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.     

Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.     

A review of HIV-1 Tat protein biological effects

The authors have reviewed some biological properties of HIV-1 Tat protein, and have also reported some personal data. This viral regulatory protein is endowed with multifunctional activities, acting as an endogenous factor in the infected cells and exogenously, on those uninfected. In particular, Tat-induced proliferation and differentiation of HIV target cells which promotes viral infection, is discussed in this review. However, exogenous Tat protein can sometimes also produce, directly or indirectly, damaging effects in different organs and host systems, such as myocardium, kidney, liver and central nervous system (CNS). For example some data also demonstrate an increase in the apoptotic index induced by Tat at various levels, including the immune system. The effective role of HIV-1 Tat protein in promoting viral replication and its high immunogenicity suggest useful employment of this protein for therapeutic or preventive vaccine preparations.     

HIV-1 Tat suppresses gp120-specific T cell response in IL-10-dependent manner

A large number of multicomponent vaccine candidates are currently in clinical evaluation, many of which also include the HIV-1 Tat protein, an important regulatory protein of the virus. However, whether Tat, a known immune effector molecule with a well-conserved sequence among different HIV subtypes, affects the immune response to a coimmunogen is not well understood. In this study, using a bicistronic vector expressing both gp120 and Tat, we have analyzed the role of Tat in elicitation of the gp120-specific immune response. The T cell responses to gp120 were greatly diminished in mice coimmunized with Tat as compared with mice immunized with gp120 alone. This immunosuppressive activity of Tat was not confined to viral Ag only because it also suppressed the immune response of unrelated Ag. Analysis of the cytokine profile suggests that Tat induces IL-10 and since IL-10 has been demonstrated to have appreciable T cell inhibitory activity, it is plausible that IL-10 could be responsible for Tat-mediated immunosuppression. Finally, the immunosuppressive effect of Tat was not observed in IL-10-deficient mice, confirming the role of IL-10 in Tat-mediated immunosuppression. Thus, our results demonstrate for the first time that the immunosuppressive effect of Tat is mediated through IL-10 and suggests that Tat-induced IL-10-mediated immune suppression seems to cripple immune surveillance during HIV-1 infection.     

Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-kappa B activity.

The Epstein-Barr virus latent membrane protein (LMP) is an integral membrane protein that is expressed in cells latently infected with the virus. LMP is believed to play an important role in Epstein-Barr virus transformation and has been shown to induce expression of several cellular proteins. We performed a series of experiments that demonstrated that LMP is an efficient transactivator of expression from the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). Mutation or deletion of the NF-kappa B elements in the LTR abolished the transactivation, indicating that the LMP effect on HIV expression was due to induction of NF-kappa B activity. Experiments in which the HIV-1 Tat protein was coexpressed in cells together with LMP showed that Tat was able to potentiate the transactivation. Surprisingly, a synergistic effect of the two proteins was observed even in the absence of the recognized target region for Tat (TAR) in the HIV-1 LTR.     

Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins

The extensive diversity of human immunodeficiency virus type 1 (HIV-1) and its capacity to mutate and escape host immune responses are major challenges for AIDS vaccine development. Ancestral sequences, which minimize the genetic distance to circulating strains, provide an opportunity to design immunogens with the potential to elicit broad recognition of HIV epitopes. We developed a phylogenetics-informed algorithm to reconstruct ancestral HIV sequences, called Center of Tree (COT). COT sequences have potentially significant benefits over isolate-based strategies, as they minimize the evolutionary distances to circulating strains. COT sequences are designed to surmount the potential pitfalls stemming from sampling bias with the consensus method and outlier bias with the most-recent-common-ancestor approach. We computationally derived COT sequences from circulating HIV-1 subtype B sequences for the genes encoding the major viral structural protein (Gag) and two regulatory proteins, Tat and Nef. COT genes were synthesized de novo and expressed in mammalian cells, and the proteins were characterized. COT Gag was shown to generate virus-like particles, while COT Tat transactivated gene expression from the HIV-1 long terminal repeat and COT Nef mediated downregulation of cell surface major histocompatibility complex class I. Thus, retrodicted ancestral COT proteins can retain the biological functions of extant HIV-1 proteins. Additionally, COT proteins were immunogenic, as they elicited antigen-specific cytotoxic T-lymphocyte responses in mice. These data support the utility of the COT approach to create novel and biologically active ancestral proteins as a starting point for studies of the structure, function, and biological fitness of highly variable genes, as well as for the rational design of globally relevant vaccine candidates.     

HIV-1 Tat directly binds to NFkappaB enhancer sequence: role in viral and cellular gene expression

HIV-1 Tat protein reprograms cellular gene expression of infected as well as uninfected cells apart from its primary function of transactivating HIV-1 long terminal repeat (LTR) promoter by binding to a nascent RNA stem–loop structure known as the transactivator response region (TAR). Tat also induces chromatin remodeling of proviral LTR-mediated gene expression by recruiting histone acetyl transferases to the chromatin, which results in histone acetylation. Furthermore several studies have shown convincing evidence that Tat can transactivate HIV-1 gene expression in the absence of TAR, the molecular mechanism of which remains to be elucidated. Here we show a direct interaction of Tat with nuclear factor kappa B (NFκB) enhancer, a global regulatory sequence for many cellular genes both in vitro and in vivo. This interaction not only provides a novel molecular basis to explain TAR-independent transactivation in HIV-1, but also points toward the potential mechanism of Tat- mediated modulation of cellular genes.     

Antibodies against a multiple-peptide conjugate comprising chemically modified human immunodeficiency virus type-1 functional Tat peptides inhibit infection

We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat(21-40)) and arginine-rich (Tat(53-68)) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat(21-40) and Tat(53-68) peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.     

Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization

DNA vaccines have been successful in eliciting potent immune responses in mice. Their efficiency, however, is restricted in larger animals. One reason for the limited performance of the DNA vaccines is the lack of molecular strategies to enhance immune responses. Additionally, genes directly cloned from pathogenic organisms may not be efficiently translated in a heterologous host expression system as a consequence of codon bias. To evaluate the influence of codon optimization on the immune response, we elected to use the Tat antigens of human immunodeficiency virus type 1 (HIV-1) (subtype C) and HIV-2, as these viral antigens are poorly immunogenic in natural infection and in experimental immunization and they are functionally important in viral infectivity and pathogenesis. Substituting codons that are optimally used in the mammalian system, we synthetically assembled Tat genes and compared them with the wild-type counterparts in two different mouse strains. Codon-optimized Tat genes induced qualitatively and quantitatively superior immune responses as measured in a T-cell proliferation assay, enzyme-linked immunospot assay, and chromium release assay. Importantly, while the wild-type genes promoted a mixed Th1-Th2-type cytokine profile, the codon-optimized genes induced a predominantly Th1 profile. Using a pepscan strategy, we mapped an immunodominant T-helper epitope to the core and basic domains of HIV-1 Tat. We also identified cross-clade immune responses between HIV-1 subtype B and C Tat proteins mapped to this T-helper epitope. Developing molecular strategies to optimize the immunogenicity of DNA vaccines is critical for inducing strong immune responses, especially to antigens like Tat. Our identification of a highly conserved T-helper epitope in the first exon of HIV-1 Tat of subtype C and the demonstration of a cross-clade immune response between subtypes B and C are important for a more rational design of an HIV vaccine.     

Immunization with a novel HIV-1-Tat multiple-peptide conjugate induces effective immune response in mice

We report here a novel, highly immunogenic synthetic, multiple-peptide conjugate comprising functional domains Tat_21–40 and Tat_53–68 from HIV-1 group M plus Tat_9–20 from HIV-1 group O of the HIV-Tat protein (HIV-1-Tat-MPC). Vaccination of mice with HIV-1-Tat-MPC induced an effective immune response to all three functional domains. The anti-HIV-1-Tat-MPC antibodies efficiently inhibited Tat-induced viral activation in monocytes infected with HIV_Ba-L as well as with various clinical HIV-1 isolates, and reduced Tat-mediated cytopathicity in infected cells by 60–75%. Our results indicate that anti-HIV-1-Tat-MPC antibodies inhibit viral pathogenesis, possibly by blocking functional determinants of Tat and disrupting autocrine and paracrine actions of secreted Tat protein. This epitope-specific, synthetic Tat construct may, therefore, provide a subunit AIDS vaccine candidate for inducing an effective immunoprophylaxis response to reduce progression of HIV infection.     

The effects of Tat protein on locomotor activity and circadian gene expressions in the mouse hypothalamus

Transactivator (Tat), a regulatory protein of HIV-1, plays a very important role in HIV-1 infection by promoting the rapid replication of HIV. Research surrounding Tat protein function has mainly focused on inhibition of the immune system, promotion of growth of vascular endothelial cells, and nervous system damage. To date, very little research has addressed the role of Tat in circadian rhythms. Previous studies in our lab have found that the concentration of Tat protein in HIV patients’ blood was positively correlated with patients’ sleep quality and melatonin concentrations. In this study, we applied a Tat expression plasmid in mice. Result demonstrated that the locomotor activities of mice and the concentration of melatonin were significantly increased. Alternatively, the expression of Clock gene was markedly decreased. On the other hand, the expression of the Cry1 gene was significantly increased, while the expression of Bmal1 and Per1 genes exhibited no significant difference.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还