Candidate HIV-1 Tat vaccine development: from basic science to clinical trials
Ensoli, Barbaraa; Fiorelli, Valeriaa; Ensoli, Fabriziob; Cafaro, Aurelioa; Titti, Faustoa; Buttò, Stefanoa; Monini, Paoloa; Magnani, Mauroc; Caputo, Antonellad; Garaci, Enricoe
From the aNational AIDS Center, Istituto Superiore di Sanità, Rome, Italy
bSan Gallicano Hospital, Rome, Italy
cUniversity of Urbino, Urbino, Italy
dUniversity of Padua, Padua, Italy
ePresident of the Istituto Superiore di Sanità, Rome, Italy.
Received 21 September, 2006
Accepted 22 September, 2006
Correspondence and reprint requests to Barbara Ensoli, National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel: +39 06 49903209; fax: +39 06 49903002; e-mail: firstname.lastname@example.org
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.
1. Zolla-Pazner S. Identifying epitopes of HIV-1 that induce protective antibodies. Nat Rev Immunol 2004; 4:199–210.
2. Humbert M, Dietrich U. The role of neutralizing antibodies in HIV infection. AIDS Rev 2006; 8:51–59.
3. Srivastava IK, Ulmer JB, Barnett SW. Neutralizing antibody responses to HIV: role in protective immunity and challenges for vaccine design. Expert Rev Vaccines 2004; 3(4 Suppl.):S33–S52.
4. Burton DR, Desrosiers RC, Doms RW, Koff WC, Kwong PD, Moore JP, et al
. HIV vaccine design and the neutralizing antibody problem. Nat Immunol 2004; 5:233–236.
5. Burton DR, Desrosiers RC, Doms RW, Feinberg MB, Gallo RC, Hahn B, et al
. Public health. A sound rationale needed for phase III HIV-1 vaccine trials. Science 2004; 303:316.
6. McNeil JG, Johnston MI, Birx DL, Tramont EC. Policy rebuttal HIV vaccine trial justified. Science 2004; 303:961.
7. Trinvuthipong C. Thailand's prime-boost HIV vaccine phase III. Science 2004; 303:954–955.
8. Belshe R, Franchini G, Girard MP, Gotch F, Kaleebu P, Marthas ML, et al
. Support for the RV144 HIV vaccine trial. Science 2004; 305:177–180.
9. Slobod KS, Bonsignori M, Brown SA, Zhan X, Stambas J, Hurwitz JL. HIV vaccines: brief review and discussion of future directions. Expert Rev Vaccines 2005; 4:305–313.
10. Graham BS, Mascola JR. Lessons from failure–preparing for future HIV-1 vaccine efficacy trials. J Infect Dis 2005; 191:647–649.
11. Flynn NM, Forthal DN, Harro CD, Judson FN, Mayer KH, Para MF. Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis 2005; 191:654–665.
12. Hel Z, Nacsa J, Tryniszewska E, Tsai WP, Parks RW, Montefiori DC, et al
. Containment of simian immunodeficiency virus infection in vaccinated macaques: correlation with the magnitude of virus-specific pre- and postchallenge CD4+ and CD8+ T cell responses 2. J Immunol 2002; 169:4778–4787.
13. Okuda K, Bukawa H, Hamajima K, Kawamoto S, Sekigawa K, Yamada Y, et al
. Induction of potent humoral and cell-mediated immune responses following direct injection of DNA encoding the HIV type 1 env and rev gene products. AIDS Res Hum Retroviruses 1995; 11:933–943.
14. Kim JJ, Ayyavoo V, Bagarazzi ML, Chattergoon MA, Dang K, Wang B, et al
. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen. J Immunol 1997; 158:816–826.
15. Negri DR, Baroncelli S, Catone S, Comini A, Michelini Z, Maggiorella MT, et al
. Protective efficacy of a multicomponent vector vaccine in cynomolgus monkeys after intrarectal simian immunodeficiency virus challenge. J Gen Virol 2004; 85:1191–1201.
16. Caputo A, Gavioli R, Altavilla G, Brocca-Cofano E, Boarini C, Betti M, et al
. Immunization with low doses of HIV-1 tat DNA delivered by novel cationic block copolymers induces CTL responses against Tat. Vaccine 2003; 21:1103–1111.
17. Caselli E, Betti M, Grossi MP, Balboni PG, Rossi C, Boarini C, et al
. DNA immunization with HIV-1 tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type Tat. J Immunol 1999; 162:5631–5638.
18. Cui Z, Patel J, Tuzova M, Ray P, Phillips R, Woodward JG, et al
. Strong T cell type-1 immune responses to HIV-1 Tat (1-72) protein-coated nanoparticles. Vaccine 2004; 22:2631–2640.
19. Marinaro M, Riccomi A, Rappuoli R, Pizza M, Fiorelli V, Tripiciano A, et al
. Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA. Vaccine 2003; 21:3972–3981.
20. Morris CB, Thanawastien A, Sullivan DE, Clements JD. Identification of a peptide capable of inducing an HIV-1 Tat-specific CTL response. Vaccine 2001; 20:12–15.
21. Gringeri A, Santagostino E, Muca-Perja M, Mannucci PM, Zagury JF, Bizzini B, et al
. Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients. J Hum Virol 1998; 1:293–298.
22. Boykins RA, Ardans JA, Wahl LM, Lal RB, Yamada KM, Dhawan S. Immunization with a novel HIV-1-Tat multiple-peptide conjugate induces effective immune response in mice. Peptides 2000; 21:1839–1847.
23. Cosma A, Nagaraj R, Buhler S, Hinkula J, Busch DH, Sutter G, et al
. Therapeutic vaccination with MVA–HIV-1 nef elicits Nef-specific T-helper cell responses in chronically HIV-1 infected individuals. Vaccine 2003; 22:21–29.
24. Osterhaus AD, van Baalen CA, Gruters RA, Schutten M, Siebelink CH, Hulskotte EG, et al
. Vaccination with Rev and Tat against AIDS. Vaccine 1999; 17:2713–2714.
25. Hel Z, Tryniszewska E, Tsai WP, Johnson JM, Harrod R, Fullen J, et al
. Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes. DNA Cell Biol 2002; 21:619–626.
26. Hejdeman B, Bostrom AC, Matsuda R, Calarota S, Lenkei R, Fredriksson EL, et al
. DNA immunization with HIV early genes in HIV type 1-infected patients on highly active antiretroviral therapy. AIDS Res Hum Retroviruses 2004; 20:860–870.
27. Mooij P, Nieuwenhuis IG, Knoop CJ, Doms RW, Bogers WM, Ten Haaft PJ, et al
. Qualitative T-helper responses to multiple viral antigens correlate with vaccine-induced immunity to simian/human immunodeficiency virus infection. J Virol 2004; 78:3333–3342.
28. Hanke T, Samuel RV, Blanchard TJ, Neumann VC, Allen TM, Boyson JE, et al
. Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen. J Virol 1999; 73:7524–7532.
29. Mwau M, Cebere I, Sutton J, Chikoti P, Winstone N, Wee EG, et al
. A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol 2004; 85:911–919.
30. Makitalo B, Lundholm P, Hinkula J, Nilsson C, Karlen K, Morner A, et al
. Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen. J Gen Virol 2004; 85:2407–2419.
31. Nilsson C, Makitalo B, Berglund P, Bex F, Liljestrom P, Sutter G, et al
. Enhanced simian immunodeficiency virus-specific immune responses in macaques induced by priming with recombinant Semliki Forest virus and boosting with modified vaccinia virus Ankara. Vaccine 2001; 19:3526–3536.
32. Asakura Y, Hinkula J, Leandersson AC, Fukushima J, Okuda K, Wahren B. Induction of HIV-1 specific mucosal immune responses by DNA vaccination. Scand J Immunol 1997; 46:326–330.
33. Dominici S, Laguardia ME, Serafini G, Chiarantini L, Fortini C, Tripiciano A, et al
. Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL. Vaccine 2003; 21:2073–2081.
34. Opi S, Peloponese JM Jr, Esquieu D, Watkins J, Campbell G, De MJ, et al
. Full-length HIV-1 Tat protein necessary for a vaccine. Vaccine 2004; 22:3105–3111.
35. Kim JJ, Yang JS, Nottingham LK, Lee DJ, Lee M, Manson KH, et al
. Protection from immunodeficiency virus challenges in rhesus macaques by multicomponent DNA immunization. Virology 2001; 285:204–217.
36. Allen TM, Mortara L, Mothe BR, Liebl M, Jing P, Calore B, et al
. Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication. J Virol 2002; 76:4108–4112.
37. Calarota S, Bratt G, Nordlund S, Hinkula J, Leandersson AC, Sandstrom E, et al
. Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 1998; 351:1320–1325.
38. Calarota SA, Leandersson AC, Bratt G, Hinkula J, Klinman DM, Weinhold KJ, et al
. Immune responses in asymptomatic HIV-1-infected patients after HIV-DNA immunization followed by highly active antiretroviral treatment. J Immunol 1999; 163:2330–2338.
39. Malkevitch N, Rohne D, Pinczewski J, Aldrich K, Kalyanaraman VS, Letvin NL, et al
. Evaluation of combination DNA/replication-competent Ad-SIV recombinant immunization regimens in rhesus macaques. AIDS Res Hum Retroviruses 2004; 20:235–244.
40. Ayyavoo V, Kudchodkar S, Ramanathan MP, Le P, Muthumani K, Megalai NM, et al
. Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes. AIDS 2000; 14:1–9.
41. Borsutzky S, Fiorelli V, Ebensen T, Tripiciano A, Rharbaoui F, Scoglio A, et al
. Efficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant. Eur J Immunol 2003; 33:1548–1556.
42. Goldstein G, Manson K, Tribbick G, Smith R. Minimization of chronic plasma viremia in rhesus macaques immunized with synthetic HIV-1 Tat peptides and infected with a chimeric simian/human immunodeficiency virus (SHIV33). Vaccine 2000; 18:2789–2795.
43. Asakura Y, Hamajima K, Fukushima J, Mohri H, Okubo T, Okuda K. Induction of HIV-1 Nef-specific cytotoxic T lymphocytes by Nef-expressing DNA vaccine. Am J Hematol 1996; 53:116–117.
44. Muthumani K, Bagarazzi M, Conway D, Hwang DS, Ayyavoo V, Zhang D, et al
. Inclusion of Vpr accessory gene in a plasmid vaccine cocktail markedly reduces Nef vaccine effectiveness in vivo resulting in CD4 cell loss and increased viral loads in rhesus macaques. J Med Primatol 2002; 31:179–185.
45. Kjerrstrom A, Hinkula J, Engstrom G, Ovod V, Krohn K, Benthin R, et al
. Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines. Virology 2001; 284:46–61.
46. Hanke T, Schneider J, Gilbert SC, Hill AV, McMichael A. DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum
: immunogenicity in mice. Vaccine 1998; 16:426–435.
47. Hinkula J, Svanholm C, Schwartz S, Lundholm P, Brytting M, Engstrom G, et al
. Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes. J Virol 1997; 71:5528–5539.
48. Hinkula J, Lundholm P, Wahren B. Nucleic acid vaccination with HIV regulatory genes: a combination of HIV-1 genes in separate plasmids induces strong immune responses. Vaccine 1997; 15:874–878.
49. Hanke T, Barnfield C, Wee EG, Agren L, Samuel RV, Larke N, et al
. Construction and immunogenicity in a prime-boost regimen of a Semliki Forest virus-vectored experimental HIV clade A vaccine. J Gen Virol 2003; 84:361–368.
50. MacGregor RR, Ginsberg R, Ugen KE, Baine Y, Kang CU, Tu XM, et al
. T-cell responses induced in normal volunteers immunized with a DNA-based vaccine containing HIV-1 env and rev. AIDS 2002; 16:2137–2143.
51. Okuda K, Xin KO, Tsuji T, Bukawa H, Tanaka S, Koff WC, et al
. DNA vaccination followed by macromolecular multicomponent peptide vaccination against HIV-1 induces strong antigen-specific immunity. Vaccine 1997; 15:1049–1056.
52. Ishii N, Fukushima J, Kaneko T, Okada E, Tani K, Tanaka SI, et al
. Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 1997; 13:1421–1428.
53. Boyer JD, Ugen KE, Wang B, Agadjanyan M, Gilbert L, Bagarazzi ML, et al
. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nat Med 1997; 3:526–532.
54. Gomez-Roman VR, Florese RH, Peng B, Montefiori DC, Kalyanaraman VS, Venzon D, et al
. An adenovirus-based HIV subtype B prime/boost vaccine regimen elicits antibodies mediating broad antibody-dependent cellular cytotoxicity against non-subtype B HIV strains. J Acquir Immune Defic Syndr
24 August 2006; E-pub ahead of print. in press.
55. Castaldello A, Brocca-Cofano E, Voltan R, Triulzi C, Altavilla G, Laus M, et al
. DNA prime and protein boost immunization with innovative polymeric cationic core-shell nanoparticles elicits broad immune responses and strongly enhance cellular responses of HIV-1 tat DNA vaccination. Vaccine 2006; 24:5655–5669.
56. Pal R, Venzon D, Santra S, Kalyanaraman VS, Montefiori DC, Hocker L, et al
. Systemic immunization with an ALVAC–HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian–human immunodeficiency virus SHIVKU2 RNA levels. J Virol 2006; 80:3732–3742.
57. Neumann J, Stitz J, Konig R, Seibold E, Norley S, Flory E, et al
. Retroviral vectors for vaccine development: induction of HIV-1-specific humoral and cellular immune responses in rhesus macaques using a novel MLV(HIV-1) pseudotype vector. J Biotechnol 2006; 124:615–625.
58. Borsutzky S, Ebensen T, Link C, Becker PD, Fiorelli V, Cafaro A, et al
. Efficient systemic and mucosal responses against the HIV-1 Tat protein by prime/boost vaccination using the lipopeptide MALP-2 as adjuvant. Vaccine 2006; 24:2049–2056.
59. Patel J, Galey D, Jones J, Ray P, Woodward JG, Nath A, et al
. HIV-1 Tat-coated nanoparticles result in enhanced humoral immune responses and neutralizing antibodies compared to alum adjuvant. Vaccine 2006; 24:3564–3573.
60. Amara RR, Smith JM, Staprans SI, Montefiori DC, Villinger F, Altman JD, et al
. Critical role for Env as well as Gag-Pol in control of a simian–human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine. J Virol 2002; 76:6138–6146.
61. Verrier B, Le GR, Taman-Onal Y, Terrat C, Guillon C, Durand PY, et al
. Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08. DNA Cell Biol 2002; 21:653–658.
62. Stittelaar KJ, Gruters RA, Schutten M, van Baalen CA, van AG, Cranage M, et al
. Comparison of the efficacy of early versus late viral proteins in vaccination against SIV. Vaccine 2002; 20:2921–2927.
63. Voss G, Manson K, Montefiori D, Watkins DI, Heeney J, Wyand M, et al
. Prevention of disease induced by a partially heterologous AIDS virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine. J Virol 2003; 77:1049–1058.
64. Patterson LJ, Malkevitch N, Zhao J, Peng B, Robert-Guroff M. Potent, persistent cellular immune responses elicited by sequential immunization of rhesus macaques with Ad5 host range mutant recombinants encoding SIV Rev and SIV Nef. DNA Cell Biol 2002; 21:627–635.
65. Amara RR, Villinger F, Altman JD, Lydy SL, O'Neil SP, Staprans SI, et al
. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Vaccine 2002; 20:1949–1955.
66. Partidos CD, Moreau E, Chaloin O, Tunis M, Briand JP, Desgranges C, et al
. A synthetic HIV-1 Tat protein breaches the skin barrier and elicits Tat-neutralizing antibodies and cellular immunity. Eur J Immunol 2004; 34:3723–3731.
67. Dale CJ, De RR, Stratov I, Chea S, Montefiori DC, Thomson S, et al
. Efficacy of DNA and fowlpox virus priming/boosting vaccines for simian/human immunodeficiency virus. J Virol 2004; 78:13819–13828.
68. Goldstein G. HIV-1 Tat protein as a potential AIDS vaccine. Nat Med 1996; 2:960–964.
69. Gallo RC. Tat as one key to HIV-induced immune pathogenesis and Tat (correction of Pat) toxoid as an important component of a vaccine. Proc Natl Acad Sci U S A 1999; 96:8324–8326.
70. Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Morgan RA, et al
. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol 1993; 67:277–287.
71. Arya SK, Guo C, Josephs SF, Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 1985; 229:69–73.
72. Fisher AG, Feinberg MB, Josephs SF, Harper ME, Marselle LM, Reyes G, et al
. The trans-activator gene of HTLV-III is essential for virus replication. Nature 1986; 320:367–371.
73. Peruzzi F. The multiple functions of HIV-1 Tat: proliferation versus apoptosis. Front Biosci 2006; 11:708–717.
74. Huigen MC, Kamp W, Nottet HS. Multiple effects of HIV-1 trans-activator protein on the pathogenesis of HIV-1 infection. Eur J Clin Invest 2004; 34:57–66.
75. Wu Y, Marsh JW. Selective transcription and modulation of resting T cell activity by preintegrated HIV DNA. Science 2001; 293:1503–1506.
76. Chen D, Wang M, Zhou S, Zhou Q. HIV-1 Tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic Bcl-2 relative Bim. EMBO J 2002; 21:6801–6810.
77. Campbell GR, Watkins JD, Esquieu D, Pasquier E, Loret EP, Spector SA. The C terminus of HIV-1 Tat modulates the extent of CD178-mediated apoptosis of T cells. J Biol Chem 2005; 280:38376–38382.
78. Bartz SR, Emerman M. Human immunodeficiency virus type 1 Tat induces apoptosis and increases sensitivity to apoptotic signals by up-regulating FLICE/caspase-8. J Virol 1999; 73:1956–1963.
79. Gibellini D, Re MC, Ponti C, Vitone F, Bon I, Fabbri G, et al
. HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity. J Cell Physiol 2005; 203:547–556.
80. Yang Y, Tikhonov I, Ruckwardt TJ, Djavani M, Zapata JC, Pauza CD, et al
. Monocytes treated with human immunodeficiency virus Tat kill uninfected CD4(+) cells by a tumor necrosis factor-related apoptosis-induced ligand-mediated mechanism. J Virol 2003; 77:6700–6708.
81. Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, Walczak H, et al
. Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature 1995; 375:497–500.
82. Ott M, Emiliani S, Van LC, Herbein G, Lovett J, Chirmule N, et al
. Immune hyperactivation of HIV-1-infected T cells mediated by Tat and the CD28 pathway. Science 1997; 275:1481–1485.
83. Li CJ, Ueda Y, Shi B, Borodyansky L, Huang L, Li YZ, et al
. Tat protein induces self-perpetuating permissivity for productive HIV-1 infection. Proc Natl Acad Sci U S A 1997; 94:8116–8120.
84. Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 1995; 268:429–431.
85. McCloskey TW, Ott M, Tribble E, Khan SA, Teichberg S, Paul MO, et al
. Dual role of HIV Tat in regulation of apoptosis in T cells. J Immunol 1997; 158:1014–1019.
86. Chang HC, Samaniego F, Nair BC, Buonaguro L, Ensoli B. HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 1997; 11:1421–1431.
87. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F. Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature 1990; 345:84–86.
88. Ensoli B, Gendelman R, Markham P, Fiorelli V, Colombini S, Raffeld M, et al
. Synergy between basic fibroblast growth factor and HIV-1 Tat protein in induction of Kaposi's sarcoma. Nature 1994; 371:674–680.
89. Marchio S, Alfano M, Primo L, Gramaglia D, Butini L, Gennero L, et al
. Cell surface-associated Tat modulates HIV-1 infection and spreading through a specific interaction with gp120 viral envelope protein. Blood 2005; 105:2802–2811.
90. Chang HK, Gallo RC, Ensoli B. Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 Tat protein. J Biomed Sci 1995; 2:189–202.
91. Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988; 55:1189–1193.
92. Shutt DC, Soll DR. HIV-induced T-cell syncytia release a two component T-helper cell chemoattractant composed of Nef and Tat. J Cell Sci 1999; 112:3931–3941.
93. Koedel U, Kohleisen B, Sporer B, Lahrtz F, Ovod V, Fontana A, et al
. HIV type 1 Nef protein is a viral factor for leukocyte recruitment into the central nervous system. J Immunol 1999; 163:1237–1245.
94. Ferrantelli F, Cafaro A, Ensoli B. Nonstructural HIV proteins as targets for prophylactic or therapeutic vaccines. Curr Opin Biotechnol 2004; 15:543–556.
95. Caputo A, Gavioli R, Ensoli B. Recent advances in the development of HIV-1 Tat-based vaccines. Curr HIV Res 2004; 2:357–376.
96. Reiss P, Lange JM, de Ronde A, de Wolf F, Dekker J, Debouck C, et al
. Speed of progression to AIDS and degree of antibody response to accessory gene products of HIV-1. J Med Virol 1990; 30:163–168.
97. Rodman TC, To SE, Hashish H, Manchester K. Epitopes for natural antibodies of human immunodeficiency virus (HIV)-negative (normal) and HIV-positive sera are coincident with two key functional sequences of HIV Tat protein. Proc Natl Acad Sci U S A 1993; 90:7719–7723.
98. Zagury JF, Sill A, Blattner W, Lachgar A, Le BH, Richardson M, et al
. Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine. J Hum Virol 1998; 1:282–292.
99. Re MC, Furlini G, Vignoli M, Ramazzotti E, Roderigo G, De Rosa V, et al
. Effect of antibody to HIV-1 Tat protein on viral replication in vitro and progression of HIV-1 disease in vivo
. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 10:408–416.
100. Re MC, Vignoli M, Furlini G, Gibellini D, Colangeli V, Vitone F, et al
. Antibodies against full-length Tat protein and some low-molecular-weight Tat-peptides correlate with low or undetectable viral load in HIV-1 seropositive patients. J Clin Virol 2001; 21:81–89.
101. Richardson MW, Mirchandani J, Duong J, Grimaldo S, Kocieda V, Hendel H, et al
. Antibodies to Tat and Vpr in the GRIV cohort: differential association with maintenance of long-term non-progression status in HIV-1 infection. Biomed Pharmacother 2003; 57:4–14.
102. Demirhan I, Chandra A, Mueller F, Mueller H, Biberfeld P, Hasselmayer O, et al
. Antibody spectrum against the viral transactivator protein in patients with human immunodeficiency virus type 1 infection and Kaposi's sarcoma. J Hum Virol 2000; 3:137–143.
103. Krone WJ, Debouck C, Epstein LG, Heutink P, Meloen R, Goudsmit J. Natural antibodies to HIV-tat epitopes and expression of HIV-1 genes in vivo
. J Med Virol 1988; 26:261–270.
104. Butto S, Fiorelli V, Tripiciano A, Ruiz-Alvarez MJ, Scoglio A, Ensoli F, et al
. Sequence conservation and antibody cross-recognition of clade B human immunodeficiency virus (HIV) type 1 Tat protein in HIV-1-infected Italians, Ugandans, and South Africans. J Infect Dis 2003; 188:1171–1180.
105. Rezza G, Fiorelli V, Dorrucci M, Ciccozzi M, Tripiciano A, Scoglio A, et al
. The presence of anti-Tat antibodies is predictive of long-term nonprogression to AIDS or severe immunodeficiency: findings in a cohort of HIV-1 seroconverters. J Infect Dis 2005; 191:1321–1324.
106. Addo MM, Altfeld M, Rosenberg ES, Eldridge RL, Philips MN, Habeeb K, et al
. The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals. Proc Natl Acad Sci U S A 2001; 98:1781–1786.
107. Novitsky V, Rybak N, McLane MF, Gilbert P, Chigwedere P, Klein I, et al
. Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for AIDS vaccine design. J Virol 2001; 75:9210–9228.
108. Cao J, McNevin J, Holte S, Fink L, Corey L, McElrath MJ. Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection. J Virol 2003; 77:6867–6878.
109. Novitsky V, Cao H, Rybak N, Gilbert P, McLane MF, Gaolekwe S, et al
. Magnitude and frequency of cytotoxic T-lymphocyte responses: identification of immunodominant regions of human immunodeficiency virus type 1 subtype C. J Virol 2002; 76:10155–10168.
110. van Baalen CA, Schutten M, Huisman RC, Boers PH, Gruters RA, Osterhaus AD. Kinetics of antiviral activity by human immunodeficiency virus type 1-specific cytotoxic T lymphocytes (CTL) and rapid selection of CTL escape virus in vitro. J Virol 1998; 72:6851–6857.
111. Jones NA, Wei X, Flower DR, Wong M, Michor F, Saag MS, et al
. Determinants of human immunodeficiency virus type 1 escape from the primary CD8+ cytotoxic T lymphocyte response. J Exp Med 2004; 200:1243–1256.
112. Cao J, McNevin J, Malhotra U, McElrath MJ. Evolution of CD8+ T cell immunity and viral escape following acute HIV-1 infection. J Immunol 2003; 171:3837–3846.
113. Allen TM, O'Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, et al
. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 2000; 407:386–390.
114. O'Connor DH, Allen TM, Vogel TU, Jing P, DeSouza IP, Dodds E, et al
. Acute phase cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus infection. Nat Med 2002; 8:493–499.
115. Tikhonov I, Ruckwardt TJ, Hatfield GS, Pauza CD. Tat-neutralizing antibodies in vaccinated macaques. J Virol 2003; 77:3157–3166.
116. Ramakrishna L, Anand KK, Mohankumar KM, Ranga U. Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol 2004; 78:9174–9189.
117. Opi S, Peloponese JM Jr, Esquieu D, Campbell G, De MJ, Walburger A, et al
. Tat HIV-1 primary and tertiary structures critical to immune response against non-homologous variants. J Biol Chem 2002; 277:35915–35919.
118. Kuiken C, Foley B, Hahn B, Korber B, McCutchan F, Marx JW, et al
. Human retroviruses and AIDS: a compilation and analysis of nucleic acid and amino acid sequences
. Los Alamos, New Mexico: Los Alamos National Laboratory.
119. Fanales-Belasio E, Moretti S, Nappi F, Barillari G, Micheletti F, Cafaro A, et al
. Native HIV-1 Tat protein targets monocyte-derived dendritic cells and enhances their maturation, function, and antigen-specific T cell responses. J Immunol 2002; 168:197–206.
120. Gavioli R, Gallerani E, Fortini C, Fabris M, Bottoni A, Canella A, et al
. HIV-1 tat protein modulates the generation of cytotoxic T cell epitopes by modifying proteasome composition and enzymatic activity. J Immunol 2004; 173:3838–3843.
121. Remoli AL, Marsili G, Perrotti E, Gallerani E, Ilari R, Nappi F, et al
. Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1. Biochem J 2006; 396:371–380.
122. Barillari G, Gendelman R, Gallo RC, Ensoli B. The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence. Proc Natl Acad Sci U S A 1993; 90:7941–7945.
123. Cafaro A, Titti F, Fracasso C, Maggiorella MT, Baroncelli S, Caputo A, et al
. Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P). Vaccine 2001; 19:2862–2877.
124. Cafaro A, Caputo A, Fracasso C, Maggiorella MT, Goletti D, Baroncelli S, et al
. Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine. Nat Med 1999; 5:643–650.
125. Caputo A, Betti M, Altavilla G, Bonaccorsi A, Boarini C, Marchisio M, et al
. Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application. Vaccine 2002; 20:2303–2317.
126. Betti M, Voltan R, Marchisio M, Mantovani I, Boarini C, Nappi F, et al
. Characterization of HIV-1 Tat proteins mutated in the transactivation domain for prophylactic and therapeutic application. Vaccine 2001; 19:3408–3419.
127. Samaniego F, Markham PD, Gallo RC, Ensoli B. Inflammatory cytokines induce AIDS–Kaposi's sarcoma-derived spindle cells to produce and release basic fibroblast growth factor and enhance Kaposi's sarcoma-like lesion formation in nude mice. J Immunol 1995; 154:3582–3592.
128. Maggiorella MT, Baroncelli S, Michelini Z, Fanales-Belasio E, Moretti S, Sernicola L, et al
. Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys. Vaccine 2004; 22:3258–3269.
129. Ensoli B, Cafaro A. HIV-1 Tat vaccines. Virus Res 2002; 82:91–101.
130. Various authors. Forum in immunology: rational vaccination strategies against AIDS. Microbes Infect
AIDS vaccine; clinical trials; HIV community; Tat; translational research
© 2006 Lippincott Williams & Wilkins, Inc.
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.