Research Activity
The main function of the immune system is to protect individuals from infective micro-organisms by producing specific antibodies and activating cells specialised in recognising and eliminating the infective agent and its products. The antibodies can prevent viruses from entering cells by recognising and binding to specific molecules on the surfaces of cells and viruses but, for reasons that are still unknown, this does not happen in the case of AIDS because the functions of the immune system are impaired and the virus manages to get the upper hand. The research being carried out at the HIV Immunobiology Unit has the aim of studying humoral (antibody) immunity as a possible means of developing new AIDS vaccines and therapies.
We are currently working on the folowing research lines:
I) Studies of risk populations (the sexual partners of seropositive subjects, prostitutes, drug addicts, the children of infected mothers, subjects receiving multiple blood transfusdions, etc.) have shown that some some individuals remain uninfected despite multiple exposures to HIV, and this has led us to investigate the possible protective mechanisms involved. The identification of the immunological mechanisms underlying this protection against HIV infection could provide tools that are useful for understanding how the immune system overcomes HIV. During the course of a number of years of observation, we have identified some specific humoral responses that are only present in "protected" subjects. These antibodies bind to a protein called CCR5, which is found on the surface of the cells of the immune system in all individuals and represents one of the most important routes of viral entry. The mechanism of action of these antibodies is very particular insofar as it modifies the CCR5 protein in such a way as to make it inaccessible to the virus, and this therefore provides natural protection against HIV infection. The production of anti-CCR5 antibodies can be induced by administering specific immunogens that should stimulate the immune system into producing its own antibodies against the protein (the vaccinal strategy) or, by means of special biotechnological techniques such as the "phage display library", the antibodies themselves can be used as a passive immunotherapy during the early phases of infection and in all subjects at risk. We have produced a recombinant antibody capable of specifically binding the CCR5 protein found on the membrane of CD4+ cells, and are currently working on its biological characterisation. Antibodies binding to CCR5 receptor can activate a cascade of intracellular events, able to induce recepto r internalization, downregulating their expression on cell surface. CCR5 endocytosis is physiologically carried out via the caveolae-mediated internalization process. Caveolae are cholesterol-enriched rafts located in the cell membrane, involved in the protein internalization and trafficking. As shown by our group, CCR5-antibodies complexes are internalized via a Clathrin-mediated process. Clathrin-mediated internalization is an alternative pathway, shown to be involved in the internalization process of a different HIV coreceptor molecule, CXCR4. Our experimental results led us to draw the following hypothesis. The anti-CCR5 antibodies binding to CCR5 receptor triggers a complex event, involving a temporary lipid platform, whose lipid components are able to take part in and modulate the binding process, via conformational changes and binding. In Figure 1, an hypothetic functional model of anti-CCR5 antibodies is presented. Characterization of anti-CC5 antibodies binding to CC5 molecule may lead us to discover new cell metabolic pathways for protein internalization , and, most of all, to identify innovatiove pharmacologic approaches able to block not only HIV infection but other inflammatory pathways relying on CCR5 signalling. In fact, CCR5 receptor is involved in the pathogenesis of inflammation, when inflammatory cytokines are released in the damaged tissues. Anti-CCR5 antibodies might play a role in preventing the CCR5 pathway, thus exerting an anti-inflammation effect.
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II) The mucosa is an important natural barrier against all sexually transmissable diseases, including AIDS. The development of new therapeutic approaches capable of protecting the mucosa aganst pathogenic micro-organisms could therefore be useful in a variety of situations. Over the last years, our group has identified another humoral immunological repertory capable of blocking HIV at mucosal level: IgA antibodies that recognise a small portion of an HIV envelope protein called gp41. This small IgA-recognised is highly conserved in all of the HIV strains in the different parts of the world, and so a therapy based on these antibodies could solve the problem of the variability of the virus, which is still one of the biggest obstacles to the development of specific vaccines.
New pharmacological approaches migh be focused on the study of small animal model, such as the mouse, has provided us encouraging results on the in vivo induction of both anti-CCR5 and anti-gp41 antibodies. Such antibodies, endowed with biological properties comparable to antibodies elicited in human natural antibodies, can be obtained in mice under systemic as well as mucosal immunization. In other words, we inoculated female mice intranasally, using the same peptides recognized by human antibodies, and we detected specific antibodies displayed on their vaginal mucosae.
These results could lead the way to set simple and smart approaches, both for prophylaxis and vaccination, able to block sexual transmission of HIV infection
III) Neutralising humoral responses (antibodies capable of blocking HIV replication) appear very late during the course of HIV infection, and their slow development may be one of the reasons underlying the virus's ability to establish a persistent infection. The introduction of highly aggressive anti-retroviral therapies (HAARTs) has considerably modified the clinical course of the disease with the consequent restoration of the immunological anomalies caused by the virus itself. Little is known about the HIV-neutralising humoral repertory in HAART-treated patients, but a large number of studies have demonstrated the highly protective role played by neutralising antibodies in seropositive patients experiencing very slow or no disease progression. We are currently carrying out a study of the role of neutralising antibodies in patients receiving HAART during the early stages of infection, and have found a highly significant statistical correlation between the treatment and the development of neutralising antibodies. These results not only confirm the protective role of humoral responses, but also highlight the possibility of adequately stimulating the immune system to control the infection..
IV) Another area of research concerns host cell-HIV interactions or, more precisely, the role of membrane proteins (other than the already known receptors and co-receptors) in the infective process. In particular, we are studying the role of some class I major histocompatibility system molecules (HLA-C class I) and the anti-HLA-C response in HIV infection and the pathogenesis of AIDS. We have identified regions of structural and immunological homology between gp 120 (an HIV envelope protein) and HLA-C, which suggests that some subjects may develop specific humoral immunity against gp120 and/or the homologous region of HLA-C. We have also observed specific antibody responses in vaccinated healthy volunteers and in individuals exposed to HIV but not infected. In our attempts to improve our understanding of the role played by HLA-C in the infection, we have demonstrated that the cell fusion induced by HIV-1 is modulated by a subset of HLA-C molecules on the surface of the receiving cell that interact with one of the regions of gp41. We have also collected evidence of two types of reaction between HIV envelope proteins and HLA-C: frontal interactions (between molecules anchored to two different cells, or between one molecule anchored to the target cell and the other to the virus) and lateral interactions (between two molecules anchored to the same membrane). In order to verify the possible role of HLA-C as a gp41 receptor, we have produced chimeric HLA-C proteins that will be used to study the specific role of HLA-C in the infection.
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