Candidate HIV-1 Tat vaccine development: from basic science to clinical trials

Introduction 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).Table 1: Reasons to use the native HIV-1 Tat protein as a vaccine candidate for HIV/AIDS.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 [105] 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 [105].Table 2: Frequencies of IgG and IgM anti-Tat antibodies in individuals stratified according to HIV status, clinical stage and CD4 T-cell counts.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 [104]. 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 [104]. 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 [119]. 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. Lesson learned 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).Fig. 1: HIV Tat vaccine development. Shown are the sequential and integrated activities for the development of the HIV-1 Tat vaccine programme from the basic research to clinical testing, including parallel activities directed at investigating the correlates of protection in natural infection and at validating laboratory testing for trial monitoring, and capacity building in developing countries for advanced clinical testing.Fig. 2: Timeline of the Tat vaccine programme. Timeline of the activities undertaken for the development and conduct of phase I clinical trials with the Tat vaccine candidate, preparatory studies in developing countries and the development of a second/third generation of Tat-based vaccine candidates. CAB, Community advisory board; CRO, contract research organization; EC, ethical committee; GMP, good manufacturing practice.Preclinical development 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).Table 3: Physicochemical, immunochemical and biological characterization of the Tat protein.Fig. 3: Physicochemical characterization of the Tat protein vaccine. Shown are the Comassie blue and silver staining as well as Western blot of the Tat protein separated by sodium dodecylsulphate–polyacrylamide gel electrophoresis. The lower panel shows the results of high-pressure liquid chromatography (HPLC) of the Tat formulated with bovine serum albumin. This product was used for in-vitro and preclinical studies.Fig. 4: Tat uptake by monocyte-derived dendritic cells. Monocyte-derived dendritic cells were incubated with serial concentrations (0.1–1000 ng/ml) of the native Tat protein, medium, or reconstitution buffer for 10 min. The intracytoplasmatic Tat content was evaluated by flow cytometry after staining with specific affinity purified rabbit anti-Tat polyclonal antibodies (or isotype control), followed by secondary fluorescein-isothiocyanate-conjugated anti-rabbit antibodies [119]. The percentages of positive cells (compared with isotype-stained samples) are reported. The bars indicate the standard deviation. Data are the mean of 86 experiments performed with cells from 71 different donors and eight different lots of the Tat protein.Preclinical testing 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 [128]. 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.Table 4: Summary of the immunological responses and post-challenge fate of Tat-vaccinated and control monkeys.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 [129]. 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). Lesson learned 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).Fig. 5: Procedures for approval of the Tat vaccine candidate for human use by regulatory agencies. are the and procedures required by the Italian regulatory authorities for approval for human use of the Tat vaccine. for 5: European guidelines for the and conduct of phase I clinical trials of on recombinant guidelines for the activity of the central learned regulatory represents a in building up research and a specific expertise and also because no training in this Therefore, training should be implemented to support scientists in this task. The of training in and human and manufacturing Tat vaccine production for phase I studies manufacturing process development of the Tat protein For the good manufacturing production of the Tat vaccine it was necessary to identify a to phase I was not in A was in the which produced and released the Tat vaccine according to current The recombinant Tat protein was produced and purified by and heparin sepharose formulated in a buffer in the presence of human serum and (Fig. studies with the product confirmed that release (Table 3). sequence and were also performed on the tests confirmed that the clinical biological activity for up to 2 years at of the clinical of the Tat vaccine candidate used in the clinical learned The to a was and the of a in to the support of and of the of a has been in for Tat vaccine production clinical trials. of laboratory and In order to for clinical trials conducted in a all clinical and laboratory as well as psychosocial and were among the good clinical procedures by specific and integrated (Fig. 2 and Fig. of the activities for trial conduct of the Tat vaccine candidate. were according to specific with the support of the contract research and the advisory Parallel preventive and therapeutic phase I clinical trials were conducted in in and of and in site in Fig. activities and support from the of data and biological and were regulated by specific the and the clinical procedures were implemented in the clinical to all activities enrollment and monitoring of the (clinical safety laboratory testing, and on and on were also for In this an Committee for the Evaluation of of clinical was by the This the and submitted and safety to the regulatory structure for the conduct of the preventive and therapeutic phase I trials. in the are the and in the structure for the conduct of the parallel preventive and therapeutic trials with the Tat vaccine candidate. 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