subtypes as well as recombinant forms. METHODS: The distribution of HIV-1 subtypes has been evaluated in 63 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the last 5 years. A modified heteroduplex mobility assay (HMA) strategy, reverse HMA (rHMA), has been developed in our laboratory, allowing rapid identification of divergent-from-B-subtype isolates, which have been subsequently characterized by detailed molecular and phylogenetic analyses. RESULTS: Five samples show, on rHMA, an electrophoretic pattern compatible with a non-B subtype classification. Their phylogenetic analysis, performed on both env and gag regions, confirms the rHMA subtyping prediction, given that 3 samples fall into the "A-family" subtype and 2 into the G subtype. The 5 non-B-subtype HIV-1 isolates have been identified among 23 variants (prevalence, 21.74%) isolated during the 2000 to 2001 period in heterosexuals. In parallel, B-subtype isolates show high levels of intrasubtype nucleotide divergence, compatible with a constant HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes, and the data indicate that rHMA represents a powerful tool for HIV-1 biomolecular screening in epidemics characterized by a mono-/dual-subtype predominance.
Abstract: Kaposi's sarcoma (KS) represents today one of the most common skin cancers in transplanted Mediterranean subjects and, since the epidemic of human immunodeficiency virus/acquired immune deficiency syndrome, in young unmarried single men. The disease has been associated with the recent identified human herpesvirus (HHV)-8 or KS herpesvirus and its incidence in the general population shows a north to south gradient that parallels the HHV-8 increasing prevalence from Nordic countries to sub-Saharan regions. The identification of the aetiopathogenetic mechanisms (viral agents and immunodeficiency) involved in the pathogenesis of KS, are relevant for identifying susceptible subjects (HHV-8 seropositive subjects), monitoring the immune levels in iatrogenic immune suppressed patients, and developing new therapeutic approaches based on antiviral and immune modulators. Learning objective: This article should enable the reader: (i) to learn about the clinical and molecular aspects of KS in order to have a multidisciplinary approach to a tumour that shows unique features; (ii) to consider the role of viral agents and immunity; and (iii) to recognize properties of an opportunistic neoplasm. The identification of the HHV-8 role in KS pathogenesis should establish a relevant tool in the clinical management of KS patients.
Abstract: Although short-course antiretroviral therapy is efficient in reducing mother-to-child transmission (MTCT) of HIV-1, it does not prevent transmission during the breastfeeding period. There is therefore an urgent need to test various approaches, including HIV-1 vaccination, to try to prevent postnatal transmission of HIV-1 in breastfeeding populations in developing countries.
Abstract: The distribution of Human Immunodeficiency Virus type 1 (HIV-1) clades is evaluated in primary HIV-1 infections (PHIs) occurring through sexual transmission in Lombardia, the Italian region with the highest prevalence/incidence of HIV-1 infections. The two primary inclusion parameters for enrollment were sexual transmission and < 1 year seroconversion. Thirty-four enrolled patients have been analysed so far at the molecular level, to characterize their infecting HIV-1 population. Two HIV-1 genomic regions with different rates of genetic variability, the hypervariable C2-V3 fragment of the env gene and the conserved 5' end of the gag p17, were amplified by Polymerase Chain Reaction (PCR) in peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. Pairwise nucleotide alignment and phylogenetic analyses show that, although with a high range of nucleotide variability, 32 out of the 34 HIV-1 isolates identified in this PHI cohort fall under the clade B genotype. The two remaining isolates, detected in a couple formed by a Nigerian woman and her Italian partner, consistently cluster with clade G standards in both sub-genomic regions. The amino acid sequences confirm this classification, showing clade-specific residues both in the V3 and p17 regions. These data suggest that the B clade is still prevalently associated with acute primary HIV-1 infections occurring in Italy through sexual transmission. However, the significant intra-clade variability and the identification of non-B clades strongly indicate the relevance of continuous molecular monitoring of the HIV-1 isolates circulating in Italy, for prognostic evaluations as well as preventive and therapeutic strategies.
Abstract: 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.
Abstract: 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.
Abstract: Human papillomavirus type 16 (HPV-16) is the predominant HPV isolate found in malignancies of male and female lower genital tracts. However, only a small percentage of individuals infected with high-risk HPVs develop a genital neoplasia, suggesting that additional events at both the cellular and the virus level are necessary for the progression to cancer, including genetic mutations/rearrangements of viral sequences involved in the oncogenic process. In this study, the genetic stability of the long control region (LCR) (nt 7289-114), which regulates expression levels of oncoproteins E6 and E7, was analysed in HPV-16 isolates from penile carcinoma (PC) biopsies of patients recruited from Uganda, one of the countries with the highest incidence of genital cancers in both men and women. Nucleotide changes within the LCR region typical of the African-1 (Af-1) lineage were observed in all HPV-16 isolates. Two out of five samples showed further rearrangements of the enhancer region. The functional activity of LCR with Af-1 mutations and/or rearrangements was evaluated by cloning each LCR into CAT expression vectors, followed by transfection in several epithelial and non-epithelial cell lines. CAT expression levels driven by a rearranged LCR were significantly higher than those driven by Af-1 or European prototype LCRs. Furthermore, in the NIH3T3 focus formation assay, the transforming activity of E6 and E7 genes, driven by a mutated or rearranged LCR, was 1.4- to 3.0-fold higher, respectively. These results indicate that rearrangements within the LCR of HPV-16 isolated from African PCs are frequently found (2 out of 5, 40%). It is also shown that increased HPV LCR activity is associated with an increased E6/E7-mediated in vitro transforming activity, suggesting that natural variants can play a major role in the pathogenesis of genital carcinomas.
Abstract: BACKGROUND: Genital cancers in Uganda have been the most frequently diagnosed cancer in men as well as in women since the 1950s. Genetic studies have detected HPV-16 variants of Af1 class and identified a new sub-class designated Af1-u. OBJECTIVES: The main goal of this study is to analyze the prevalence of HPV strains and HPV variants in anogenital lesions of Ugandan male and female subjects in order to possibly determine their role in the pathogenesis of such lesions and to develop an Ugandan preventive HPV vaccine program. STUDY DESIGN: The study is planning to enroll male and female subjects affected by genital lesions, in particular to collect 200 scrapes/biopsies from women with normal ectocervical epithelium as well as with all different degrees of ectocervical lesions (from CIN 1/LSIL to cervical carcinoma). All samples are analyzed by PCR amplification of the L1 conserved region (nt 6584-7035) and the E6/E7 genes (nt 34-880), nucleotide sequence analysis, homology and phylogenetic studies. Variant distribution studies will be followed by serological studies of prevalence and incidence in 1000 women. PRELIMINARY RESULTS AND CONCLUSIONS: Penile cancers from the Kyadondo County have been analyzed for the presence of HPV sequences. More recently 16 ectocervical scrapes and three biopsies have been received from women attending the Nsambya Hospital and analyzed for the presence and type of HPVs. Our results, obtained by PCR and sequencing analysis, allowed the identification of HPV-16 Af1 sequences in 100% of tumor tissue and in 6.25% of scrapes. HPV 45 was identified only in one tumor together with HPV 16 infection. HPV 33 and HPV 58 were present in 20% and 40%, respectively of HPV positive benign samples. The results are showing a narrowing of the HPV pattern in more advanced lesions, suggesting that mainly HPV-16 Af1 patients are progressing to cancer.
Abstract: Sequence variations in the E6/E7 (nt 34-880) and the L1-(nt 6584-7035) ORFs, and in the long control region (LCR) (nt 7289-93) of human papillomavirus type 16 (HPV-16) were analysed in five penile carcinoma biopsies obtained from Ugandan patients. Uganda is a country with a high incidence of genital cancers. All five isolates were classified as members of African-1 lineage (Af1) by phylogenetic analysis based on LCR sequences. The E6 gene phylogenetic analysis, however, showed that four isolates fell into a new subclass designated Af1-u. This subclass, characterized by three point mutations located at the 5' end of the E6 gene with resulting changes in amino acids at positions 10 and 14, is distinguishable from the Af1 class by the absence of synonymous mutations at nt 286 and 289. The nonsynonymous substitution at nt 335 was present in three out of five samples. The E6 Af1 mutation pattern was present in only a single Ugandan HPV-16 isolate. Nucleotide sequence analysis of the E7 and L1 regions did not allow any Af1 subclass identification. The physical state of the viral DNA in these samples was characterized by PCR and Southern blot analysis. Oligonucleotides which enable amplification of the full length E2 region (nt 2734-3872) failed to amplify the target sequence in four out of five samples, suggesting disruption of the E2 ORF and integration of the HPV genome into the human DNA. Southern blot analysis confirmed the virus integration status. Our results contribute to the characterization of the HPV-16 'African lineages' with the identification of the Af1-u subclass; furthermore, this is also the first report showing that in male genital cancers HPV-16 is integrated into the human genome with disruption of the E2 ORF.
Abstract: The identification of Kaposi's sarcoma (KS) clusters in sub-equatorial 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.
Abstract: We analyzed peripheral blood mononuclear cells from 19 asymptomatic seropositive pregnant women from the district of Gulu in northern Uganda. A 700-bp fragment of the human immunodeficiency virus type 1 (HIV-1) env gene, including the V3-V5 region, was successfully amplified by PCR from 10 samples (52.6%) and was subsequently subjected to both a heteroduplex mobility assay for genetic screening and subtyping and DNA sequence analysis (approximately 300 bp) for nucleotide comparison and phylogenetic studies. The results show the presence of HIV-1 A and D subtypes (or clades) in this rural area, with the prevalence of the A subtype (8 of 10) being greater than that of the D (2 of 10) subtype, which is unlike what was previously reported for Uganda. By pairwise comparison analysis, the percentage of sequence divergence among samples within each subtype is low (the average intrasubtype divergence is 15.8%), but it is significantly higher between the two subtypes (the average intersubtype divergence is 23%). At the amino acid level, the two HIV-1 subtypes show distinct tetramers at the apex of the V3 loop and, in particular, GPGQ in clade A and GPGR in clade D. In addition, 10 of the 19 viral samples (52.6%) have been isolated in vitro. Nine of the samples have been classified as rapid/high, which reflects a high in vitro replication capacity for the HIV-1 field isolates from this country, even for those obtained from seropositive asymptomatic individuals. These observations, despite being made on the basis of a limited sample size, show a modest degree of genetic divergence among samples isolated in the last 4 years in this country by comparison with those based on the 1990 data on HIV-1 isolates from Kampala. The results reported here are, therefore, extremely relevant for Uganda, which is one of the selected World Health Organization field sites for future HIV-1 vaccine evaluation programs.
Abstract: We have previously shown that the Tat protein of human immunodeficiency virus type 1 (HIV-1) transactivates tumor necrosis factor alpha and beta (TNF alpha and TNF beta) gene expression in HIV-1-infected and in tat-transfected T-lymphocytic and monocytic cell lines. The product encoded by the first exon of the tat gene (amino acids 1 to 72) is sufficient for this transactivation. Here we show that (i) the NF-kappa B and Sp1 binding sites of the TNF beta promoter are required for Tat-mediated transactivation and (ii) a predicted stem-loop structure in the TNF beta mRNA leader region, which resembles the Tat-responsive element of the HIV-1 long terminal repeat (TAR) and which is therefore termed TAR-like, is essential for TNF beta transactivation by Tat. These data suggest that similar promoter regulatory elements are necessary for Tat-mediated transactivation of both TNF beta and HIV-1 gene expression. This represents the first demonstration of a cellular gene with a regulatory element downstream of the transcriptional initiation site that, like TAR, may function as an RNA element.
Abstract: To investigate the possible direct/indirect role of Human immunodeficiency virus (HIV) as a cofactor in human papillomavirus (HPV) oncogenesis, cotransfection experiments were carried out in which a recombinant plasmid containing the HPV16 long control region (LCR) linked to the chloramphenicol acetyltransferase (CAT) gene was cotransfected into cultured cells with a plasmid expressing HIV-1 Tat protein. Tat expression efficiency and transactivation activity were evaluated in different cell lines by cotransfecting plasmids containing the HIV tat gene and HIV LTR-driven CAT-coding sequences. HeLa and CaSki cell lines represented the most appropriate recipient cells for Tat-directed transactivation of both the HIV LTR and the HPV LCR promoters. Furthermore, HIV tat was transfected into HeLa cells (containing 10-20 copies per cell of HPV18), and HPV18 E7 protein expression was evaluated by a radioimmunoprecipitation assay using polyclonal antibodies against the E7 protein. Our results show that the Tat protein can transactivate the HPV LCR and increase HPV18 E7 expression in HeLa cells.
Abstract: Biopsies of 13 penile cancers (PC), from patients living in regions of Uganda with a high incidence of genital cancers, were studied for the presence, molecular characteristics and physical state of DNA related to that of human papillomavirus (HPV) types 6, 11, 16, 18, 31 and 33. HPV DNA sequences were detected in all PC specimens by dot/Southern blot analyses and by gene amplification of DNA sequences highly conserved among several HPVs. HPV 16 DNA sequences were found in one PC; DNA sequences with low homology to HPV16 or HPV18 were present in all other samples. Viral DNA is primarily integrated in the cellular DNA. To isolate and characterize a possible highly oncogenic HPV, a genomic library of the DNA extracted from the PC-8 biopsy has been constructed in the EcoRI arms of the EMBL4 phage. A single phage containing 8.30-kb HPV16-related sequences has been identified and the 3 segments of 0.45, 0.65 and 7.2 kb, released by EcoRI digestion, have been independently subcloned in pUC18 for further analysis.
Abstract: Etiopathogenetic mechanisms involved in the development of Kaposi's sarcoma and the possible role of viruses, such as CMV, BKV, HBV, HHV-6 and retroviruses are reviewed. An etiopathogenetic model is also presented.
Abstract: Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro. By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated. Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences. Two cell lines which retained viral DNA were chosen for further study. They were capable of anchorage independent growth and formed tumors in nude mice. Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting. A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences. A complex rearrangement between pCM4127 and pSV2neo had occurred. DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence.
Abstract: NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.
Abstract: Incidences of neoplastic transformation of 10T 1/2 cells, derived from a C3H mouse embryo, were measured following irradiation of cells with 60Co gamma-rays delivered in single total doses, from 25 to 300 rads, or in different numbers of fractions and with different time intervals between fractions. Throughout the range of total doses that was examined (25-300 rads) exposure of asynchronous, actively growing 10T 1/2 cells to a dose of gamma-rays in five equal daily fractions, rather than in a single exposure, results in a significant reduction in transformation frequency. The initial part of the induction curve fits a straight line quite well. The slope of the line for five daily fractions is approximately 3-fold less steep than that for single exposures. A further increase in the interfraction interval, to 48 h, or further reductions in the dose per fraction, led to additional decreases in the incidence of neoplastic transformation. Our observations are consistent with a net 'error-free' repair of subtransformation damage as a result of protracted irradiation with 60Co gamma-rays.
Abstract: When 0.5 M sodium thiocyanate is added to uterine cytosol previously labeled with excess [3H]-17 beta-estradiol, no change can be detected in the steady-state cytosol concentration of [3H]estradiol-receptor complex for at least 20 h at 4 degrees C. However, the rate of exchange of bound estradiol in the presence of NaSCN was found to be substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increases about 10-fold (K-1 SCN = 1.10 x 10(-2) min-1 vs. K-1 = 1.07 X 10 (-3)min-1) while the rate of association increases about 2-fold (K1 SCN = 1.2 X 10(7) min-1M-1 vs.K1= 7.4 X 10(6) min-1 M-1). The Kd changes 6.4-fold (Kd SCN = 9 X 10(-10) M vs. Kd = 1.4 x 10(-10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN can be exploited to assay preformed estrogen-receptor complex by exchange with [3H]estradiol at low temperature. When the sample containing preformed complex is incubated overnight (16 h) at 4 degrees C with excess [3H]estradiol in the presence of 0.5 M NaSCN, there is a quantitative exchange of nonlabeled for estradiol without loss of binding sites. Hormonal steroids other than estrogens do not interfere, and the exchange estradiol is bound with high affinity. Precision, accuracy, and linearity of the method are highly satisfactory.
