Candidate HIV-1 Tat vaccine development: from basic science to clinical trials.
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AIDS Vaccines↗
Biomedical subjects
Antonella Caputo
Publications and source records attributed to Antonella Caputo.
Expression of human immunodeficiency virus type 1 tat from a replication-deficient herpes simplex type 1 vector induces antigen-specific T cell responses.
Herpes simplex type-1 virus (HSV-1) based vectors have been widely used in different gene therapy approaches and also as experimental vaccines against HSV-1 infection. Recent advances in the HSV-1 technology do support the use of replication defective HSV-1 as vaccine vectors for delivery of foreign antigens. We have examined the ability of a recombinant replication-defective HSV-1 vector expressing the HIV-1 Tat protein to induce long-term Tat-specific immune responses in the Balb/c murine model. The results showed that vector administration by the subcutaneous route elicits anti-Tat specific T-cell mediated immune responses in mice characterized by the presence of the Tat-specific cytotoxic activity and production of high levels of IFN-gamma.
AIDS Vaccines↗
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.
Novel biocompatible core-shell cationic nanoparticles, composed of an inner hard core of poly(methylmethacrylate) (PMMA) and a hydrophilic tentacular shell bearing positively charged groups and poly(ethyleneglycol) chains covalently bound to the core, were prepared by emulsion polymerization and characterized in vitro and in vivo for DNA vaccine applications. The nanoparticles reversibly adsorbed large amounts of DNA, mainly through electrostatic interactions, preserved its functional structure, efficiently delivered it intracellularly, and were not toxic in vitro or in mice. Furthermore, two intramuscular (i.m.) immunizations (4 weeks apart) with a very low dose (1 microg) of the plasmid pCV-tat delivered by these nanoparticles followed by one or two protein boosts induced significant antigen-specific humoral and cellular responses and greatly increased Th1-type T cell responses and CTLs against HIV-1 Tat.
AIDS Vaccines↗
Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DeltaV2 Env.
The promising results obtained with the HIV-1 Tat-based vaccines in mice, monkeys and humans, a better understanding of Tat immunomodulatory functions, as well as evidence that vaccination with trimeric V2 loop-deleted HIV-1 Env induces cross-clade neutralizing antibodies led to the rational design of a novel vaccine based on the combination of Tat and V2-deleted Env.
AIDS Vaccines↗
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.
The human immunodeficiency virus (HIV) type 1 Tat protein plays a key role in the life cycle of the virus and in pathogenesis and is highly conserved among HIV subtypes. On the basis of this and of safety, immunogenicity, and efficacy findings in monkeys, Tat is being tested as a vaccine in phase 1 trials. Here, we evaluated the incidence and risk of progression to advanced HIV disease by anti-Tat serostatus in a cohort of 252 HIV-1 seroconverters. The risk of progression was lower in the anti-Tat-positive subjects than in the anti-Tat-negative subjects. Progression was faster in the persistently anti-Tat-negative subjects than in the transiently anti-Tat-positive subjects, and no progression was observed in the persistently anti-Tat-positive subjects.
Acquired Immunodeficiency Syndrome↗
Core-shell microspheres by dispersion polymerization as promising delivery systems for proteins.
Functional poly(methyl methacrylate) core-shell microspheres were prepared by dispersion polymerization. An appropriate selection of experimental parameters and in particular of the initiator and stabilizer amount and of the medium solvency power allowed a monodisperse sample as large as 600 nm to be prepared. To this purpose, low initiator concentration, high steric stabilizer amount and a low solvency power medium were employed. The microspheres present a core-shell structure in which the outer shell is constituted by the steric stabilizer which affords carboxylic groups able to interact with basic proteins, such as trypsin, whose adsorption is essentially driven by the carboxylic group density in the microsphere shell. Finally, fluorescent microspheres were prepared for biodistribution studies and shown to be readily taken up by the cells both in vitro and in vivo. These results suggest that these microspheres are promising delivery systems for the development of novel protein-based vaccines.
Acrylic Resins↗
HIV-1 tat protein modulates the generation of cytotoxic T cell epitopes by modifying proteasome composition and enzymatic activity.
Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.
Antigen Presentation↗
Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.
Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.
AIDS Vaccines↗
Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application.
Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.
AIDS Vaccines↗
Recent advances in the development of HIV-1 Tat-based vaccines.
Over the last two decades most of the efforts in HIV vaccine development have been based on the use of the HIV Env with the goal to induce sterilizing immunity. However, as a result of Env variability disappointing results have been obtained in preclinical and phase III clinical trials. Although the objective of a preventive immunity still remains a priority, secondary endpoints (e.g. block of virus replication and disease onset) are being considered at the present as more achievable end-points in HIV vaccine development. This is based on accumulating evidence indicating that low viral load correlates with maintenance of immune functions and slow progression to disease, and that cell-mediated immunity plays a major protective role in the absence of sterilizing immunity. The promising results obtained in non-human primates with a vaccine based on a native Tat protein (B-clade), which is an early regulatory protein key for HIV replication and AIDS pathogenesis, highlights the importance of targeting the virus very early after infection. In particular, the immune response against Tat appears to modify the virus-host interactions at the very beginning of infection, thus containing the depletion of critical immune cells and the progression of infection. Moreover, since Tat targets and induces maturation of dendritic cells, has immunomodulatory activities and drives Th-1 and CTL responses, immunization with Tat may drive or increase these immune responses also against other HIV antigens to support an effective, long-lasting and hopefully even sterilizing antiviral immunity. Finally, Tat B-clade is similarly recognized by sera from individuals infected by different virus clades (A, B, C, D) supporting the concept of a cross-clade vaccine. Therefore, the Tat-vaccine should contain virus replication protecting from disease progression (non-sterilizing immunity) or even favoring an abortive infection. Although only a phase III clinical trial will establish the efficacy of this vaccine strategy, the Tat-vaccine has recently entered preventive and therapeutic phase I clinical testing in Italy to establish safety (primary-end-point) and immunogenicity (secondary end-point) and phase II studies are being prepared.
AIDS Vaccines↗
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.
We determined immune cross-recognition and the degree of Tat conservation in patients infected by local human immunodeficiency virus (HIV) type 1 strains. The data indicated a similar prevalence of total and epitope-specific anti-Tat IgG in 578 serum samples from HIV-infected Italian (n=302), Ugandan (n=139), and South African (n=137) subjects, using the same B clade Tat protein that is being used in vaccine trials. In particular, anti-Tat antibodies were detected in 13.2%, 10.8%, and 13.9% of HIV-1-infected individuals from Italy, Uganda, and South Africa, respectively. Sequence analysis results indicated a high similarity of Tat from the different circulating viruses with BH-10 Tat, particularly in the 1-58 amino acid region, which contains most of the immunogenic epitopes. These data indicate an effective cross-recognition of a B-clade laboratory strain-derived Tat protein vaccine by individuals infected with different local viruses, owing to the high similarity of Tat epitopes.
AIDS Vaccines↗
Tumor-host interaction mediates the regression of BK virus-induced vascular tumors in mice: involvement of transforming growth factor-beta1.
Several sexually transmitted viruses have been associated with the development of Kaposi's sarcoma (KS), a highly vascularized multi-focal neoplasm, characterized by the presence of spindle-shaped and endothelial cells, fibroblasts and macrophages. As BK virus (BKV) sequences were found in 100% of primary KS and 75% of KS cell lines, we established an experimental model to test whether BKV may be involved in the pathogenesis of KS. For this purpose, we transformed primary and spontaneously immortalized murine endothelial cells with BKV or with a plasmid containing BKV early region, which encodes BKV T antigen. Murine endothelial cells lost endothelial markers after transformation by BKV and, when inoculated s.c. in nude mice, induced tumors which regressed 7-30 days after onset, whereas spontaneously immortalized murine endothelial MHE cells induced progressing tumors, which brought the animals to death. Histologic examination showed an initial formation of vessels around the tumors, followed by the appearance of a dense population of fibroblasts and mononuclear cells in the peritumoral tissue. Subsequently, tumors appeared to be infiltrated by mononuclear cells and surrounded by a thick fibrous wall with scattered fibroblasts and without vessels. Areas of necrosis developed in the tumor mass and finally the neoplastic tissue completely degenerated. The medium conditioned by BKV-transformed cells induced proliferation and migration of human fibroblasts and NIH3T3 cells. These effects were inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Northern blot analysis revealed that BKV-transformed cells express a greater amount of TGF-beta1 RNA than normal murine endothelial cells. Besides, TGF-beta1 was not expressed in progressing tumors induced by spontaneously immortalized endothelial MHE cells, whereas it was highly expressed during the regression of tumors induced by BKV-transformed MHE and primary endothelial cells. Over-expression of TGF-beta1 may be responsible for the mononuclear cell infiltration, inhibition of angiogenesis and formation of the fibrotic wall around tumors, inducing tumor regression through tumor cell necrosis and nutritional starvation. These results prompt us to test whether production of TGF-beta1 is associated with spontaneous KS regression in human patients. In this case, KS regression could be induced or accelerated by any means that enhances TGF-beta1 production at the tumor site.
Animals↗
Interaction of Sp1 transcription factor with HIV-1 Tat protein: looking for cellular partners.
The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates HIV-1 transcription by functionally interacting with a number of cellular proteins, among which the Sp1 transcription factor. We recently demonstrated that Tat does not directly interact with Sp1 either in vitro or in vivo, and we suggested that other protein(s) could indirectly mediate Tat-Sp1 interaction. In keeping, here we showed that addition of HeLa cell nuclear extracts to purified Tat and Sp1 proteins allows the formation of a Tat/Sp1 complex in in vitro binding assays. In an attempt to identify the partner(s) that bridge Tat and Sp1, we developed a yeast multi-protein system, in which cellular proteins recently shown to play a relevant role in Tat function, namely TATA box-binding protein, cyclin T1, CDK9, and cyclin T1/CDK9 complex, were coexpressed, individually or in pair-wise combination, with Tat and Sp1 hybrids. We demonstrated that none of these candidate partners bridges Tat and Sp1. However, our yeast multi-protein system, which allows simple and rapid detection of interactions among up to four proteins, will be most helpful to further dissect the interaction of Tat and Sp1 with other candidate partners that participate in the assembly of transcriptionally active complexes at the HIV-1 LTR.
Gene Products, tat↗
Immunization with low doses of HIV-1 tat DNA delivered by novel cationic block copolymers induces CTL responses against Tat.
Cytotoxic T cell responses are key to the control of intracellular pathogens including HIV-1. In particular, HIV-1 vaccines based on regulatory proteins, such as Tat, are aimed at controlling HIV-1 replication and at blocking disease development by inducing cytotoxic T cell responses. Naked DNA is capable of inducing such responses but it requires several inoculations of high amounts of DNA, and/or prime-boost regimens. Here, we show that a novel class of cationic block copolymers protect the DNA from DNAse I digestion, and improve DNA delivery to antigen-presenting cells (APCs) after intramuscular (i.m.) vaccination. In particular, three cationic block copolymers (K1, K2 and K5) were used to deliver the HIV-1 pCV-tat DNA vaccine in BALB/c mice. The results indicate that vaccination with a very low dose (1 microg) of pCV-tat delivered by the cationic block copolymer K2 is safe and, as compared to naked DNA (up to 30 microg), greatly increases the CTL response against Tat, which was detected in all animals in the absence or in the presence of re-stimulation.
AIDS Vaccines↗
The Sp1 transcription factor does not directly interact with the HIV-1 Tat protein.
The role of Sp1 in regulating the trans-activating activity of the human immunodeficiency virus type 1 (HIV-1) Tat protein has not yet been clearly defined. In fact, studies on the physical and functional interaction between Sp1 and Tat have yielded contradictory results. Here we investigated whether a physical interaction between Sp1 and Tat indeed occurs, exploiting both biochemical and genetic techniques that allow detection of direct protein-protein interactions. Studies performed with the yeast two-hybrid system indicate that Sp1 does not directly interact with the HIV-1 Tat protein. Control experiments demonstrated that both proteins are functionally expressed in the yeast cells. In vitro binding assays further confirmed that Sp1 does not physically bind Tat. These data suggest that in vivo Tat and Sp1 most likely take part of a multicomponent complex and thus encourage the search of the molecule(s) which mediate Tat-Sp1 interaction.
Adsorption↗
Inhibition of HIV-1 Tat activity correlates with down-regulation of bcl-2 and results in reduction of angiogenesis and oncogenicity.
The Tat protein of the human immunodeficiency virus type 1 promotes survival and growth and inhibits apoptosis of different cell types. These effects of Tat are attributed to the induction of bcl-2 gene expression. In this study we show that the blocking of both intracellular and extracellular Tat correlates with a decrease of bcl-2 transcripts, leading in vitro to a lower growth rate and attenuation of the transformed phenotype and in vivo to a reduced angiogenic and oncogenic activity of Tat-expressing cells. These results support the notion that bcl-2 is an effector of Tat-induced angiogenesis and oncogenesis and indicate that the blocking of Tat functions by immunoprophylactic, pharmacological, and gene therapy approaches may help to control oncogenesis during AIDS.
Animals↗
Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application.
A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1-5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.
AIDS Vaccines↗
HIV-1 Tat-based vaccines: from basic science to clinical trials.
Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.
AIDS Vaccines↗