Over the past 20 years most of the efforts in HIV vaccine development have focused on sterilizing immunity by targeting the Envelope protein (Env). However, results from preclinical and clinical trials have been largely disappointing [1–11]. Therefore, current vaccine strategies are not only aimed at preventing virus infection but also at blocking virus replication and disease onset. In particular, the control of virus replication should provide protection from disease development and reduce virus transmission, halting the HIV epidemic. This objective may be achieved by targeting virus regulatory genes, which are expressed early after infection, are essential for virus replication and pathogenesis, and are more conserved among HIV clades. This approach may be effective for both preventive and therapeutic vaccine strategies [12–68]. In this article we review the characteristics of Tat and why it was selected for use in a vaccine. We also cite the lesson learned in the development of this anti-Tat vaccine for use in human clinical trials.
Why HIV-1 Tat?
Tat represents an optimal candidate for a vaccine controlling virus replication and blocking disease progression (Table 1).
Role of Tat in the virus life cycle
Tat is a key viral regulatory protein produced very early after infection, even before virus integration, and is necessary for viral gene expression, cell-to-cell virus transmission and disease progression [69–85]. Furthermore, Tat is released by acutely infected cells [70,86–89] promoting HIV-1 replication [70,90,91], as well as the recruitment and activation of uninfected cells, providing new targets for HIV spread [61,70,87,90,92–95].
Cross-sectional and longitudinal studies of Tat immune response in natural infection
The presence of anti-Tat antibodies appears to play a protective role from disease progression [96–101]. In particular, a higher prevalence of anti-Tat antibodies has been detected in asymptomatic HIV-infected individuals compared with progressed patients [98,100,102,103].
In addition, a cross-sectional assessment in 302 HIV-1-infected patients showed that anti-Tat antibodies are more frequent at an early stage (A) compared with symptomatic stages (B or C) (Table 2), whereas no differences are observed for antibodies directed against structural proteins. Furthermore, a study performed in a cohort of 252 individuals with known dates of seroconversion and a medium follow-up of 7.2 years  indicated a strong association of anti-Tat antibodies with slower disease progression. Moreover, none of the individuals who were persistently anti-Tat positive progressed to AIDS, whereas AIDS occurred in anti-Tat-negative individuals .
Anti-Tat cytotoxic T lymphocytes are frequently found in natural infection [24,106–109]. In particular, CD8 T-cell responses to Tat are more frequent in patients controlling viraemia [106,110], and correlate with early virus control both in humans [111,112] and monkeys [113,114].
Tat sequence conservation among HIV clades
The immunogenic regions of Tat are conserved among the HIV-1 M group [115–118]. Cross-clade recognition of Tat B clade (BH-10) is observed with sera from Ugandan, South African and Italian patients who are infected with different subtypes . In addition, the predicted Tat amino acidic sequence (1–86) is well conserved in its first 58 amino acids among the circulating virus clades and in the BH-10 Tat sequence, which derives from the first isolate of two decades ago, providing evidence that a Tat vaccine may be used in different geographical areas of the world .
Immunoregulatory properties of biologically active Tat protein
Active Tat protein possesses immunomodulant and adjuvant properties that are highly advantageous in vaccine development. Native, but not oxidized, Tat protein is selectively and very efficiently taken up by monocyte-derived dendritic cells (MDDC) promoting cell maturation and T helper type 1 polarization, leading to a more efficient presentation of both allogeneic and exogenous soluble antigens . Furthermore, Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells, leading to a more efficient presentation of subdominant MHC-I-binding cytotoxic T-lymphocyte epitopes of heterologous antigens both in vitro and in vivo [120,121, R. Gavioli, paper in preparation].
Absence of seroconversion in vaccinees
Being devoid of structural HIV proteins, the Tat vaccine does not induce seroconversion, facilitating trial recruitment as well as the monitoring of vaccinees.
Taken together, these data suggest that vaccination with Tat may modify the virus–host dynamics and control HIV-1 replication both in primary infection (preventive strategy) and in infected individuals (therapeutic strategy). Therefore, the active Tat protein was chosen as a vaccine candidate against HIV/AIDS for the development of both preventive and therapeutic strategies.
Studies performed both at the level of basic and clinical research are essential to address antigen selection and to design innovative strategies for vaccine development. Dissecting the role of Tat in HIV pathogenesis, exploring its biological properties, and investigating the anti-Tat immune response in natural infection gave a twofold gain by both directing our attention to this regulatory protein and providing the necessary know-how for its development as a vaccine candidate.
Creating the structure for HIV Tat vaccine development
The development of the Tat vaccine candidate required a complex multidisciplinary approach, accomplished by multiple milestones and regulated by national and international authorities (Fig. 1). These activities included the production of the vaccine candidate, an evaluation of its safety, immunogenicity and efficacy in preclinical models, dossier preparation, and approval for human use and clinical trials. Parallel activities consisted of: (a) studies aimed at defining the role of Tat and the Tat immune response in natural infection to identify correlates of protection and to validate tests to monitor vaccinees, and (b) capacity building to conduct advanced clinical trials in developing countries (Fig. 1). The activities undertaken for Tat vaccine development from basic research to clinical testing required the build up of ‘ad hoc’ structures and expertise within the Italian public sector, which represented the focus of a 10-year-long effort (Fig. 2).
Tat vaccine production and characterization
The active substance of the Tat vaccine is the biologically active recombinant Tat protein (HTLV-IIIB strain, clone BH-10), produced in Escherichia coli and purified by heparin sepharose chromatography followed by high-pressure liquid chromatography [70,86,122]. This product was used for in-vitro and preclinical studies. A set of tests, which include the determination of physicochemical, immunochemical and biological properties, was selected to confirm the quality and stability of the protein (Table 3 and Fig. 3). Performing these assays is particularly relevant because Tat contains seven cysteines and is very sensitive to oxidation [70,86], which induces conformational changes, hampering its biological activity as well as recognition by conformational antibodies. For these reasons, the activity of the product was evaluated by two assays: the rescue of a Tat-defective provirus (rescue assay) and the uptake by MDDC [70,86,119]. As a result of the higher level of reproducibility and sensitivity, the uptake by MDDC has then been selected for the release of the Tat protein batches. The reliability of this test has been confirmed by comparing the results obtained by testing several lots of Tat with MDDC from a large number of normal blood donors (Fig. 4).
Safety and immunogenicity studies were conducted in mice and monkeys with both the biologically active Tat protein or tat DNA. The results indicated that both approaches are safe because no local nor systemic toxicity was detected [17,88,123–127].
Efficacy studies in cynomolgus monkeys demonstrated that vaccination with active Tat protein can elicit a specific and broad immune response, and can control viral replication blocking disease progression after challenge with the highly pathogenic cynos-grown SHIV89.6P cy243 (Table 4) [123,124]. Of note was the fact that no residual virus hidden in resting cells was detected in the protected monkeys either in blood or lymph nodes, upon two boosts with tetanus toxoid, a stimulus known to induce virus replication . Long-term protection (up to 2 years) correlated with the presence of high and stable humoral and cellular (CD4 and CD8 T-cell-mediated) responses against Tat. Vaccination with the native Tat protein thus contained viral replication in peripheral blood and tissues, preventing the development of AIDS.
Immunization with native Tat was also safe in monkeys with AIDS and no increase in viral replication nor a further decrease in CD4 T-cells was observed .
On the basis of these data, the active Tat protein was chosen for the conduct of preventive and therapeutic phase I clinical trials (Fig. 2).
To guarantee translation to the clinical level, all preclinical activities must be conducted in compliance with regulations and procedures ensuring safety and data quality. For example, a process of production compliant with regulatory guidelines for human use should be adopted early in the developmental pipeline. Specific training programmes should be implemented to support scientists in this task.
Regulatory approval by the national agency within the European Union
In order to proceed to phase I clinical trials of a new vaccine in Italy, an application must be submitted to the Committee for the Evaluation of the Safety and Quality of New Drugs at Istituto Superiore di Sanità (ISS) and to the Italian Ministry of Health (Fig. 5). The process is regulated by guidelines and laws issued by European and Italian regulatory authorities (Table 5). Therefore, a dossier termed ‘Expert Report’ containing the required information on the quality, safety, immunogenicity and efficacy of the Tat vaccine and the clinical protocols was submitted to this Committee, which approved the use of the Tat vaccine candidate in both healthy and HIV-infected individuals (Fig. 2). After that, all the relevant documentation (clinical protocols, psychosocial protocol, investigator brochure, informed consent, clinical sites, insurance policy) (Table 6) was submitted and approved by the central (ISS) and local Ethics Committees/Institutional Review Boards (Fig. 5). Competitive enrollment was then started in each clinical site for the conduct of both the preventive and therapeutic phase I trials (Fig. 2).
Approaching regulatory issues represents a fundamental step in building up translational research programmes, and requires a specific expertise while being extremely time-consuming and frustrating also because no academic training in this matter exists. Therefore, training should be implemented to support scientists in this task. The implementation of training will help in properly planning timelines and organizing human and economic resources.
Good manufacturing practice Tat vaccine production for phase I studies
Good manufacturing practice-grade process development of the Tat protein
For the good manufacturing practice (GMP) production of the Tat vaccine it was necessary to identify a validated facility adequate to sustain phase I trials, which, however, was not available in Italy. A contractor was finally identified in the United Kingdom, which produced and released the Tat vaccine according to current regulations.
The recombinant Tat protein was produced and purified by diethylaminoethyl and heparin sepharose chromatography, formulated in a suitable buffer in the presence of human serum albumin and vialed (Fig. 6). Comparability studies with the research-grade product confirmed that release specifications remained unchanged (Table 3). Amino acid terminal sequence and mass spectrometry were also performed on the GMP product. Stability tests confirmed that the vialed clinical lot retained full biological activity for up to 2 years at −80°C.
The need to find a contractor outside Italy was extremely costly and time consuming, underlining the necessity of a dedicated small-scale GMP facility in Italy. Thanks to the support of ISS and of the University of Urbino such a facility (AVITECH) has been built in Italy for Tat vaccine production following clinical trials.
Establishment of clinical, laboratory and social–behavioural platforms
In order to ensure comparable read-outs for clinical trials conducted in a multicentre context, all clinical and laboratory activities, as well as psychosocial and behavioural assessments, were harmonized among the participants along common good clinical practice procedures by establishing specific and integrated platforms (Fig. 2 and Fig. 7).
Parallel preventive and therapeutic phase I clinical trials were conducted in three sites in Rome (L. Spallanzani Hospital, San Gallicano Hospital and University of Rome ‘La Sapienza’), and in one site in Milan (S. Raffaele Hospital; Fig. 8). Clinical activities and responsibilities, financial support from the sponsor, property of data and biological samples and confidentiality were regulated by specific contracts between the sponsor and the clinical sites. Standard operating procedures were implemented in the clinical sites to standardize all activities encompassing prescreening, enrollment and monitoring of the volunteers (clinical evaluation, safety laboratory testing, risk assessment, and counseling on risk reduction and on avoiding pregnancy). Clinical sites were also responsible for adverse event reporting. In this regard, an independent Committee for the Evaluation of Adverse Events, composed of external clinical experts, was appointed by the sponsor. This committee held periodic meetings during the study, and submitted interim and final safety reports to the regulatory authorities.
A dedicated Core Laboratory for Immunology and Virology was created at the San Gallicano Hospital in Rome as a joint unit with ISS (Fig. 8), and validated upon an international standard of quality (ISO 9001). Immunomonitoring was performed by a two-step strategy with a first line of testing, assessing the strength and breadth of Tat-specific B- and T-cell responses (antibody detection and mapping by enzyme-linked immunosorbent assay, Tat-specific peripheral blood mononuclear cell proliferation and γ-IFN and IL-4 production), and a second line of testing focusing at multiparametric antigen-specific profiles (proliferation coupled with an assessment of T helper types 1/2 cytokine production, multiplexed enzyme-linked immunosorbent assay for cytokines and chemokines and protein microarray), directed at validating novel methodologies for future clinical testing.
Psychological and behavioural platform
Participation in HIV vaccine clinical trials involves intimate matters, repeated HIV testing and exposure to scientific and medical concepts that may cause anxiety, stress and depression, and may also contribute to dropouts. A specific platform integrating experts from the clinical sites was therefore created (Fig. 8), and a psychosocial protocol was implemented for the assessment of psychological and sociobehavioural parameters to support volunteers throughout critical points during the study (enrollment, conclusion of the study, follow-up, screening failure or adverse events).
Communication and enrollment
Information from the sponsor/investigators must provide a good understanding of the nature of the trial to enable potential volunteers to weigh accurately the risks and the benefits of trial participation. To this goal a specific enrollment procedure was developed. In particular, ISS announced the starting of the enrollment with a press release, which referred to the AIDS Helpline at ISS for both general information on AIDS, vaccine clinical trials and specific information on Tat vaccine trial participation (Fig. 9). The AIDS Helpline operators gave to individuals willing to participate in the trial a dedicated telephone number for each clinical site, which was chosen by the volunteers, and an alpha-numeric code needed for the first visit appointment (Fig. 9).
Contract research organization
To guarantee the quality control and quality assurance of the clinical trials, a contract research organization was hired to provide the following services: study preparation (preparation of case report forms, submission to ethical committee, investigator qualification visits, generation and distribution of randomization codes), study initiation (study-specific monitoring visits, site initiation visits), study monitoring (routine monitoring visits, drug accountability and drug returns for destruction, resolution of queries with sites, termination visits), quality assurance (clinical site audit, database audit), data management (database design and testing, data transfer, data entry, validation and query resolution, quality control of database), analysis and reporting (statistical analysis plan design, statistical programming, statistical analysis, International Conference on Harmonization good clinical practice compliant preparation of clinical and statistical reports (Fig. 2).
Community advisory board
A community advisory board (CAB) comprising the most representative Italian non-governmental organizations involved in all issues relating to HIV/AIDS was established to provide a communication network among communities, scientists, community care providers and the sponsor (Fig. 8). The CAB contributed to establishing the methodology for ethical information, and provided activity of counseling and communication to the volunteers. The CAB also cooperated with ISS in approaching critical situations such as confidentiality issues with trial participants.
All the activities performed by the different platforms, contract research organization and CAB were implemented and coordinated by the sponsor via numerous ad hoc meetings conducted before and during the trials.
For the conduct of preventive and therapeutic phase I studies, a network was created as a highly motivated team. Networking greatly helped the process of the harmonization of procedures and allowed an important ‘exchange’ of expertise among the different platforms, to the full benefit of the volunteers. In particular, the psychological platform and the CAB represented a major support to the volunteers' wellbeing.
Parallel preventive and therapeutic phase I trial conduct
Clinical trials were conducted in healthy HIV-uninfected adults at low risk of infection (preventive protocol) and in HIV-1-infected adult asymptomatic volunteers not in therapy (i.e. CD4 T-cell counts ≥ 400 cells/μl and viral loads ≤ 50 000 copies/ml; therapeutic protocol). The endpoints were to qualify the biologically active Tat protein as safe (primary endpoint) and immunogenic (secondary endpoint) in both healthy and HIV-infected individuals for its further evaluation in phase II trials (Fig. 8).
Both studies were randomized, placebo-controlled, and double-blinded. Volunteers were randomly assigned to one of two treatment arms with different routes of administration and blinded to the dosage group. In arm A, volunteers received Tat subcutaneously with alum at a dose of 7.5, 15 or 30 μg, at weeks 0, 4, 8, 12, and 16. One group of volunteers received alum plus saline solution as placebo. In arm B, volunteers received Tat intradermally without adjuvant at a dose of 7.5, 15 or 30 μg at weeks 0, 4, 8, 12, and 16; one group of volunteers received saline solution as placebo.
The study structure is described in Table 7 and all clinical, laboratory and psychological and sociobehavioural evaluations performed during the trial are shown in Table 8 and Table 9. Evaluations were conducted during the treatment phase, the 6-month follow-up and are continuing for an additional 3 years. An assessment of clinical and laboratory safety was performed at several timepoints during the study and was monitored by the Committee for the Evaluation of Adverse Events.
The studies have been successfully completed. Both primary and secondary endpoints were fully achieved for both the preventive and the therapeutic trials (manuscripts in preparation), sustaining the advancement of the Tat vaccine candidate to phase IIA trials both in Italy and South Africa. On the basis of the results obtained in phase IIA, an extended ‘proof-of-concept’ phase IIB trial will be conducted in South Africa (preventive protocol) and in Italy (therapeutic protocol) for a preliminary evaluation of efficacy (Fig. 10).
The volunteers have established close relationships between themselves during the trial, providing an additional level of care and support. Their participation was so enthusiastic that it was proposed to the sponsor that a working group should be created to share their experience with the volunteers of the following clinical trials.
Of note is the fact that this is the first time that the same vaccine product has been tested in parallel in preventive and therapeutic trials, allowing a comparison of the safety and immunogenicity in two different populations. In particular, trials in infected subjects may give key information on the impact of vaccination on HIV infection and pathogenesis and a fast readout on vaccine efficacy, providing insights for the development of a non-sterilizing vaccine.
Preparatory studies in Africa for the conduct of advanced clinical trials
Strengthening and building up the local clinical and laboratory capacity as well as community involvement are crucial steps that must be undertaken before starting clinical testing in African countries. Preparatory studies are also essential to estimate HIV incidence and prevalence in the populations targeted by vaccination, and to evaluate the immune cross-recognition of the vaccine antigen. To this goal, preparatory studies are ongoing in Africa (Fig. 2). In particular, cooperation with South Africa has been established with the HIV/AIDS Vaccine Division at the Perinatal HIV Research Unit at the Chris Hani Baragwanath Hospital in Soweto (Johannesburg, South Africa) within bilateral as well as European Union-funded vaccine programmes. A similar platform is being established in Swaziland.
Feasibility studies for the advanced clinical testing of a vaccine in developing countries have to be started well in advance, because a number of issues must be resolved before starting clinical trials. Priority issues are: (i) evaluating the willingness of both local political and scientific authorities, as well as key stakeholders of the community to host vaccine trials; (ii) building up laboratory and clinical capacity and identifying suitable cohorts for vaccine testing; and (iii) performing background immunological and virological field studies.
Sponsorship of Tat vaccine clinical development
The ISS is a governmental agency with functions of the Centers for Disease Control and Prevention, National Food and Drug Administration and National Institutes of Health. As such, the ISS is strongly involved in basic and applied research in areas that represent a threat to national health, including HIV/AIDS. On the basis of the promising results from preclinical studies with the Tat vaccine, the ISS has sponsored, through the allocation of specific funds, preventive and therapeutic phase I clinical trials of the Tat vaccine. These trials represent the first public, fully government-supported phase I trials of a vaccine against HIV/AIDS in Italy. On the basis of the data obtained, the Italian government has committed to fund phase IIA and IIB preventive and therapeutic trials in Italy and South Africa.
Full sponsorship by a government agency such as the ISS represents a guarantee of the no-profit nature of the programme, while providing protection of the intellectual properties of the Tat vaccine.
Institutional sponsorship of the Italian vaccine programme was very favourably perceived by all players, including volunteers, non-governmental organizations and developing countries. At the same time, both the protection of intellectual properties as well as the advancement to phase IIB trials with public resources greatly reduce the financial risk of the private enterprises willing to develop the vaccine further.
National and international HIV/AIDS vaccine networks
The National AIDS Centre at ISS has established networks with national and international public and private institutions focused on the development of new preventive and therapeutic vaccine strategies to curb the HIV-1 pandemic. Among them is the AIDS Vaccine Integrated Project (AVIP; http://avip-eu.org), which is a European Union-funded 5-year project involving 16 institutions from the public and private sectors from Italy, Sweden, France, Germany, Finland, United Kingdom and South Africa. The design of HIV vaccines within AVIP (Table 10) is based on two general ideas. One is a ‘minimalistic’ approach combining regulatory HIV proteins (Tat or Nef) with a modified (V2 deleted) Env (ΔV2 Env). The other approach aims at ‘imitating’ a live attenuated vaccine using as many HIV genes as necessary (‘maximalistic’ approach). The specific objective and activities of AVIP are described in Table 11 .
The Italian Concerted Action on HIV/AIDS Vaccine Development (ICAV) has been established under the National AIDS Programme coordinated by the National AIDS Centre and consists of a network of approximately 70 Italian centres. The activities of the ICAV programme are described in Table 12.
Through these and the other networks in which ISS participates, several vaccines and formulations by different participants are in the pipeline. These novel vaccine candidates also include second-generation Tat-based vaccines such as the Tat/ΔV2Env combination [AVIP, Mucosal vaccines for poverty-related diseases (MUVAPRED), very innovative AIDS vaccine (VIAV) and ISS/Novartis–Chiron agreement] and Tat alone or in combination with other HIV products delivered by micro/nanoparticles (ICAV) as well as herpesvirus vectors (ICAV) and replication-competent adenovirus vectors (Italy–USA, ISS/National Cancer Institute–National Institutes of Health) for parenteral and mucosal vaccination strategies.
The establishment of national and international networks, including private companies, public and academic institutions, is essential for vaccine development and should always include training programmes such as the AVIP International School (www.avip-eu.com), which is proving to be an optimal forum to train students, scientists and clinicians in the difficult aspects of HIV/AIDS vaccine development. Although creating these networks has been a very challenging task, particularly for management, the intellectual, scientific, and human interactions among the participants have generated true cooperative teams adding a synergistic value to research conduct.
In conclusion, the development of the Tat vaccine programme required a multidisciplinary approach, adequate economic resources, training and a great effort of managing and coordination. The programme has been fully funded and conducted by the ISS, which is the Italian health governmental agency. A great effort was, therefore, dedicated to build up a structure capable of translational research. The accomplishment of this task took 10 years and taught us important lessons (Table 13), at the same time resulting in key achievements. This structure is now ready to run the following clinical phases of the Tat vaccine, as well as new vaccine programmes. In addition, such organization offers the flexibility to update all the different areas of the programme rapidly in response to scientific needs and innovation, with no interference from private/profit interests or ‘fashioned’ scientific agendas, which have undermined targeting regulatory genes as well as conducting therapeutic vaccine trials that may offer new opportunities in HIV treatment. In particular, the parallel conduct of preventive and therapeutic trials with the Tat vaccine candidate has provided important insights into HIV pathogenesis and for the development of a preventive vaccine based on virus control and not on sterilizing immunity. Finally, the creation of networks for vaccine development is greatly helping in this task and provides a suitable forum for training programmes, which are greatly needed in the field.
The authors wish to thank all the personnel at the National AIDS Centre, ISS, and particularly: S. Moretti, M.R. Pavone-Cossutt, F. Nappi, A. Borsetti, M.T. Maggiorella, L. Sernicola, R. Belli, I. Macchia, P. Leone, O. Longo, F. Ferrantelli, S. Bellino, C. Sgadari, D. Bernasconi, E. Fanales-Belasio, L. Tavoschi, and all the personnel from the animal facilities and technical services; the AHL (Division of Epidemiology, MIPI, ISS): A. Luzi, P. Gallo, B. De Mei, A. Colucci, A. Santoro, A. D'Agostini, R. Valli, G. Rezza; Division of Biologic Products, MIPI, ISS: C. Pini; the Joint ISS/S. Gallicano Laboratory site: A. Tripiciano, A. Scoglio, B. Collacchi, M. Ruiz-Alvarez, V. Francavilla, G. Paniccia, A. Fazio, P. Cordiali-Fei, G. Prignano, A. Arancio, F. Stivali; the Institute of Biochemistry, University of Urbino: M.E. Laguardia; the clinical sites: A. Lazzarin, G. Tambussi, R. Visintini (S. Raffaele Hospital, Milan); P. Narciso, A. Antinori, G. D'Offizi, M. Giulianelli (L. Spallanzani Hospital, Rome); A. Di Carlo, G. Palamara, M. Giuliani (S. Gallicano Hospital); M. Carta (University of Rome ‘La Sapienza’); the Adverse Events Monitoring Committee: P. Popoli (Istituto Superiore di Sanità); M. Galli (L. Sacco Hospital, Milan); M. Picardo (San Gallicano Hospital, Rome); and C.F. Perno (University of Rome ‘Tor Vergata’); the Community Advisory Board: R. Iardino (NPS); S. Marcotullio (I-CAB); A. Vatrella (LILA); C. Valvo (GITA); G. Bevacqua (Positifs Onlus); S. Lombardo (M. Mieli); R. Gavioli and P. Marconi (University of Ferrara); S. Barnett (Novartis, Emeryville, USA); M. Robert-Guroff (NCI–NIH, Bethesda, USA); E. Vardas (University of the Witwatersrand, Soweto, South Africa); F.M. Regini and P. Sergiampietri for technical and secretarial support and A. Carinci and S. Ceccarelli for editorial assistance. A particular thanks to all trial volunteers.
Sponsorship: This work was funded by Istituto Superiore di Sanità special funding for Tat vaccine trials, the Italian Concerted Action on HIV-AIDS Vaccine Development (ICAV; National AIDS Programme), the AIDS Vaccine Integrated Project (AVIP; European Commission, grant LSHP-CT-2004-503487), and the Italian Ministry of Health and Foreign Affairs.
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