Meetings

09.0 Welcome addresses :

S. Natoli (INMI "L. Spallanzani")
T. Carettoni (UNESCO National Commission)
E. Garaci (MURST)
L. Montagnier (World Foundation AIDS Research & Prevention)
G. Rotilio (CNR)

09.30 Opening Lecture: (G. Tocchini Valentini) "New perspectives in Biology"
presentation of the Lecture by G. Ippolito (INMI "L. Spallanzani")

Molecular bases of the microbial escape mechanisms

10.15 Chairmen: F. Bistoni (University of Perugia)
S. Vella (ISS, Rome)

10.30 V. Erfle (GSF, Institute of Molecular Virology): Modulation of nuclear trafficking of HIV-Rev
10.45 L. Romani (University of Perugia): Fungi at the interface with the innate immune system: implications for pathogenesis and immunity
11.00 A. Santoni (University of Rome "La Sapienza"): Viral escape mechanisms from natural killer cell-mediated response
11.15 M. Capobianchi (INMI "L. Spallanzani, Rome): Comparative analysis of cell derived membrane proteins present on circulating HIV-1 in patients before initiation of HAART+IL 2 and after controlled therapy suspensions.

11.30 Coffee Break

Strategies for development of immunotherapeutics in infectious diseases

11.45 Chairmen: A. Salerno (University of Palermo)
M. Fiorilli (University of Rome "La Sapienza)

12.00 T. Lehner (University of London): "A novel HIV vaccine strategy targeting both the CCR5 coreceptor and HIV".
12.15 F. Poccia (INMI "L. Spallanzani & ICAERI, Rome): Involvement of intrahepatic lymphocytes in HCV and HIV immunopathology
12.30 A. Cassone: (ISS, Rome) Novel immunotherapeutic approaches for control of opportunistic infections
12.45 G. Antonelli (University of Rome "La Sapienza") Host & viral predictive markers of response to interferon alpha in hepatitis C patients.
13.00 G. D'Offizi (INMI"L. Spallanzani, Rome): STI in HIV infection
13.15 C. F. Perno (University of Rome "Tor Vergata" & INMI "L. Spallanzani") Strategies for the selective elimination of HIV reservoirs in the body

13. 30Lunch

The biomedical research & transfer technology in Africa

14.45 Chairmen: T. Carettoni (UNESCO National Commission)
G. Rotilio (CNR, Rome)

15.30 H.Chenal (CIRBA, Abidjan): HAART therapy in Africa: experiences and problematics
15.50 V. Colizzi (University of Rome "Tor Vergata" & ICAERI): Immune response to HIV-1 Nef peptides as model for the anti-AIDS therapeutic vaccine: preliminary steps in Abidjan
16.10 G. Del Prete (University of Florence): A study of genetic and environmental risk factors for tuberculosis in West Africa
16.30 F.Esposito (University of Camerino): Research and Training on Malaria in Burkina Faso
16.50 G. Magliano (MAE, Rome): The Italian Co-operation in Africa

20.00 Social Dinner

o Scientific Committee: V. Colizzi, E. Garaci, G. Ippolito, L. Montagnier, A. Sanduzzi.

o Organizing Committee: C. Costa, V. Kouzminov, T. Persichini, P. Vagliani,

o Scientific and financial sponsorships: UNESCO Venice Office, CNR, INMI "L. Spallanzani", Monaldi Hospital, University of Rome "Tor Vergata".

Modulation of HIV-1 Rev function

Volker Erfle, Ruth Brack-Werner and Markus Neumann, GSF-Institute for Molecular Virology, Neuherberg, Germany (e-mail: erfle@gsf.de).

Many viral infections of the central nervous system usually do not result in fulminant virus replication and often lack signs of inflammation., probably to protect this organ from rapid destruction. Nevertheless several viruses such as HIV-1 can infect brain cells and do exert long term negative effects. Additionally such latently infected cells form potentially very large reservoirs for the virus making therapeutic attempts of virus eradication difficult if not impossible.
In our work we studied the replication of HIV in astrocytes, which make up about 40% of the total cell mass in the brain of which between 0.1 and 2% can become infected (Brack-Werner, 1999). We found, that virus replication is reduced dramatically shortly after infection and cannot be significantly induced by know stimulators of HIV-expression. This interesting feature could be largely attributed to a lack of efficient stimulation of the synthesis of structural virus proteins by the essential viral regulatory protein Rev. In addition, Rev, which shuttles between the nucleus and the cytoplasm of cells, is aberrantly localized to the cytoplasm of astrocytes compared to a usually nuclear localization in Rev-permissive cell types. Using a new live-cell-assay allowed us to demonstrate a strongly reduced rate of nuclear accumulation of Rev in astrocytes.
This is one of the first documented examples of cell specific downregulation of virus replication by interefering with an essential mechanism of viral replication and by this control virus spread..

Luigina Romani
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università degli Studi di Perugia, Via del Giochetto, 06100 Perugia.

The fungus Candida albicans can switch from a unicellular yeast form into various filamentous forms, all of which can be found in infected tissues. The ability to reversibly switch between these forms is thought to be important for Candida's virulence. Although recent studies have clearly shown that the ability to switch from yeast to filamentous form is required for virulence, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still an open question. One possibility is that the filamentous growth form is required to evade the cells of the immune system, whereas the yeast form may be the mode of proliferation in infected tissues. For this to be possible, a cell must exist that finely discriminates between the two forms of the fungus in terms of class of immune response elicited. Yeasts and, to some extent, short filamentous forms of C. albicans can be ingested by neutrophils and macrophages through a variety of mechanisms and opsonic requirements, ultimately affecting the antifungal effector functions of the cells. Recent evidence indicates that neutrophils discriminate between the two forms of the fungus, being able to produce IL-12 in response to C. albicans yeasts, and IL-10 in response to C. albicans hyphae. However, the induction of T-dependent immunity against the fungus necessitates prior pathogen-nonspecific triggering of antigen presenting cells capable to activate antigen-specific Th cells. Dendritic cells (DC) fulfill this requirement, being uniquely able to initiate responses in naive T cells and to participate in Th cell education. Immature myeloid dendritic cells rapidly and efficiently phagocytose both yeasts and hyphae of the fungus. Phagocytosis occurs through different phagocytic morphologies and receptors, resulting in phagosome formation. However, hyphae escape the phagosome and are found lying free in the cytoplasm of the cells. In vitro, ingestion of yeast activates DC for IL-12 production and priming of Th1 cells, while ingestion of hyphae inhibits IL-12 and Th1 priming, and induces IL-4 production. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. These results indicate that DC fulfill the requirement of a cell uniquely capable of sensing the two forms of C. albicans in terms of type of immune responses elicited. Moreover, the finding that the internalization and the intracellular localization of the different forms of the fungus occur differently in effector phagocytes and DC, suggests a distinction of labor between different phagocytic cells in the overall resistance to the fungus. Upon contact with the fungus, effector macrophages and neutrophils rapidly activate oxidative and nonoxidative pathways of killing through phagocytic and nonphagocytic mechanisms. In contrast, the antifungal activity of DC appears to be more tightly regulated. This may ultimately have an impact on fungal antigen presentation by DC. Indeed, yeast degradation inside phagolysosomes may result in an efficient release of fungal peptides for class II-restricted antigen presentation, while hyphae surviving free in the cytosol may eventually intersect the class I-restricted antigen presentation pathway. As both MHC class II-and class I-restricted T cell responses have been detected in mice with candidiasis, our results suggest that DC are uniquely qualified to serve as APC in antifungal host immune responses.

VIRAL ESCAPE MECHANISMS FROM NATURAL KILLER CELL-MEDIATED RESPONSE
Angela Santoni and Gabriella Palmieri

Department of Experimental Medicine and Pathology
University "La Sapienza", Rome, Italy

Natural Killer (NK) cells, by their constitutive cytolytic activity against virus-infected cells, and their ability to rapidly synthesize and secrete a wide array of cytokine (mainly IFNg, TNFa, GM-CSF) and chemokines, constitutes one of the most important effector mechanisms that control the early phases of viral infections, and also regulate the following adaptive anti-viral responses.
The importance of the loco-regional vs. systemic immune responses, and the temporal relationships between the activation of different cell populations, are recently becoming more and more appreciated as crucial parameters for the successful control of viral infections. Chemokine-driven and integrin-supported NK cell migration to the target organ, NK cell activation upon interaction with activating and/or costimulatory receptors on infected cells, and with the organ microenvironment (local cytokines, components of the extracellular matrix, other immune cells), lead to activation of a complex functional program, consisting of the discharge of perforin- and granzyme-containing cytotoxic granules, the expression/upregulation of ligands for the TNF receptor family, and the production of cytokines and chemokines. These NK functional activities result in the death of infected cells, and in the activation and recruitment of further effectors in the anti-viral response.
Examples of different strategies adopted by a vast array of viruses aimed at interfering with NK cell recruitment, recognition/costimulation and induction of apoptotic death will be discussed. In particular:
a) viral cytokines or chemokines, viral homologs of cyto/chemokine receptors, viral proteins interfering with the cytokine-activated intracellular signalling pathways can alter either the localization of NK cells, and their activation and ability to activate other cell populations in a cascade. Moreover, m144 MCMV protein, putatively interacting with an inhibitory receptor on NK or other cells, can alter NK cell redistribution;
b) the modulation of CD94/NKG2-A inhibitory receptor recognition of HLA-E specific ligand by UL-40 HCMV protein, the downregulation of ICAM-1 and B7-2 ligands for activating/costimulatory receptors on NK cells by Kaposi sarcoma-associated Herpes virus, represent two different strategies for inducing the functional inactivation or preventing the triggering of NK cells;
c) the expression of anti-apoptotic proteins by several viruses can efficiently block either the granzyme A/B initiated caspase activation, or interfere with the formation of the DISC by TNF and/or FasL-induced triggering of different death receptors; moreover, the perturbation of activation-induced apoptosis of effectors themselves can alter the termination of the immune response, and contribute to immunopathology.

1. Recruitment to infection site:
CMV or LCMV - the importance of the local vs systemic response. The importance of the extravasation, tissue microenvironment, ECM, and cell/cell adhesion contact.
The importance of local production of cytokines by NK cells (i.e. IFNg, to activate other effector cells - Mig) and chemokines (to recruit the relevant cell populations).
Escape: cytokine competitors (IFNg), viral chemokines (vMIP) which interfere with activation or migration or determine a subversion of the response (Th1/Th2).
MCMV viral chemokine m131. Its mutation leads to a more rapid clearance which is dependent on both Nk and CD4/8 T cells.

2. Recognition/activation
1. Downregulation of MHCI by many viruses
Example: HCMV US2/US11 (redirect to cytoplasm HLA-A and B, not C)
US3 (retention)
US6 (blocks TAP)
HIV (Nef) downregulates HLA-A and B, but not C and E
Impairment of CTL recognition, but leave intact NK inhibitory interactions.Expression of MHC homologs (ILT-2 - part of NK cells, B and Mo/UL18; 1000-fold higher affinity than classical MHC I); upregulation of endogenous MHC (NKG2-A/HLA-E+UL-40 (TAP-independent)). But ILT-2 is on only a subset of NK ceells, so it could be more inhibtory for other cell populations. The inhibitory activity of UL18 on Nk cells not confirmed (Leong reports augmentation of lysis by NK clones of HCMV-infetcted HFF expressing UL18 and in UL18 transfected cells) - upregulation of ICAM-1)
MCMV: resistance in spleen but not in liver maps to NKC (Cmv-1 locus) and is NK1.1-dependent. Disruption of m144 gene (UL18 homolog) decreases in vivo infectivity.
MCMV infection modifies NK distribution in spleen/peritoneum, and proportion of different Ly49; m144 transfected in a lymphoma regulates accumulation and activation of NK cells (direct or indirect effect, through other cells expressing inhibitory receptors?)

Kaposi Herpesvirus K3 and K5 proteins downregulate MHC-1
K5 protein also downregulates B7-2 and ICAM-1; resistance to NK lysis, impairment of activating (CD28-like) and costimulatory (LFA-1) signals.
Herpes virus and HCMV downregulation of HLA-C and upregulation of NK lysis

3. Effector mechanisms:
Example: MCMV, and many other viruses are more susceptible to perforin and granzyme lytic mechanisms (from CTL/CD8 and NK)
Escape: resistance to apoptosis vFLIP and others

COMPARATIVE ANALYSIS OF CELL-DERIVED MEMBRANE PROTEINS PRESENT ON CIRCULATING HIV-1 IN PATIENTS BEFORE INITIATION OF HAART+IL-2 AND AFTER CONTROLLED THERAPY SUSPENSION.

Capobianchi MR*., Turriziani O.***, D'Offizi G.* , Pandolfi F. §, Dianzani F.§§, Spanò A.**, Cappiello G.*, Longo R.**, Abbate I**.

*National Institute for Infectious Diseases "L.Spallanzani", *** Dep. of Experimental Medicine "La Sapienza" University ", §Institute of Internal Medicine, Catholic University, §§ Libera Università Campus Biomedico, ** Laboratory of Microbiology, "S.Pertini" Hospital, Rome, Italy.

OBJECTIVES: To determine the profile of cell-derived membrane proteins (CMP) on HIV-1 circulating in the plasma of asymptomatic patients and to analyze possible changes after a cycle of highly active anti retroviral therapy (HAART) plus IL-2.
METHODS: Plasma samples from eight drug-naïve asymptomatic subjects were tested to detect CMP by an immobilized antibody capture assay (IAC), followed by quantitative RT-PCR. Patients were sampled before the initiation of HAART plus IL-2, and after controlled therapy suspension, at the time of viral rebound. Lymphocyte subset markers (CD45RO and CD45RA), activation markers (HLA-DR), adhesion molecules (LFA-3), costimulatory molecules (B7-2), lymph node homing receptors (CD62L) and pro-apoptosis molecules (Fas-L) were considered in the study.
RESULTS: LFA-3 and CD45RO and HLA-DR are the most represented CMP on the surface of HIV-1 present in the plasma of drug-naïve asymptomatic patients, whereas CD45RA, CD62L, B7-2 and FasL are detected in a minority of cases, and to a low extent. After therapy suspension, at the time of viral rebound, a significant reduction in both HLA-DR and CD45RO embedded in virion envelope is observed, whereas LFA-3 content is virtually not affected. At the same time, CD45RA, CD62L, B7-2 and FasL remain not detectable on circulating HIV-1.
CONCLUSIONS: Based on the assumption that CMP present on HIV-1 envelope represent a footprint of the cell actually replicating the virus, activated memory T-cell appear to be the main source of plasma HIV-1 in asymptomatic patients. After therapy suspension, when naïve T cells population is significantly expanded, CD45RA is still virtually absent on circulating virions, indicating that these cells do not became a major source of virus replication. The decreased presence of HLA-DR on HIV-1 after therapy is in agreement with a reduced activation status of the virus-producing cells. These findings can help to identify viral reservoirs responsible for virus rebound after therapy interruption.

Involvement of intrahepatic lymphocytes in HCV and HIV immunopathology

C.Agrati, C.Selva, P.Narciso, G.Ippolito, V.Colizzi, L.Pucillo, G.D'Offizi, and F.Poccia,
National Institute for Infectious Diseases "Lazzaro Spallanzani", Rome, Italy

Background: HIV and HCV co-infection is frequently associated with rapid progression of HCV-related disease, resulting in higher risk of cirrhosis. Recent data indicate that natural T cells expressing the Vd1 TCR rearrangement are recruited in the liver of chronic HCV-infected patients (Agrati et al., Mol. Med. 7; 2001) and are increased in the peripheral blood of HIV-infected persons (Bouiller S. et al., Immunol., 1995, Martini et al., Immunol 2000). Thus, we analyzed the distribution and functional activation of gd T cell subsets in HIV/HCV co-infected persons during highly active antiretroviral therapy (HAART) and the influence of this T cell subset on liver disease progression.
Materials and Methods: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection and from 13 patients with HIV and HCV coinfection, were compared in terms of T cell subset distribution in the peripheral blood and in the liver. Moreover, we analyzed whether these immunological parameters were associated with liver inflammation measured by Ishak index and the influence of highly active antiretroviral therapy (HAART).
Results: A specific compartmentalization of Vd1+ T cells releasing Th1 cytokines was observed in the liver of HCV+ patients and HIV/HCV coinfected persons (p<0.001). Interestingly, HIV/HCV coinfected patients and HCV-infected persons show a different distribution of peripheral and intrahepatic Vd1 T lymphocytes, resulting in a higher degree of liver inflammation when compared to healthy donors or HIV- patients with other liver diseases. Finally, HAART was not able to restore the distribution of Vd1 T cells to normal levels.
Conclusions: In the liver of HCV-infected persons we observed a polyclonal localization of T cells expressing the Vd1 TCR rearrangement. The Vd1 T cells showed a memory/effector phenotype and the percentage of intrahepatic Vd1 T cells releasing IFN-g was higher in the liver of HCV-infected patients with necroinflammatory process. The increased percentage of Vd1 T cells in the liver of HCV-infected and HIV/HCV co-infected persons was associated with a higher degree of liver inflammation. Finally, HAART was unable to restore gd T cell circulation to normal levels.

Immunoevasion strategies and novel immunotherapeutic approaches in the control of opportunistic infections

Antonio Cassone
Department of Bacteriology, Istituto Superiore di Sanità, Rome, Italy

Fighting infections in an era of emerging diseases and rise of antibiotic-resistance substantially means the provision of novel tools for prevention and therapy. Among these tools, those based on specificity and potency of immune recognition are seen with renewed interest. A non-approximate knowledge of the mechanisms whereby infectious agents circumvent natural and/or adaptive immunity is however needed before attempting to generate vaccines or terapeutics. The human opportunistic agent Candida albicans is a special case in point. It is a dimorphic agent capable of growing as commensal prevalently under yeast form and as host tissue invader as mycelial (hyphal) form. Recent evidence from various source demonstrates the fungus largely exploitates this dimorphic transition to create an immunoevasion niche. For instance, hyphal cells were shown to be much less efficient than yeast cells in stimulating production by human monocytes of critical defensive host constituents such as IL-12 and chemokines (macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, interleukin-8 (IL-8), and particularly, monocyte chemotactic protein-1 (MCP-1)) (Torosantucci, Chiani and Cassone, J. Leukocyte Biol. 68, 923-932). This different stimulation did not depend on the monocyte inability to ingest the hyphae nor did it imply hyphal resistance to the extracellular killing by the monocytes. Purified hyphal and yeast cell walls reproduced the differences shown by the whole cells, and chemical-enzymatic dissection of cell wall components suggested cell wall beta-1,6, rather than beta-1,3 glucan was the main chemokine inducer. Coherently, immunofluorescence studies with an anti beta-1,6 glucan serum that the surface expression of this polysaccharide was much lower on hyphae than on yeast cells. In addition, hyphal cells induced much less production of IL-12 (even in the presence of IFN-g) as compared to yeast cells and LPS. For this cytokine, however, phagocytosis was an essential step in the production process by monocytes. After intraperitoneal inoculation of identical number of yeast and hyphal cells in mice, the latter induced in the peritoneal exudates an early chemokine response significantly lower than that generated by the former cells. This was coupled with the influx of a lower number of inflammatory cells in the peritoneal exudate. Overall, the data strongly suggest that the formation of hyphal filaments by C. albicans invading host tissues effectively minimizes cytokine induction, and, as such, it may facilitate fungal escape from host immunity. These data also suggest that b 1-6 glucan could be usefulls exploited as an immunomodulator capable of generating an appropriate cyto-chemokine milieu at the site of infection.

Supported by the National AIDS Program, Contract N° 50/CB

HOST- AND VIRAL- PREDICTIVE MARKERS OF RESPONSE TO INTERFERON ALPHA IN HEPATITIS C PATIENTS.

Guido Antonelli

Department of Experimental Medicine and Pathology, Virology Section, University "La Sapienza", Rome , Italy

Interferon (IFN) alpha treatment of patients with chronic active hepatitis C is only beneficial in a proportion of patients, and some way of identifying those likely to respond to therapy is urgently needed. We tried to address such issue by verifying whether the early measurement of some virus- or host-markers could be predictive of the degree of the IFN alpha-induced decrease of the viral load and, consequently, of the clinical outcome of the therapy. Specifically the following markers have been considered: Hepatitis C virus (HCV) genotype, HCV quasispecies, ALT levels, plasma and PBL-associated viral load, expression of IFN alpha-induced proteins, level of circulating IFN. The above markers have been analyzed very early during the therapy and specifically in a group of 30 chronic hepatitis C patients at 0, 24, 48, 72 hrs from the first IFN alpha injection (3x106IU) and in a restricted group of 10 patients at baseline and at 2, 4, 6, 8, 12, 16, 24, 48 hrs from the first injection of IFN alpha.
The study is still in progress but the results so far obtained revealed that: in 75% of the patients examined a significant reduction (more than 0.3 Log) of HCV viral load could be recorded after 24 hrs and/or 48 hrs (range 0.36-2.54 Log); the baseline viral load levels and ALT levels do not influence the level of early HCV RNA reduction measured in PBL and plasma. Importantly, the HCV reduction measured in PBL and in plasma at 48 hrs from the first injection of IFN alpha was correlated with the harbouring genotype and, in a preliminary analysis, with the outcome of 6 months therapy. Importantly, some patients (40%) after the administration of the single dose of IFN alpha exhibited changes in plasma HCV quasispecies composition that were clearly evident by SSCP analysis, thus indicating that IFN alpha can produce very rapidly profound perturbations in the genetic heterogeneity of circulating HCV.
Furthermore the results indicated that while serum IFN alpha concentration peaks in most of the patients (80%) at 6 hrs since the injection, the expression of mRNA of MxA, which is a protein specifically induced by IFN alpha, in PBL varies considerably on individual basis, the peak of expression being between 4 (30%) and 18 hrs (20%). Importantly, considering the expression of IFN alpha-MxAmRNA, the results also showed that MxA mRNA was expressed, though to varying degrees, in the PBMC of all 27 patients we have examined before they started treatment IFN alpha. The level of mRNA increased in 19 patients when IFN alpha was administered, but the increase was only significant (p<0.001) in patients classified as end of treatment responders.
All together the above findings suggest that the early clearance of HCV viral load and the clinical outcome of the IFN alpha therapy might be more complex than expected from the data in the literature, and may depend on still unknown host factors correlated or not with the harbouring strain of HCV.

Reintroduction of HAART after drug holidays.

G D'Offizi, L Vincenzi, C Selva, V Galati, F Poccia, C Gioia, C Agrati, F Martini, L Pucillo, P Narciso

National Institute for Infectious Diseases "Lazzaro Spallanzani" I.R.C.C.S. Rome

Therapeutic approach with Highly Active Antiretroviral Therapy (HAART) can lead to suppression of HIV-1 plasma viremia to undetectable levels. However, adherence to complex drug regimens with the occurrence of several side effects can be problematic, and patients may temporarily discontinue HAART.
The aim of our study was to evaluate the safety of antiretroviral therapy interruption, and the immunological changes following the reintroduction of HAART. In addition, we are going to evaluate whether boosting HIV-1 specific immune response should be considered a novel strategy for HIV-1 patients on HAART.
We enrolled 18 patient with HIV-1 chronic infection (12 female) that suspended HAART for severe lipodystrophy (12), hypertransaminase (1) pregnancy (1). All patients gave written informed consent approved by the Ethical Committee. All patients have been on HAART for > 1 year with undetectable HIV RNA copies for 6 months prior to study entry. We evaluated clinical immunologic and virologic parameters, at the suspension of HAART (t0), after 1 month from the suspension (t1), to the resumption of therapy (t2) and after 15 (t3) and 30 days (t4). 10 patients completed the study , 6 patients are completing it, while 2 patients have not still restarted HAART according to the international guidelines on HIV therapy. In 10 patients that have completed the cycle we observed: median t0 CD4+ 625 (SD±132), t1 CD4+ 401 (SD±117), t2 CD4+ 380 (SD±89), t3 CD4+ 411 (SD+263), t4 CD4+ 563 (SD±225). Plasma viremia median value (NASBA) was: t0 HIVRNA: 80 (SD±538), t1 HIVRNA 42,000 (SD± 61,172), t2 HIVRNA 42,000 (SD±439,457), t3 HIVRNA 1700 (SD±21,989), t4 HIVRNA 470 (SD±275). In the 2 patients still out of therapy, we have observed: median t0 CD4+ 878 (SD±323), t1 CD4+ 838 (SD±191). After 4 months from the suspension (t1c) CD4+ was 841 (SD±82). Median of HIVRNA was: T0 80 (SD±0), t1 210 (SD±33,981). After 4 months of suspension (t1c) HIVRNA was 300 (SD±12592). Only a modest reversal of the body shape abnormalities was referred by the patients after HAART withdrawal. In 4 patient a mild cervicoaxillary lymphadenopathy has been observed.
Discontinuation of HAART was not associated with clinical events or deleterious effects in this group of patients after 1 and 4 month of suspension. The interuption of HAART did not seem to modify the fat distribution in patients with lipodystrophy. Viral load did not rebound in 2 patients that showed a marked immuno recovery. The reintroduction of HAART shows viral kinetics and a pattern of immune reconstitution similar to that of naive patients. Further analysis on HIV specific T-helper cell response are under investigation.

Programma nazionale di ricerca sull'AIDS-1999
Accordo di collaborazione scientifica n. 30C/23

Strategies for the selective elimination of HIV reservoirs in the body

CF. Perno

INMI L. Spallanzani for Infectious Diseases, Rome, Italy

The presence of reservoirs where HIV replicates under limited control of drugs and/or immune system makes not feasible the eradication of the virus from the body. Central nervous system and testis represent tissue reservoirs (otherwise named sanctuaries) not efficiently permeated by antiviral drugs. There is substantial evidence that the virus circulating in the central nervous system is mainly of local origin, and is genetically different that that circulating in the blood. For this reason, therapeutic attempts aimed to achieve high and long-lasting efficacy against virus replication should take into consideration the presence of such tissue reservoirs, in order to select drugs able to penetrate physiological barriers and to inhibit locally replicating virus.
Cellular reservoirs also represent a great challenge for antiretroviral therapy. Cells latently infected by HIV (such as resting-CD4+ lymphocytes) do not replicate HIV, therefore no antiviral efficacy should be expected, even with highly potent regimens. At the same time, persistently-infected cells harbor actively-replicating HIV. These cells (such as macrophages) have biological characteristics different than those typical of activated CD4-lymphocytes, sustain long-lasting HIV replication, and are poorly susceptible to the cytopathic effect induced by HIV. All these cellular components play a role in the pathogenesis of HIV infection, therefore they should be taken into consideration in view of the current requirement of long-term (up to life-long) therapies.
On the basis of these pathogenetic concepts, therapeutic attempts against HIV in reservoirs should consider not only strategies aimed to decrease virus replication, but also those dedicated to the selective elimination of cells chronically-infected by the virus. Therefore, the activity of antiviral drugs in reservoirs, their penetration into sanctuaries, as well as new attempts to eliminate chronically-infected macrophages, will be presented and discussed.

Immune response to HIV-Nef peptides as model for the anti-AIDS therapeutic vaccine

Colizzi V., G. Cappelli, C. Montesano, A. Sacchi, M. Amicosante, S. Vendetti,
H. Chenal, M. Enouf, L. Montagnier

The recent introduction of the antiretroviral therapy, and the subsequent reduction of the viral load and the partial reconstitution of the immune response allow now to develop suitable models for therapeutic vaccines. Although there is a consensus view that such vaccines may contain several constituents of the HIV-1, each laboratory all over the world is focusing on particular HIV-1 gene or protein. We have focused our attention to HIV-1 Nef for the following reasons:
- Nef is strictly linked to pathogenesis, in particular this gene/protein enables the virus to escape by the specific immune response by down regulating CD4 and HLA molecules;
- the sequence variability of Nef is less than other viral proteins, and conserved aminoacid sequences have been identified on Nef;
- SIV-Nef vaccine has been shown to protect monkey from the infection.
However, preliminary results have shown that preincubation of recombinant Nef with antigen presenting cells inhibit presentation of microbial recall antigens (PPD and TT) to CD4 T cells. These findings suggest us to use synthetic peptides to boost the immune response against Nef. Considering that HIV-1 Nef contains conserved aminoacid sequences responsible for pathogenesis, it may be possible to designed a therapeutic vaccine able exert a selective pressure on high pathogenetic HIV-1 isolates. By bioinformatic approaches and algorithms more than 2000 HIV-Nef sequences have been screened and common T cell epitopes able to bind the more representative HLA class I and II designed. Particular attention has been focused to the HIV-1 sequenced isolated in Africa, and the finding that only 0.5% of the sequences present in the data bank (HIV WEB Lanl.Gov) originate from african isolates, strongly suggest that more information on HIV-1 Nef from african patients are need before develop vaccination in Africa.
Preliminary results have shown that antigen presenting cells (macrophages and dendritic cells) from AIDS patients carry HIV and are able to transfer the virus T cells in the course of antigen presentation. Experiments are in progress to ascertain i) whether dendritic cells precursors are infected during HAART therapy, ii) whether in vitro differentiated dendritic cells transfer HIV during presentation of Nef peptides, iii) whether is possible to kill infected dendritic cells by microenviromentally activated natural T killer cells. The interest on dendritic cells is based on the consideration that these cells are widely used for the vaccination against tumour in experimental animals and human (phase II and III). In particular IDM (Immuno-Designed Molecules, inc, Paris) have developed a suitable system of cellular vaccine based on the use of "dendritophage" carrying synthetic peptides. A collaborative project for preclinical cellular vaccination with HIV-Nef peptides is in preparation with IDM.
The CIRBA (Centre Integree de Recherché Bioclinique d'Abidjan) follow one of the major cohort of AIDS patients under HAART therapy in Ivory Coast. This African country has more than 10% infected population with a great HIV-1 genetic variability. A collaborative project for extensive HIV-I Nef sequencing with RETRO-CI (CDC, Atlanta) is in preparation in Abidjan, Ivory Coast, while the HLA class I and II will be typed by the Institute of Pathology of the University of Palermo. This information will allow to confirm the aminoacid sequences of the synthetic peptides that could be used for vaccination in Africa.

A study of genetic and environmental risk factors for tuberculosis in West Africa

Gianfranco Del Prete,3 and the TB-GENENV Group

Tuberculosis (TB) is a complex multifactorial disorder, in which several genes interact with the environment to contribute to the overall phenotype. The joint investigation of genetic, immunological and environmental factors at play in susceptibility to TB represents an innovative and challenging goal for better understanding of the pathogenesis of TB, with particular reference to West Africa. The study, funded in the frame of INCO-DC Projects of the European Union, is being carried out in three countries: The Gambia, Guinée Conakry and Guinea Bissau, using the same methodology and standardised questionnaires. The design combines two case-control studies, a prospective household study and family genetic studies. Laboratory investigations are conducted with the assistance of the european partners. The duration of the study allows 12 to 18 months for recruitment of index cases and controls and 2 years for follow-up of their household contacts. In each country, the study has been reviewed and approved by the national ethical committee. Newly diagnosed, smear positive, TB cases are recruited at a TB clinic. Their household is visited and demographic information collected from all members. Two types of controls are selected at random for each case: one within the household of the TB case (internal control), and one living in the neighbourhood (healthy community control). Then members of the household of the TB case (i.e. exposed to TB) and of the community control household (i.e. not exposed to TB) are followed up over 2 years to detect newly occurring ("secondary") TB cases. The host related factors under investigation are: BCG vaccination, previous history of TB, infection with M. tuberculosis, smoking, alcohol intake, drug use, nutritional status, intercurrent infectious diseases (including HIV infection) and conditions triggering Th2 response (such as parasitic diseases or atopy). Environmental factors include the number of people living in the household, number of people per room (crowding), type of house, hygiene, water supply, sanitation, presence of animals and socio-economic status. The objectives of the genetic study are to determine the risks associated with allelic variants of candidate genes, to map regions of the genome already thought to be linked to disease susceptibility ("candidate regions") and to contribute cases to a genome-wide search for susceptibility genes. Candidate genes include various cytokines and other mediators known to be of importance in the pathogenesis of tuberculosis, such as NRAMP 1, Vit D3 receptor, CD40 ligand, IFN-g, interleukin-1b (IL-1b), IL-12, TNFa, IL-4, and IL-10, their receptors and the transcription factors that regulate their expression.
Since IL-4 and IFN-g are short-range cytokines that are rapidly bound by their receptors on cells or inactivated by serum proteases, the assessment of their levels in the serum is of poor or no value. Rather, IgE, soluble CD30 and monocyte-derived chemokine (MDC) induced by Th2 cytokines or soluble LAG-3 released by IFN-g-producing Th1 cells, represent reliable markers for the in vivo assessment of the actual Th1/Th2 balance in a large cohort of TB patients and their controls. The use of the triplets (index TB case, H contact and X community control) is an useful model for the natural history of TB, from exposure to disease. In addition, the follow-up of index TB cases during treatment allows to investigate the impact of treatment on Th1/Th2 balance and possibly to associate certain changes with disease persistence or healing. In a preliminary study, all HIV infected individuals were not considered. Th1/Th2 parameters were assessed in a cohort of 538 newly diagnosed smear positive index (I) TB cases, 498 household (H) and 549 external (X) controls. At diagnosis, TB patients showed significantly (p < 0.0001) lower levels of sLAG-3 (Th1 activity) and higher levels of Th2 parameters than their corresponding H or X controls. Interestingly, H controls (exposed to M. tuberculosis but still healthy) showed higher levels of sLAG-3 and lower levels of IgE, sCD30 and MDC not only in comparison with TB patients, but also with X controls. These data indicate that TB disease is associated with poor Th1 and high Th2 activity of T-cell responses, whereas exposure to M. tuberculosis is associated with an opposite Th1/Th2 balance, i.e. high Th1 and reduced Th2 activity.
Measurement of Th1 and Th2 parameters in TB patients during the follow up of their treatment (2-3 months and 6-8 months) showed that healing and/or at least completion of treatment were both associated with a significant (p < 0.0001) change in the Th1/Th2 balance represented by remarkable increase of Th1 and concomitant decrease of Th2 activity. In contrast, treatment failure or interruption of therapy resulted in poor Th1 polarization of T-cell responsiveness, with maintenance of high Th2 activity.

The TB-GENENV Group members include Christian Lienhardt,1 Jackson Syllah, 1 Keith McAdam,1 Adrian Hill,2 Mario M. D'Elios,3 Annalisa Azzurri,3 Amedeo Amedei,3 Marisa Benagiano,3 Roberto Manetti,3 Oumou Bah Sow,4 Boubacarr Bah,4 Kebba Maneh,5 Peter Aaby,6 Victor Gomez,6 Katherine Fielding7 and S. Bennet7

1MRC Laboratories, The Gambia; 2Wellcome Trust Centre for Human Genetics, Oxford; 3Department of Internal Medicine University of Florence, Florence; 4Programme National de Lutte Anti-Tuberculeuse, République de Guinée; 5National Leprosy/TB Control Programme, Ministry of health, The Gambia; 6Projecto de Saude de Bandim, Statens Serum Institut, Copenhagen; 7 London School of Hygiene and Tropical Medicine.

Research and training on malaria in Burkina Faso

Fulvio Esposito

Dipartimento di Biologia Molecolare, Cellulare e Animale, Università di Camerino, 62032 Camerino (MC) - Italy

In Burkina Faso, an example of a country severely hit by malaria, out of ~10 million inhabitants, at least 50 children in the age group 0.5 to 5 years die every day for malaria. Even darker perspectives are proposed by the fast spread of Plasmodium resistance to the cheapest drugs, and of Anopheles resistance to the most convenient insecticides. The shortcomings of conventional control methods point to vaccines as the ultimate breakthrough in the fight against malaria. Unfortunately, the way towards this achievement is far from being straight.
Under the auspices and with the financial support of the Italian Direzione Generale per la Cooperazione allo Sviluppo (then Dipartimento) a programme was started in the '80s to establish a malaria research and training facility in Ouagadougou, the capital city of the country. Over these almost 30 years, several research programmes based at the Centre National de Lutte contre le Paludisme (now Centre National de Recherche et Formation sur le Paludisme) have contributed to the advancement of knowledge in the fields of both, basic and applied malaria research (Esposito F et al, 1988, Trans R Soc Trop Med Hyg, 82: 827-832).
A large commitment of resources was devoted in recent years to a project aimed to assess the impact of insecticide treated curtains on child mortality. The project involved over 150 villages in a rural area of Burkina Faso with a surface of about 1,000 km2 and a population of ca. 100,000. The intervention resulted in a dramatic impact on malaria transmission, reduced to less than 1/10 of the initial level (Habluetzel A et al, 1999, Trop Med Intern Health, 4: 557-564). The starting level was however so high, that interruption of transmission could not be achieved, and a modest impact was recorded on the proportion of children infected by Plasmodium falciparum (Cuzin-Ouattara N et al., 1999, Trans R Soc Trop Med Hyg, 93: 473-479).
Due to the difficulties in measuring malaria-specific mortality, the overall mortality was measured in children aged 6 months to 5 years. Over two years, a reduction of mortality of about 15% was observed by a randomised controlled study design. This reduction was the product of a dramatic impact in the 1st year (-27%), followed by a marginal impact in the 2nd year. We cannot conclude whether this reflects real phenomena, as a loss of immunity followed by a shift of mortality towards a higher age, or simply chance oscillation of mortality, or interference with causes of death different from malaria, like e.g. microepidemic foci of meningitis (Habluetzel A et al., 1997, Trop Med Intern Health, 2: 855-862; Ilboudo-Sanogo E, accepted for publication in Trans R Soc Trop Med Hyg).

The author research is supported by grants from Ministero degli Affari Esteri (Dir. Gen. Cooperazione allo Sviluppo), Ministero dell'Università e della Ricerca Scientifica e Tecnologica (cofin. 1999), Consiglio Nazionale delle Ricerche, the European Commission,

List of Chairmen & Invited speakers:

· G. Antonelli (ICAERI & University of Rome "La Sapienza")
· F. Bistoni (University of Perugia)
· M.Capobianchi (INMI "L. Spallanzani", Rome)
· T. Carrettoni (UNESCO National Commission, Rome)
· A. Cassone (Istituto Superiore di Sanità, Rome)
· H. Chenal (CIRBA, Abidjan)
· V. Colizzi (ICAERI & University of Rome "Tor Vergata")
· G. De Libero (University of Basel)
· G. Del Prete (University of Florence)
· G. D'Offizi (INMI "L. Spallanzani, Rome)
· V. Erfle (GSF, Neuherberg, Germany)
· F. Esposito (University of Camerino)
· M. Fiorilli (University of Rome "La Sapienza)
· E. Garaci (University of Rome "Tor Vergata")
· G. Ippolito (INMI "L. Spallanzani", Rome)
· T. Lehner (University of London)
· G.Magliano (MAE)
· L. Montagnier (World Foundation AIDS Research and Prevention)
· S. Natoli (INMI L.Spallanzani)
· G. Palmieri (University of Rome "La Sapienza")
· F. Perno (University of Rome "Tor Vergata" & INMI L.Spallanzani)
· F. Poccia (INMI"L. Spallanzani" & ICAERI)
· L. Romani (University of Perugia)
· G. Rotilio (CNR, Rome)
· Salerno (University of Palermo)
· Saltini (INMI "L. Spallanzani" & University of Rome "Tor Vergata")
· A.Santoni (University of Rome "La Sapienza")
· G. Tocchini Valentini (CNR, Rome)
· P.Vagliani (World Foundation AIDS Research and Prevention)
· S.Vella (Istituto Superiore di Sanità, Rome)
· S. Zappacosta (University of Naples "Federico II")