Engineered dendritic cells-derived exosomes harboring HIV-1 Nefmut-Tat fusion protein and heat shock protein 70: A promising HIV-1 safe vaccine candidate.

Antigen presenting cells (APCs)-derived exosomes are nano-vesicles that can induce antigen-specific T cell responses, and possess therapeutic effects in clinical settings. Moreover, dendritic cells (DCs)-based vaccines have been developed to combat human immunodeficiency virus-1 (HIV-1) infection in preclinical and clinical trials. We investigated the immunostimulatory effects (B- and T-cells activities) of DCs- and exosomes-based vaccine constructs harboring HIV-1 Nefmut-Tat fusion protein as an antigen candidate and heat shock protein 70 (Hsp70) as an adjuvant in mice. The modified DCs and engineered exosomes harboring Nefmut-Tat protein or Hsp70 were prepared using lentiviral vectors compared to electroporation, characterized and evaluated by in vitro and in vivo immunological tests. Our data indicated that the engineered exosomes induced high levels of total IgG, IgG2a, IFN-γ, TNF-α and Granzyme B. Moreover, co-injection of exosomes harboring Hsp70 could significantly increase the secretion of antibodies, cytokines and Granzyme B. The highest levels of IFN-γ and TNF-α were observed in exosomes harboring Nefmut-Tat combined with exosomes harboring Hsp70 (Exo-Nefmut-Tat + Exo-Hsp70) regimen after single-cycle replicable (SCR) HIV-1 exposure. Generally, Exo-Nefmut-Tat + Exo-Hsp70 regimen can be considered as a promising safe vaccine candidate due to high T-cells (Th1 and CTL) activity and its maintenance against SCR HIV-1 exposure.

New insights into pathogenesis point to HIV-1 Tat as a key vaccine target.

Despite over 30 years of enormous effort and progress in the field, no preventative and/or therapeutic vaccines against human immunodeficiency virus (HIV) are available. Here, we briefly summarize the vaccine strategies and vaccine candidates that in recent years advanced to efficacy trials with mostly unsatisfactory results. Next, we discuss a novel and somewhat contrarian approach based on biological and epidemiological evidence, which led us to choose the HIV protein Tat for the development of preventive and therapeutic HIV vaccines. Toward this goal, we review here the role of Tat in the virus life cycle as well as experimental and epidemiological evidence supporting its key role in the natural history of HIV infection and comorbidities. We then discuss the preclinical and clinical development of a Tat therapeutic vaccine, which, by improving the functionality and homeostasis of the immune system and by reducing the viral reservoir in virologically suppressed vaccinees, helps to establish key determinants for intensification of combination antiretroviral therapy (cART) and a functional cure. Future developments and potential applications of the Tat therapeutic vaccine are also discussed, as well as the rationale for its use in preventative strategies. We hope this contribution will lead to a reconsideration of the current paradigms for the development of HIV/AIDS vaccines, with a focus on targeting of viral proteins with key roles in HIV pathogenesis.

HIV-1 Tat: An update on transcriptional and non-transcriptional functions.

Over the past decades, much have been learned about HIV-1 virus and its molecular strategies for pathogenesis. However, HIV-1 still remains an enigmatic virus, particularly because of its unique proteins. Establishment of latency and reactivation is still a puzzling question and various temporal and spatial dynamics between HIV-1 proteins itself have given us new way of thinking about its pathogenesis. HIV-1 replication depends on Tat which is a small unstructured protein and subjected to various post-translational modifications for its myriad of functions. HIV-1 Tat protein modulates the functions of various strategic cellular pathways like proteasomal machinery and inflammatory pathways to aid in HIV-1 pathogenesis. Many of the recent findings have shown that Tat is associated with exosomes, cleared from HIV-1 infected cells through its degradation by diverse routes ranging from lysosomal to proteasomal pathways. HIV-1 Tat was also found to be associated with other HIV-1 proteins including Vpr, Nef, Nucleocapsid (NC) and Rev. Interaction of Tat with Vpr and Nef increases its transactivation function, whereas, interaction of Tat with NC or Rev leads to Tat protein degradation and hence suppression of Tat functions. Research in the recent years has established that Tat is not only important for HIV-1 promoter transactivation and virus replication but also modulating multiple cellular and molecular functions leading to HIV-1 pathogenicity. In this review we discussed various transcriptional and non-transcriptional HIV-1 Tat functions which modulate host cell metabolism during HIV-1 pathogenesis.

Anti-Tat immunity defines CD4+ T-cell dynamics in people living with HIV on long-term cART.

BACKGROUND: Low-level HIV viremia originating from virus reactivation in HIV reservoirs is often present in cART treated individuals and represents a persisting source of immune stimulation associated with sub-optimal recovery of CD4+ T cells. The HIV-1 Tat protein is released in the extracellular milieu and activates immune cells and latent HIV, leading to virus production and release. However, the relation of anti-Tat immunity with residual viremia, persistent immune activation and CD4+ T-cell dynamics has not yet been defined. METHODS: Volunteers enrolled in a 3-year longitudinal observational study were stratified by residual viremia, Tat serostatus and frequency of anti-Tat cellular immune responses. The impact of anti-Tat immunity on low-level viremia, persistent immune activation and CD4+ T-cell recovery was investigated by test for partitions, longitudinal regression analysis for repeated measures and generalized estimating equations. FINDINGS: Anti-Tat immunity is significantly associated with higher nadir CD4+ T-cell numbers, control of low-level viremia and long-lasting CD4+ T-cell recovery, but not with decreased immune activation. In adjusted analysis, the extent of CD4+ T-cell restoration reflects the interplay among Tat immunity, residual viremia and immunological determinants including CD8+ T cells and B cells. Anti-Env immunity was not related to CD4+ T-cell recovery. INTERPRETATION: Therapeutic approaches aiming at reinforcing anti-Tat immunity should be investigated to improve immune reconstitution in people living with HIV on long-term cART. TRIAL REGISTRATION: ISS OBS T-002 ClinicalTrials.gov identifier: NCT01024556 FUNDING: Italian Ministry of Health, special project on the Development of a vaccine against HIV based on the Tat protein and Ricerca Corrente 2019/2020.

The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

Physicochemical studies on the structural stability of the HIV-1 vaccine candidate recombinant Tat protein.

HIV-1 transactivator of transcription protein is one of the most promising AIDS vaccine candidates and plays central roles in the virus life cycle and pathogenesis. Understanding structural properties of vaccine candidate antigens leads to rational design of vaccines which improves their presentation to immune system and facilitates their manufacturing and storage. This study aims to investigate structural properties and stability of one variant of HIV-1 Tat recombinant protein using different spectroscopic, electrophoretic, and microscopic methods. Therefore, after the gene transformation, protein expression was optimized in E. coli cells and the C-terminal His6-tagged protein was purified using Ni-NTA resin. The structural stability of the pure protein was then investigated under different conditions including pH, Zn2+ ions, thermal and chemical stress. Acidic and alkaline pHs affects spectroscopic properties of the vaccine in different ways. The structure unfolding experiment shows relatively poor stability of the zinc-free protein sample compared to the ion-containing one. According to the quenching experiment and also thermal stability study results, the protein has attained more structural compactness in the presence of Zn2+. Secondary structure of the protein is mainly disordered and didn't significantly affect under various conditions. Finally, different degrees of oligomerization and aggregation were found under physiological conditions.

Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes.

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40-70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/ß-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit ß-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to ß-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of ß-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of ß-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect ß-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development.

Boosting Tat DNA vaccine with Tat protein stimulates strong cellular and humoral immune responses in mice.

The aim of the present study was to evaluate the efficacy of a novel DNA priming-protein boosting regimen in simultaneous enhancing humoral and cellular immunogenicity of the HIV-1-Tat-based candidate vaccines in mice. The experimental BALB/c mice were successfully immunized with the HIV-1-Tat DNA vaccine and boosted with the corresponding protein vaccine over a two-week interval and the elicitation of cellular and humoral immune responses were simultaneously assessed. The results showed that the prime-boost immunization has significantly given rise to lymphocyte proliferation and CTL responses, as well as the levels of both IgG and IgG antibodies compared to the other candidate vaccines. The results of the Th polarization also revealed that the Th1: Th2 indexes in the mice vaccinated with the HIV-1 Tat protein, Tat DNA, and the prime-boost vaccines were 1.03, 1.19, and 1.25, respectively. The results suggest that co-administration of the HIV-1-Tat DNA with the corresponding protein may serve as a potential formulation for enhancing of Tat vaccineinduced immunity and has measurable effects on shaping vaccines' induced Th polarization.

HIV-1 Tat protein attenuates the clinical course of experimental autoimmune encephalomyelitis (EAE).

A growing body of evidence has shown that the human immunodeficiency virus (HIV) infection is associated with a significantly decreased risk of developing multiple sclerosis (MS) in patients with acquired immunodeficiency virus (AIDS). It is thought that two mechanisms are in charge of protection against MS, which include immunosuppression induced by chronic HIV infection (depletion of CD4 + T cells) and antiretroviral medications. HIV-1 encodes several regulatory (Tat and Rev) and accessory (Vpr, Vif, Vpu, and Nef) proteins that have immunosuppressive and immunomodulatory properties. HIV-1 Tat protein is a strongly immunosuppressive agent and can cross the blood-brain barrier (BBB). In this study, we examined the effect of HIV-1 Tat, which is classified into clade B and C, on inflammation, gliosis, apoptosis, and behavioral function in a murine model of MS called experimental autoimmune encephalomyelitis (EAE). For this aim, mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), followed by pertussis toxin to induce paralysis in EAE mice. After the induction of EAE in mice, the animals intraperitoneally received serial doses of HIV-1 Tat clade B and C (5, 10, and 20 µg/kg body weight) when the early clinical manifestations of EAE were initiated. The results showed that the administration of both clades of the Tat protein led to a marked decrease in the clinical score of EAE mice, as well as improvement in motor-neuron functions. In line with this, Tat considerably reduced the number of apoptotic cells in the sacral region of the spinal cord through the upregulation expression of the Bcl-2 protein. Besides, proinflammatory cytokines such as, IFN-γ, TNF-α, IL-6, and IL-17 were significantly diminished in the serum and spinal cord of EAE mice receiving HIV-1 Tat clade B and C. Conversely, anti-inflammatory cytokines, including IL-10 and IL-4 were elevated in the serum and spinal cord of EAE mice receiving HIV Tat clade B and C when compared with the control group. The immunohistochemical analysis indicated that HIV-1 Tat clade B and C mitigated microgliosis and astrogliosis. The flow cytometry analysis demonstrated that the number of Th1 and Th17cells was significantly decreased in response to TAT administration while the frequency of Th2 cells was markedly increased in the peripheral blood of mice with EAE without influencing the number of T regulatory cells (CD4 + CD25 + forkhead box protein 3 + ). It seems that HIV-1 Tat could be a bona fide therapeutic protein for the alleviation of MS since it has beneficial roles in the suppression of neuroinflammation in MS pathology.

HIV-1 Tat protein inhibits the hematopoietic support function of human bone marrow mesenchymal stem cells.

Most HIV-1-infected patients experience hematopoiesis suppression complications. Bone marrow mesenchymal stem cells (BMSCs) are involved in regulation of hematopoietic homeostasis, so we investigated the role of Tat, a protein released by infected cells in bone marrow and impacted differentiation potential of mesenchymal stem cells, in the BMSC hematopoietic support function. BMSCs were treated with HIV-1 Tat protein (BMSCTat-p), transfected with HIV-1 Tat mRNA (BMSCTat-m) or treated with solvent (PBS) (BMSCcon) for 20 days. Then, the hematopoietic support function of BMSCTat-p, BMSCTat-m and BMSCcon was analyzed via ex vivo expansion of hematopoietic stem cells (HSCs) grown on the BMSCs and via in vivo cotransplantation of HSCs and BMSCs. In addition, the hematopoiesis-supporting gene expression patterns of BMSCTat-p, BMSCTat-m and BMSCcon were compared. The results showed that BMSCTat-p and BMSCTat-m displayed reduced expansion, a decline in the number of colony forming units (CFUs) and a decreased proportion of the primitive subpopulation of hematopoietic stem cells under coculture conditions compared with BMSCcon. The ability of BMSCTat-p to support hematopoietic recovery was also impaired, which was further confirmed by the patterns in gene expression analysis. In conclusion, Tat treatment reduced the function of BMSCs in hematopoietic support, likely by downregulating the expression of a series of hematopoietic cytokines.

Continued Decay of HIV Proviral DNA Upon Vaccination With HIV-1 Tat of Subjects on Long-Term ART: An 8-Year Follow-Up Study.

Introduction: Tat, a key HIV virulence protein, has been targeted for the development of a therapeutic vaccine aimed at cART intensification. Results from phase II clinical trials in Italy (ISS T-002) and South Africa (ISS T-003) indicated that Tat vaccination promotes increases of CD4+ T-cells and return to immune homeostasis while reducing the virus reservoir in chronically cART-treated patients. Here we present data of 92 vaccinees (59% of total vaccinees) enrolled in the ISS T-002 8-year extended follow-up study (ISS T-002 EF-UP, ClinicalTrials.gov NCT02118168). Results: Anti-Tat antibodies (Abs) induced upon vaccination persisted for the entire follow-up in 34/92 (37%) vaccinees, particularly when all 3 Ab classes (A/G/M) were present (66% of vaccinees), as most frequently observed with Tat 30 µg regimens. CD4+ T cells increased above study-entry levels reaching a stable plateau at year 5 post-vaccination, with the highest increase (165 cells/µL) in the Tat 30 µg, 3 × regimen. CD4+ T-cell increase occurred even in subjects with CD4+ nadir ≤ 250 cells/uL and in poor immunological responders and was associated with a concomitant increase of the CD4+/CD8+ T-cell ratio, a prognostic marker of morbidity/mortality inversely related to HIV reservoir size. Proviral DNA load decreased over time, with a half-life of 2 years and an estimated 90% reduction at year 8 in the Tat 30 µg, 3 × group. In multivariate analysis the kinetic and amplitude of both CD4+ T-cell increase and proviral DNA reduction were fastest and highest in subjects with all 3 anti-Tat Ab classes and in the 30 µg, 3 × group, irrespective of drug regimens (NNRTI/NRTI vs. PI). HIV proviral DNA changes from baseline were inversely related to CD4+/CD8+ T-cell ratio and CD4+ T-cell changes, and directly related to the changes of CD8+ T cells. Further, HIV DNA decay kinetics were inversely related to the frequency and levels of intermittent viremia. Finally, Tat vaccination was similarly effective irrespective of the individual immunological status or HIV reservoir size at study entry. Conclusions: Tat immunization induces progressive immune restoration and reduction of virus reservoirs above levels reached with long-term cART, and may represent an optimal vaccine candidate for cART intensification toward HIV reservoirs depletion, functional cure, and eradication strategies.

Synaptic transmission may provide an evolutionary benefit to HIV through modulation of latency.

Transmission of HIV is known to occur by two mechanisms in vivo: the free virus pathway, where viral particles bud off an infected cell before attaching to an uninfected cell, and the cell-cell pathway, where infected cells form virological synapses through close contact with an uninfected cell. It has also been shown that HIV replication includes a positive feedback loop controlled by the viral protein Tat, which may act as a stochastic switch in determining whether an infected cell enters latency. In this paper, we introduce a simple mathematical model of HIV replication containing both the free virus and cell-cell pathways. Using this model, we demonstrate that the high multiplicity of infection in cell-cell transmission results in a suppression of latent infection, and that this modulation of latency through balancing the two transmission mechanisms can provide an evolutionary benefit to the virus. This benefit increases with decreasing overall viral fitness, which may provide a within-host evolutionary pressure toward more cell-cell transmission in late-stage HIV infection.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

Hindsiipropane B alleviates HIV-1 Tat-induced inflammatory responses by suppressing HDAC6-NADPH oxidase-ROS axis in astrocytes.

Human immunodeficiency virus-1 (HIV-1) transactivator of transcription (Tat) is an important viral factor in neuroinflammation. Hindsiipropane B, present in Celastrus hindsii, possesses various biological mechanisms including antiinflammatory activity. In this report, we explored the regulatory activity of hindsiipropane B on HIV-1 Tat-mediated chemokine production and its mode of action in astrocytes. Hindsiipropane B significantly alleviated HIV-1 Tat-mediated production of inflammatory chemokines, CCL2, CXCL8, and CXCL10. Hindsiipropane B inhibited expression of HDAC6, which is important regulator in HIV-1 Tat-mediated chemokine production. Hindsiipropane B diminished HIV-1 Tat-mediated reactive oxygen species (ROS) generation and NADPH oxidase activation/expression. Furthermore, hindsiipropane B inhibited HIV-1 Tat-mediated signaling cascades including MAPK, NF-κB, and AP-1. These data suggest that hindsiipropane B exerts its inhibitory effects on HIV-1 Tat-mediated chemokine production via down-regulating the HDAC6-NADPH oxidase-MAPK-NF-κB/AP-1 signaling axis, and could serve as a therapeutic lead compound against HIV-1 Tat-associated neuroinflammation. [BMB Reports 2018; 51(8): 394-399].

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

BACKGROUND: DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. METHODS: In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). RESULTS: We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. CONCLUSION: These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Proceedings of the 2017 ISEV symposium on "HIV, NeuroHIV, drug abuse, & EVs".

Despite the success of combination antiretroviral therapy (cART), there is increased prevalence of HIV-associated neurocognitive disorders (HAND) in HIV-1-infected individuals on cART, which poses a major health care challenge. Adding further complexity to this long-term antiretroviral use is the comorbidity with drugs of abuse such as morphine, cocaine, and methamphetamine, which can in turn, exacerbate neurologic and cognitive deficits associated with HAND. Furthermore, HIV proteins, such as the transactivator of transcription (Tat) and the envelope protein (gp120), as well as antiretrovirals themselves can also contribute to the progression of neurodegeneration underlying HAND. In the field of NeuroHIV and drug addiction, EVs hold the potential to serve as biomarkers of cognitive dysfunction, targets of therapy, and as vehicles for therapeutic delivery of agents that can ameliorate disease pathogenesis. Based on the success of a previous Satellite Symposium in 2015 at the ISEV meeting in Washington, experts again expanded on their latest research findings in the field, shedding light on the emerging trends in the field of Extracellular Vesicle (EV) biology in NeuroHIV and drug abuse. The satellite symposium sought to align experts in the fields of NeuroHIV and drug abuse to share their latest insights on the role of EVs in regulating neuroinflammation, neurodegeneration, peripheral immune response, and HIV latency in HIV-infected individuals with or without the comorbidity of drug abuse.

HIV and HIV-Tat inhibit LPS-induced IL-27 production in human macrophages by distinct intracellular signaling pathways.

Monocyte-derived Mϕs (MDMs) from HIV-infected patients and MDM infected in vitro with HIV exhibit a reduced ability to secrete various cytokines, including IL-12. Recently, IL-27, an IL-12 family cytokine, was shown to inhibit HIV replication in Mϕ. Whether HIV infection or HIV accessory protein(s) impact IL-27 production in Mϕs remains unknown. Herein, we show that in vitro HIV infection, as well as intracellular HIV-Tat (Tat) and Tat peptides, inhibit LPS-induced IL-27 production in human MDMs, suggesting impairment of the TLR4 signaling pathway. To understand the signaling pathways governing HIV or Tat-mediated inhibition of LPS-induced IL-27 production, we first demonstrated that p38 MAPK, PI3K, Src-homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1), and Src kinases regulate LPS-induced IL-27 production in MDMs. Tat caused down-regulation of TNFR-associated factor (TRAF)-6 and inhibitor of apoptosis 1 (cIAP-1) and subsequently decreased phosphorylation of downstream PI3K and p38 MAPKs, which were implicated in LPS-induced IL-27 production. Whereas SHP-1 and Src kinases regulated LPS-induced IL-27 production, Tat did not inhibit these kinases, suggesting that they were not involved in Tat-mediated inhibition of LPS-induced IL-27 production. In contrast to Tat, in vitro HIV infection of MDM inhibited LPS-induced IL-27 production via inhibition of p38 MAPK activation. Overall, HIV and Tat inhibit LPS-induced IL-27 production in human macrophages via distinct mechanisms: Tat through the inhibition of cIAP-1-TRAF-6 and subsequent inhibition of PI3K and p38 MAPKs, whereas HIV through the inhibition of p38 MAPK activation.

Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes.

Histone deacetylase 6 (HDAC6) likely is important in inflammatory diseases. However, how HDAC6 exerts its effect on inflammatory processes remains unclear. HIV-1 transactivator of transcription (Tat) activates NADPH oxidase resulting in generation of reactive oxygen species (ROS), leading to extensive neuro-inflammation in the central nervous system. We investigated the correlation of HDAC6 and NADPH oxidase in HIV-1 Tat-stimulated astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced ROS generation and NADPH oxidase activation. HDAC6 knockdown suppressed HIV-1 Tat-induced expression of NADPH oxidase subunits, such as Nox2, p47phox, and p22phox. Specific inhibition of HDAC6 using tubastatin A suppressed HIV-1 Tat-induced ROS generation and activation of NADPH oxidase. N-acetyl cysteine, diphenyl iodonium, and apocynin suppressed HIV-1 Tat-induced expression of HDAC6 and the pro-inflammatory chemokines CCL2, CXCL8, and CXCL10. Nox2 knockdown attenuated HIV-1 Tat-induced HDAC6 expression and subsequent expression of chemokines. The collective results point to the potential crosstalk between HDAC6 and NADPH oxidase, which could be a combined therapeutic target for relief of HIV-1 Tat-mediated neuro-inflammation.

Human Rhinovirus-A1 as an Expression Vector.

Expression vectors that are based on live human rhinoviruses (HRVs) are attractive, yet often overlooked in vaccine development due to their limited capacity for foreign gene inserts and poor genetic stability. This chapter describes a novel methodology to engineer a replication-competent genetically stable recombinant HRV (rHRV) without affecting viral replication capability. We have previously used these methods to generate live, genetically stable recombinant HRVs encoding HIV Gag and Tat proteins (rHRV-Gag-Tat), a potential mucosally targeted HIV vaccine.

HIV-1 Tat Primes and Activates Microglial NLRP3 Inflammasome-Mediated Neuroinflammation.

Neuroinflammation associated with HIV-1 infection is a problem affecting ∼50% of HIV-infected individuals. NLR family pyrin domain containing 3 (NLRP3) inflammasome has been implicated in HIV-induced microglial activation, but the mechanism(s) remain unclear. Because HIV-1 Transactivator of Transcription (Tat) protein continues to be present despite antiretroviral therapy and activates NF-kB, we hypothesized that Tat could prime the NLRP3 inflammasome. We found a dose- and time-dependent induction of NLRP3 expression in microglia exposed to Tat compared with control. Tat exposure also time-dependently increased the mature caspase-1 and IL-1ß levels and enhanced the IL-1ß secretion. These in vitro findings were validated in archival brain tissues from Simian Immunodeficiency Virus (SIV)-infected and uninfected rhesus macaques. Further validation of NLRP3 priming in vivo involved administration of lipopolysaccharide (LPS) to HIV transgenic (Tg) rats followed by assessment of IL-1ß mRNA expression and inflammasome activation (ASC oligomers and mature IL-1ß). Intriguingly, LPS potentiated upregulation of IL-1ß mRNA and inflammasome activation in HIV-Tg rats compared with the wild-type controls. Interestingly, we found an inverse relationship in the expression of NLRP3 and its negative regulator, miR-223, suggesting a miR-223-mediated mechanism for Tat-induced NLRP3 priming. Furthermore, blockade of NLRP3 resulted in decreased IL-1ß secretion. Collectively, these findings suggest a novel role of Tat in priming and activating the NLRP3 inflammasome. Therefore, NLRP3 can be envisioned as a therapeutic target for ameliorating Tat-mediated neuroinflammation.SIGNIFICANCE STATEMENT Despite successful suppression of viremia with increased longevity in the era of combined antiretroviral therapy, chronic inflammation with underlying neurocognitive impairment continues to afflict almost 50% of infected individuals. Viral, bacterial, and cellular products have all been implicated in promoting the chronic inflammation found in these individuals. Understanding the molecular mechanism(s) by which viral proteins such as HIV-1 Transactivator of Transcription (Tat) protein can activate microglia is thus of paramount importance. Herein, we demonstrate a novel role of Tat in priming and activating NLR family pyrin domain containing 3 (NLRP3) inflammasomes in microglial cells and in HIV-Tg rats administered lipopolysaccharide. Targeting NLRP3 inflammasome pathway mediators could thus be developed as therapeutic interventions to alleviate or prevent neuroinflammation and subsequent cognitive impairment in HIV-positive patients.