Role of HIV-1 Tat Protein Interactions with Host Receptors in HIV Infection and Pathogenesis.

Each time the virus starts a new round of expression/replication, even under effective antiretroviral therapy (ART), the transactivator of viral transcription Tat is one of the first HIV-1 protein to be produced, as it is strictly required for HIV replication and spreading. At this stage, most of the Tat protein exits infected cells, accumulates in the extracellular matrix and exerts profound effects on both the virus and neighbor cells, mostly of the innate and adaptive immune systems. Through these effects, extracellular Tat contributes to the acquisition of infection, spreading and progression to AIDS in untreated patients, or to non-AIDS co-morbidities in ART-treated individuals, who experience inflammation and immune activation despite virus suppression. Here, we review the role of extracellular Tat in both the virus life cycle and on cells of the innate and adaptive immune system, and we provide epidemiological and experimental evidence of the importance of targeting Tat to block residual HIV expression and replication. Finally, we briefly review vaccine studies showing that a therapeutic Tat vaccine intensifies ART, while its inclusion in a preventative vaccine may blunt escape from neutralizing antibodies and block early events in HIV acquisition.

HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication.

Previous work has shown that the Tat protein of Human Immunodeficiency Virus (HIV)-1 is released by acutely infected cells in a biologically active form and enters dendritic cells upon the binding of its arginine-glycine-aspartic acid (RGD) domain to the α5ß1, αvß3, and αvß5 integrins. The up-regulation/activation of these integrins occurs in endothelial cells exposed to inflammatory cytokines that are increased in HIV-infected individuals, leading to endothelial cell dysfunction. Here, we show that inflammatory cytokine-activated endothelial cells selectively bind and rapidly take up nano-micromolar concentrations of Tat, as determined by flow cytometry. Protein oxidation and low temperatures reduce Tat entry, suggesting a conformation- and energy-dependent process. Consistently, Tat entry is competed out by RGD-Tat peptides or integrin natural ligands, and it is blocked by anti-α5ß1, -αvß3, and -αvß5 antibodies. Moreover, modelling-docking calculations identify a low-energy Tat-αvß3 integrin complex in which Tat makes contacts with both the αv and ß3 chains. It is noteworthy that internalized Tat induces HIV replication in inflammatory cytokine-treated, but not untreated, endothelial cells. Thus, endothelial cell dysfunction driven by inflammatory cytokines renders the vascular system a target of Tat, which makes endothelial cells permissive to HIV replication, adding a further layer of complexity to functionally cure and/or eradicate HIV infection.

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.

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.

Control of human herpes virus type 8-associated diseases by NK cells.

The "natural killer" (NK) cells preferentially kill targets lacking surface major histocompatibility complex class I (MHC-I) molecule expression. NK cells recognize these targets through membrane receptors, which can trigger activating or inhibitory signals for killing. Several tumors or virus-infected cells downregulate MHC-I expression as a mechanism to evade recognition and killing by cytotoxic T lymphocytes (CTL). They, however, become targets for NK cells cytotoxic activity. NK cell activity is reduced during disease progression in human immunodeficiency virus (HIV) infection, and in individuals with AIDS-associated tumors linked with infection by the oncogenic human herpes virus type-8 (HHV8), including Kaposi's sarcoma (KS) and primary effusion lymphomas (PEL). We have demonstrated that AIDS-related KS (AIDS-KS) is characterized by an increased expression of inhibitory receptors by T lymphocytes, and that HIV-non-infected patients with KS (classic KS, C-KS) have a substantial number of NK cells bearing these same receptors. NK cells from patients with C-KS are normally equipped with cytolytic molecules including granzyme A and perforin. However, the cytotoxic activity of NK cells is reduced in patients with C-KS, AIDS-KS, or PEL patients, who are all infected by the HHV8, and this correlates with disease severity. Moreover, we have found that HHV8-infected cell lines established from PELs have a reduced surface expression of MHC-I molecules and are sensitive to the lysis mediated by NK cells. Since PEL cells express the same HHV8 latency program as KS cells, these data point to MHC-I downregulation by HHV8 as a primary immune evasion mechanism against CTL responses, further reinforced by upregulation of inhibitory receptors on T and NK cells in the setting of HIV and/or HHV8 infection. Thus, studies on killing receptor regulation and signaling in T and NK cells may shed light on the pathogenesis of HHV8-associated tumors both in HIV-infected or -noninfected patients.

Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART.

UNLABELLED: Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+) T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+) and CD8(+) cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+) T cells and B cells with reduction of CD8(+) T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+) and CD8(+) T cells were accompanied by increases of CD4(+) and CD8(+) T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the OBS population. These findings support the use of Tat immunization to intensify HAART efficacy and to restore immune homeostasis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00751595.

Downregulation of the major histocompatibility complex class I molecules by human herpesvirus type 8 and impaired natural killer cell activity in primary effusion lymphoma development.

Primary effusion lymphomas (PELs) are invariably infected by human herpesvirus type 8 (HHV8) and often co-infected by Epstein-Barr virus (EBV). We found that expression of major histocompatibility complex class I (MHC-I) surface molecules was significantly decreased in PEL cells when compared with HHV8 negative lymphomas, irrespective of EBV infection. MHC-I downregulation rendered PEL cells sensitive to recognition and killing by natural killer (NK) cells. Intriguingly, analysis of MHC-I non-restricted cytotoxicity in two PEL patients indicated a reduced NK cell activity when compared with healthy individuals. These data suggest that PEL outgrowth may require an impaired NK cell function.