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LABORATORY OF: Viral Immunology - GASLINI INSTITUTE
CONTACT PERSON: Dr. Fabrizio Manca
Phone +39 010 5737370 E-mail: manca@email.it

Description of Laboratory and Expertise:

Cellular immunity specific for relevant human viruses is the main focus of this Unit. Therefore the laboratory of Fabrizio Manca has developed tissue culture facilities for the functional analysis of human T lymphocytes in vitro. Immunochemical, cytometric and molecular biology techniques are currently used for functional studies and for the definition of cytokine profiles. T cell expansion and selection methods allow to produce established T lymphocyte lines with defined specificities, that are analyzed for clonal diversity and fine specificities. These lines are essential tools to test antigen preparations (synthetic or recombinant) as potential vaccine candidates.

Abstract of Activities:

The Laboratory of Viral Immunology is focused on the analysis of pathogen-specific cellular immunity as a relevant mechanism in protection from viral infections. In fact, the combined action of CD4 and CD8 T lymphocytes complements with antibody responses. Two viruses relevant for human pathology are being investigated: cytomegalovirus (CMV) and HIV. Immunity to the former virus is highly effective to confer protection, while immunity to the latter is apparently ineffective. We have developed CD4 and CD8 T cell lines specific for the two viruses as tools to test in vitro antigenicity of vaccine candidates, to test optimal modes of antigen delivery to antigen presenting cells and to screen immunodominant peptides that can be included in vaccine preparations or be used as diagnostic tools to assess cellular immunity in immunocompromised or immunocompetent subjects.
Our work is based on collaborative projects with other laboratories at ABC (Li Pira, Quarto) and with other partners with specific expertise in the development of vaccine candidates, either synthetic or recombinant.

Detailed Research Activities:

One of the major goals of vaccinology is to produce vaccine candidates that enhance cellular immunity, i.e. responses of pathogen-specific CD4 and CD8 T lymphocytes. Different tools for antigen delivery are currently available and are continuously being developed. Immunogenicity of such preparations can be easily tested in animal models, but in vitro systems need to be developed in order to test antigenicity with human immunocompetent cells. Therefore, we have produced a panel of human T cell lines with different phenotypes and with specificities for different pathogens, keeping our focus on CMV and HIV.
These established T cell lines are valuable tools to assess antigenicity of different vaccine candidates. In particular, the T cell lines can be used as readouts for antigen processing and presentation. In fact, depending on the different carriers that display antigenic proteins or peptides, antigens can be taken up by presenting cells with different efficiency. These events can be related to higher or lower efficiency of antigen delivery in vivo and thus are important features to be defined. As an example, microparticulated antigens, virus like particles of antigens that are made more palatable for antigen presenting cells, are preferentially or selectively captured by APC. This leads to more effective antigen presentation to specific T cells present in the naïve repertoire, resulting in improved priming. The same mechanisms may facilitate secondary responses with recruitment of memory T cells of different phenotype.

For the construction of antigen carriers (either recombinant or synthetic), we rely on the collaboration of different groups with specific expertise. Virus derived epitopes can be expressed on viral vectors (e.g. MVA vectors encoding for CMV or HIV epitopes, retroviral vectors encoding for HIV epitopes, virus like particles expressing HIV peptides) that can be proposed for human use. Such vectors can encode for viral antigens synthesized within the APC and thus the epitopes can be optimally presented to CD8 lymphocytes. This is tested by using our virus specific CD8 T cell lines as readouts. If viral antigens are synthesized and secreted by vector infected cells, they can be taken up by APC as exogenous antigens, thereby favouring presentation to CD4 lymphocytes. Also these events can be dissected using our virus specific CD4 T cell lines.

An accurate epitope mapping of viral proteins has been undertaken using a miniaturized method developed in collaboration with the Unit of Cellular Immunology. The high-throughput method is now applied to screen response profiles in terms of fine protein-peptide specificities and cytokine patterns in several cohorts, in collaboration with other clinical units, in HIV infected patients. Tuberculosis patients are also under screening with dedicated plates that contain antigenic panels (proteins and peptides) focused on the relevant pathogen.

By using culture methods for T cells based on repeated stimulation cycles with autologous antigen presenting cells and antigenic proteins or peptides, we have successfully selected and expanded T cells obtained from the naïve repertoire in non-immune subjects. This protocol mimics in vitro the events occurring in vivo when the immune system of a naïve subject is challenged with antigen for the first time, as in the case of a primary infection or of a prophylactic vaccination. We are currently improving the efficiency of these culture systems, by enhancing interactions between T cells and presenting cells, using additional accessory cells (e.g endothelial, stromal) to mimic the anatomical compartments of the lymph-nodes where these events take place in vitro. For a better simulation, we are also testing 3D structures in collaboration with the Stem Cell Laboratory (R. Quarto). We are also committed at comparing the fine specificities of the responses induced by in vitro priming with responses that can be observed after deliberate vaccination. It may be helpful, in fact, to predict the quality of the response in a vaccine candidate, expecially if peptide based vaccines will be used in the future.

Since antigen specific T cell responses can be highly oligoclonal when a single epitope is recognized, we are defining clonal diversity and clonal identity in human T cells selected in vitro according to their specificity. Preliminary observations in HIV specific and CMV specific T cell lines have shown that the same T cell clonotypes persist for years in vivo even in the absence of an antigenic stimulus. Clonotypes are identified by sequencing the variable regions of the T cell receptors and, based on these sequences, clone-specific primers have been designed for molecular identification of the relevant clones in the heterogenous population of circulating lymphocytes. Panels of such primers that define antigen specific T cells will be constructed as molecular probes for antigen specific T cells in peripheral blood or in inflammatory sites.

Since we are involved in research projects focused on HIV vaccine candidates, we are also committed to the development of in vitro assays to test cellular immunity in vaccinees participating in vaccination trials. Evaluation of cellular immunity, in fact, poses a serious challenge for accurate monitoring during the trial. In fact, while antibodies are a robust analyte that can be measured in serum samples collected and frozen locally, specific T cells are ideally appreciated on fresh blood specimens using functional assays. Therefore we are developing 1. panels of viral antigens derived from CMV and HIV in the form of proteins or immunodominant peptides, 2. methods for simple, fast, inexpensive and robust analysis of T cell immunity, to be applied in resource poor settings, as those in which HIV vaccination trials are generally carried out. In collaboration with the Laboratory of Cellular Immunology (G. Li Pira) at ABC, we have developed novel assay systems to functionally quantitate T cell immunity. These assays have been refined in high-throughput formats and are being validated in primate vaccination trials (coll. G. Lewis, Baltimore and G. Koopman, Rijswijk). For these trials, we shall ship pre-formatted, frozen microculture plates in which antigenic preparations have already been dispensed. Lymphocytes from the vaccinated animals will be dispensed and cultured for 24 hours. After culture, the plates will be frozen and shipped back to our laboratory for development (miniaturized assays for detection and profiling of secreted cytokines). This approach can obviously be proposed for human trials carried out in developing countries.

Applications and Developments:

Since it is well established that cellular immunity plays a relevant role in protection from viral infections, its assessment by standardized in vitro methods, its potentiation by appropriate vaccine candidates and its reconstruction by adoptive cellular immunotherapies are important perspectives and biotechnological challenges. Therefore, applications are foreseen at the diagnostic and therapeutic level. Novel functional assays will be developed with our antigen specific T cell lines, that can be taken as gold standards. Oligonucleotide primers based on the sequences of hypervariable T cell receptor genes will be applied to the molecular definition of antigen specific T cells.

Ongoing collaborations:

International
Florian Kern, Univ. Brighton, UK. Detection of antigen specific T cells and epitope mapping
Frances Gotch, Imperial College London, UK. Cytokine profiles of specific T cells
George Lewis, Inst. Human Virology, Baltimore, US. Cytokine profiling in primates
Susan Zolla Pazner, VA Hospital, New York, US. HIV specific human monoclonal antibodies
Jan Gratama, Erasmus Med. School, Rotterdam, NL. MHC-peptide multimers
Mario Roederer, Vaccine Res. Ctr., NIH, Bethesda, US. Antigen specific T cells

Domestic
Luisa Bracci, Univ. Siena. Production of panels of synthetic peptides for epitope mapping
Martino Introna, City Hospital, Bergamo. Generation of GMP grade therapeutic T cell lines
Raffaele de Palma, II Univ. Naples. Analysis and sequencing of T cell receptor genes
Paolo Rossi, Univ. Tor Vergata, Roma. Cellular immunity in immunocompromised children

Local
Gino Tripodi, Gaslini Inst. Genova. Selection of immunocompetent donors for a T-cell bank
Claudio Viscoli, Infect. Dis. Univ. Genoa. Cellular immunity in HIV patients
Giusi Li Pira, Cellular Immunology, ABC Genoa. Epitope mapping and T cell immunity
Rodolfo Quarto, Stem Cell Unit, Univ.-ABC, Genoa. T lymphocyte and mesenchymal stem cells

Most recent and significant publications:

Dander E, Li Pira G, Biagi E, Perseghin P, Renoldi G, Gaipa G, Introna M, Marin V, Manca F, Biondi A, D’Amico G. Characterization of migratory activity and cytokine profile of helper and cytotoxic CMV-specific T cell lines expanded by a peptide library. 2008, Exp. Haematol. In press

Li Pira G, Ivaldi F, Bottone L, Manca F. High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays. J Immunol Methods. 2007,326,22-32

Li Pira G, Kern F, Gratama J, Roederer M, Manca F. Measurement of antigen specific immune responses: 2006 update. Cytometry B Clin Cytom. 2007,72,77-85

D'Apice L, Sartorius R, Caivano A, Mascolo D, Del Pozzo G, Di Mase DS, Ricca E, Li Pira G, Manca F, Malanga D, De Palma R, De Berardinis P. Comparative analysis of new innovative vaccine formulations based on the use of procaryotic display systems. Vaccine. 2007,25,1993-2000

Li Pira G, Ivaldi F, Bottone L, Koopman G, Manca F. Li Pira G, Ivaldi F, Bottone L, Koopman G, Manca F. Helper function of cytolytic lymphocytes: switching roles in the immune response. Eur J Immunol. 2007,37,:66-77.

Li Pira G, Ivaldi F, Bottone L, Quarto R, Manca F. Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells. Hum Immunol. 2006,67,976-85

Li Pira G, Bottone L, Ivaldi F, Risso M, Tripodi G, Manca F. A sealed and unbreached system for purification, stimulation and expansion of CMV specific human CD4 and CD8 lymphocytes. Transfusion 2006, 46,2053-62

Li Pira G, Bottone L, Ivaldi F, Accolla R, Koopman G, De Palma R, Manca F. Human naive CD4 T cell clones specific for HIV env persist for years in vivo in the absence of antigenic challenge. J. AIDS, 2005,40,132-139

Kern F, Li Pira G, Gratama J, Manca F, Roederer M. Measuring antigen specific immune responses: understanding immunopathogenesis and improving diagnostics in infectious diseases, autoimmunity and cancer. Trends Immunol. 2005,9,477-484

Li Pira G, Bottone L, Ivaldi F, Barbano G, Manca F. Generation of Cytomegalovirus specific CD4 T cell lines devoid of alloreactivity using a cocktail of CMV-pp65 peptides for reconstitution of the Th repertoire. J. Infect. Dis., 2005,191,215-226

Patent

Method for determining antigen-specific T cell responses in high throughput format
06012034.2, June 2, 2006

Inventors: Fabrizio Manca, Giusi Li Pira