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Upon the introduction of the combination antiretroviral therapy (cART), HIV infection has become a chronic disease. However, cART is unable to eradicate the virus and fails to restore the CD4 counts in about 30% of the treated individuals. Furthermore, treatment is life-long, and it does not protect from morbidities typically observed in the elderly. Therapeutic vaccines represent the most cost-effective intervention to intensify or replace cART.Here, we briefly discuss the obstacles to the development and evaluation of the efficacy of therapeutic vaccines and review recent approaches evaluated in clinical trials.Although vaccines were generally safe and immunogenic, evidence of efficacy was negligible or marginal in most trials. A notable exception is the therapeutic Tat vaccine approach showing promising results of cART intensification, with CD4 T-cell increase and proviral load reduction beyond those afforded by cART alone. Rationale and evidence in support of choosing Tat as the vaccine target are thoroughly discussed.

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.

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.

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 &alpha, 5&beta, 1, &alpha, v&beta, 3, and &alpha, 5 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-&alpha, 1, -&alpha, 3, and -&alpha, 5 antibodies. Moreover, modelling&ndash, docking calculations identify a low-energy Tat-&alpha, 3 integrin complex in which Tat makes contacts with both the &alpha, v and &beta, 3 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.

The development of therapeutic strategies to control the reactivation of the Herpes Simplex Virus (HSV) is an unaddressed priority. In this study, we evaluated whether Tat, a HIV-1 protein displaying adjuvant functions, could improve previously established HSV-specific memory responses and prevent viral reactivation. To this aim, mice were infected with non-lethal doses of HSV-1 and, 44 days later, injected or not with Tat. Mice were then monitored to check their health status and measure memory HSV-specific cellular and humoral responses. The appearance of symptoms associated with HSV-reactivation was observed at significantly higher frequencies in the control group than in the Tat-treated mice. In addition, the control animals experienced a time-dependent decrease in HSV-specific Immunoglobulin G (IgG), while the Tat-treated mice maintained antibody titers over time. IgG levels were directly correlated with the number of HSV-specific CD8+ T cells, suggesting an effect of Tat on both arms of the adaptive immunity. Consistent with the maintenance of HSV-specific immune memory, Tat-treated mice showed a better control of HSV-1 re-infection. Although further studies are necessary to assess whether similar effects are observed in other models, these results indicate that Tat exerts a therapeutic effect against latent HSV-1 infection and re-infection by favoring the maintenance of adaptive immunity.

Introduction: Although successful at suppressing HIV replication, combination antiretroviral therapy (cART) only partially restores immune functions and fails to reduce the latent HIV reservoir, th...

Objective:HIV infection is characterized by several immune dysfunctions, such as chronic activation of the immune system, premature aging and loss of CD4(+) T cells, in particular within the naive compartment. The Tat protein of HIV is released extracellularly and enters neighboring cells affecting their functionality, for instance impacting on CD8(+) T-cell programs and activity. As the presence and/or induction of anti-Tat immune responses is associated with reduced T-cell dysfunction and CD4(+) T-cell loss, we investigated whether Tat impacts human resting or activated CD4(+) T cells.Methods:Purified CD4(+) T cells were activated by T cell receptor engagement in the presence or absence of Tat. Cytokine production, surface phenotype and expression of transcription factors important for T-cell programing were measured. Purified naive CD4(+) T cells were cultured in nonpolarizing conditions in the presence or absence of Tat and their proliferation and differentiation was evaluated.Results:Tat favors the secretion of IL2, IFN and TNF in CD4(+) T cells, as well as the upregulation of T-bet and Eomes expression. Naive CD4(+) T cells cultured in the presence of Tat showed enhanced expansion and differentiation toward memory phenotype, showing in particular recruitment into the effector memory T-cell pool.Conclusion:Tat affects the programing and functionality of CD4(+) T lymphocytes favoring the differentiation of naive CD4(+) T cells.

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.

Classical approaches aimed at targeting the HIV-1 envelope as well as other structural viral proteins have largely failed. The HIV-1 transactivator of transcription (Tat) is a key HIV virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. Notably, anti-Tat Abs are uncommon in natural infection and, when present, correlate with the asymptomatic state and lead to lower or no disease progression. Hence, targeting Tat represents a pathogenesis-driven intervention.Here, we review the rationale and the translational development of a therapeutic vaccine targeting the Tat protein. Preclinical and Phase I studies, Phase II trials with Tat in anti-Tat Ab-negative, virologically suppressed highly active antiretroviral therapy-treated subjects in Italy and South Africa were conducted. The results indicate that Tat-induced immune responses are necessary to restore immune homeostasis, to block the replenishment and to reduce the size of the viral reservoir. Additionally, they may help in establishing key parameters for highly active antiretroviral therapy intensification and a functional cure.We propose the therapeutic setting as the most feasible to speed up the testing and comparison of preventative vaccine candidates, as the distinction lies in the use of the vaccine in uninfected versus infected subjects and not in the vaccine formulation.

Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/TatΔ51–59virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA) of A/WSN/33 virus. The TatΔ51–59insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replicationin vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/TatΔ51–59virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines.

Background Although combined antiretroviral therapy (cART) has saved millions of lives, it is incapable of full immune reconstitution and virus eradication. The transactivator of transcription (Tat) protein is a key human immunodeficiency virus (HIV) virulence factor required for virus replication and transmission. Tat is expressed and released extracellularly by infected cells also under cART and in this form induces immune dysregulation, and promotes virus reactivation, entry and spreading. Of note, anti-Tat antibodies are rare in natural infection and, when present, correlate with asymptomatic state and reduced disease progression. This suggested that induction of anti-Tat antibodies represents a pathogenesis-driven intervention to block progression and to intensify cART. Indeed Tat-based vaccination was safe, immunogenic and capable of immune restoration in an open-label, randomized phase II clinical trial conducted in 168 cART-treated volunteers in Italy. To assess whether B-clade Tat immunization would be effective also in patients with different genetic background and infecting virus, a phase II trial was conducted in South Africa. Methods The ISS T-003 was a 48-week randomised, double-blinded, placebo-controlled trial to evaluate immunogenicity (primary endpoint) and safety (secondary endpoint) of B-clade Tat (30 μg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART (randomised 1:1) with CD4+ T-cell counts ≥200 cells/µL. Study outcomes also included cross-clade anti-Tat antibodies, neutralization, CD4+ T-cell counts and therapy compliance. Results Immunization was safe and well-tolerated and induced durable, high titers anti-Tat B-clade antibodies in 97 % vaccinees. Anti-Tat antibodies were cross-clade (all vaccinees tested) and neutralized Tat-mediated entry of oligomeric B-clade and C-clade envelope in dendritic cells (24 participants tested). Anti-Tat antibody titers correlated positively with neutralization. Tat vaccination increased CD4+ T-cell numbers (all participants tested), particularly when baseline levels were still low after years of therapy, and this had a positive correlation with HIV neutralization. Finally, in cART non-compliant patients (24 participants), vaccination contained viral load rebound and maintained CD4+ T-cell numbers over study entry levels as compared to placebo. Conclusions The data indicate that Tat vaccination can restore the immune system and induces cross-clade neutralizing anti-Tat antibodies in patients with different genetic backgrounds and infecting viruses, supporting the conduct of phase III studies in South Africa. Trial registration ClinicalTrials.gov NCT01513135, 01/23/2012 Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0261-1) contains supplementary material, which is available to authorized users.

A workshop entitled: “Revisiting HIV inactivation, elite controllers, immunogenetics and new strategies for developing HIV vaccines” took place during a Eurovaccine Conference held in Rome in June 2016. The purpose of this workshop was to revisit old and new concepts and strategies in HIV vaccinology in the light of novel, and sometimes unexpected, data from recent preventative and therapeutic vaccine approaches that could guide future vaccine research. Panelists were asked to respond to five questions regarding key points and critical issues and problems in current HIV/AIDS vaccine research. Their responses are summarized.

Mauritian cynomolgus macaques (MCM) are widely used in human immunodeficiency virus research because of their restricted major histocompatibility complex (MHC) diversity which provides the opportunity to address the influence of host factors on vaccine studies. We herein report the impact of MHC haplotype on the outcome of 21 MCM infections with the CCR5-tropic simian/human immunodeficiency virus (SHIV)(SF162P4cy). MCM were susceptible to SHIV(SF162P4cy) infection as shown by viremia and loss of CD4+ T cells. A significant association between haplotype M7 (class IA, IB, II) and persistent viremia was observed in chronic phase, whereas recombinant class IA haplotype was associated with a reduction of viral RNA during acute infection. Class IB M4 haplotype displayed significantly lower acute phase provirus copy numbers. In addition, statistical analysis indicated a detrimental effect of haplotype M4 (class IA, IB) on the course of infection as indicated by lower CD4+ T-cell levels during chronic infection. A decrease in post-acute phase CD4+ T-cell numbers was also observed in haplotype M2 animals. This is the first report that documents the effects of host MHC class I and II molecules on the SHIV(SF162P4cy) infection in MCM, particularly with regard to the association between recombinant class IA, M4, and M7 haplotypes and the dynamic of viral replication and level of CD4+ T cells.

HIV native Tat and V2 loop-deleted Env (EnvΔV2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV/AIDS vaccine based on the combination of Tat and EnvΔV2 proteins in cynomolgus macaques against homologous intrarectal challenge with 35 MID(50) (monkey infectious dose 50) of an R5 simian-human immunodeficiency virus (SHIV(SF162P4cy)). Upon challenge, three of four macaques immunized with Tat and EnvΔV2, and two of three monkeys immunized with EnvΔV2 alone were protected from infection. In contrast, all three control animals, which had been either administered with the adjuvants only or left untreated, and an additional monkey immunized with Tat alone became systemically infected. Protection of the macaques vaccinated with EnvΔV2 or Tat/EnvΔV2 correlated with higher peak titers of pre-challenge neutralizing antibodies obtained during the immunization period (between 70 and 3 weeks before challenge) and with anti-Env V3 loop binding antibodies assessed 3 weeks before challenge. Compared to EnvΔV2 alone, the Tat and EnvΔV2 combined vaccine elicited faster antibody responses (IgM) with a trend, early in the vaccination schedule, after the second immunization including EnvΔV2, towards broader anti-Env IgG epitope specificity and a higher ratio of neutralizing to Env-binding antibody titers. As the number of immunizations increased, vaccination with EnvΔV2 approached the immune response assessed after two inocula with the Tat/EnvΔV2 combined vaccine, even though some differences remained between groups, as indicated by anti-Env IgG epitope mapping. In fact, three weeks before challenge, plasma IgG of animals in the EnvΔV2 group showed a trend towards stronger specificity for the V1 loop and V5 loop-C5 regions of Env, whereas the Tat/EnvΔV2 group displayed an overall higher reactivity for epitopes within the Env V3 loop throughout the immunization period. Although differences in terms of protection rate were not found between the EnvΔV2 or Tat/EnvΔV2 vaccination groups in this pilot study, vaccination with Tat/EnvΔV2 appeared to accelerate the induction of potentially protective antibody responses to Env. In particular, antibodies to the Env V3 loop, whose levels at pre-challenge correlated with protection, were already higher early in the vaccination schedule in monkeys immunized with Tat/EnvΔV2 as compared to EnvΔV2 alone. Further studies including larger vaccination groups and fewer immunizations with these two vaccine candidates are needed to confirm these findings and to assess whether the Tat/EnvΔV2 vaccine may afford superior protection against infection.

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated ( n = 67) or not vaccinated ( n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P cy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID 50 ]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load ( P = 0.0280) and CD4 loss ( P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID 50 ( P < 0.0001), and contained acute CD4 loss at 15 MID 50 ( P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility ( P = 0.0199) and resistance ( P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion ( P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.

Protection afforded by HIV Tat-based vaccines has differed in Indian rhesus and Mauritian cynomolgus macaques. We evaluated native Tat and Ad-HIVtat priming/Tat-boosting regimens in both species. Both vaccines were immunogenic. Only the Ad-tat regimen modestly reduced acute viremia in rhesus macaques after SHIV(89.6P) challenge. Confounding variables uncovered in Mauritian macaques included significant associations of susceptibility to infection with MHC class IB and class II H2 and H5 haplotypes, and resistance to infection with class IB haplotypes H3 and H6. Although protection here was limited, Tat-based vaccines incorporating other HIV components have shown greater efficacy. Combination strategies should be further explored.

We have previously shown that the biologically active Tat protein targets and efficiently enters dendritic cells, and increases the proteolytic activities of the immunoproteasome, thereby favoring the generation and presentation of the subdominant MHC-I binding CTL epitopes of heterologous antigens. In the present study, we demonstrate that Tat broadens in vivo epitope-specific T cell responses directed to heterologous antigens including HIV structural proteins. Specifically, co-immunization of mice with OVA and Tat proteins induces CTL responses against subdominant and cryptic OVA-derived epitopes, which are not detected in mice vaccinated with OVA alone. Similarly, mice vaccinated with the HIV-1 Gag, Env or V2-deleted Env antigens in combination with Tat show Th1-type and CTL responses directed to a larger number of T cell epitopes, as compared to mice vaccinated with these proteins in absence of Tat. In contrast, Tat did not affect Th2-type responses to these structural HIV proteins. These results indicate that Tat is not only an antigen but also a novel Th1-type adjuvant capable of broadening in vivo the spectrum of epitopes recognized by T cells, and suggest that Tat can be considered an optimal co-antigen in the development of novel vaccination strategies against AIDS.

Simian-human immunodeficiency virus (SHIV) 89.6P is considered to be one of the most pathogenic chimeric viruses in rhesus macaques. However, when crossing from one to another species of monkeys the pathogenicity of this virus may be affected. By using SHIV-89.6P(cy243), a virus obtained by passaging SHIV-89.6P in cynomolgus macaques, we investigated the dynamics of viral replication and the impact of the inoculum size (from 10 up to 50 monkey infectious dose) on the progression of the infection in 22 cynomolgus macaques. SHIV-89.6P(cy243 )caused massive depletion of CD4+ T-cells within 4 weeks of the inoculum, followed by an irreversible immune deficiency in a high proportion of the infected monkeys. This study demonstrates that SHIV-89.6P(cy243) is pathogenic in cynomolgus macaques and that the dynamics of the viral replication and the rate of clinical progression depend on the size of the inoculum. Our findings provide unique and relevant data, particularly with regard to the value of the in vivo titration used to select the most appropriate infectious dose to study the "virus-host" interplay.

Pre-clinical HIV-1 vaccine protocols, using multiple vaccine modalities and a potent adjuvant were assessed for vaccine efficacy in an experimental HIV-1 challenge model. C57Bl/6 mice were immunized with DNA plasmids encoding HIV-1 gp140, Gag and Tat alone or in combination with the corresponding recombinant proteins formulated in the adjuvant MF59. HIV-1 DNA alone or a DNA prime protein boost schedule resulted in complete protection against challenge with HIV-1/MuLV-infected murine cells. Although HIV-1 protein immunization in combination with MF59 resulted in partial protection, the DNA priming seemed to be crucial for obtaining full protection against the challenge. It is likely that the partial protection seen after immunization with protein alone is, to a certain extent, due to effects of the adjuvant since some animals that received the adjuvant MF59 alone were protected from the challenge. For the most part, antigen-specific cell-mediated immune responses as detected in the spleen (in contrast to responses detected in peripheral blood) of immunized animals appeared to be associated with protection in this study.

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIVmac251. Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV89.6Pchallenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV)tatand boosting with the Tat protein would elicit protection against SHIV89.6P. We also evaluated a Tat/Env regimen, adding an Ad-HIVenvrecombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIVtat, -HIVenv, -SIVgag, and -SIVnefrecombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV89.6Pchallenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P= 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic,P= 0.0003; Tat/Env,P< 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P= 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV89.6P-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.

Mucosal HSV infection remains a public health issue in developing and developed world. However, an effective vaccine is still missing, partly because of the incomplete knowledge of correlates of protection. In this study we have investigated the kinetics and quality of immunity elicited by an attenuated HSV1 vector expressing the immunomodulatory Tat protein of HIV-1 (HSV1-Tat). Animals were immunized by intravaginal (IVag) or intradermal (ID) route with HSV1-Tat or with a control HSV1 vector expressing the LacZ gene (HSV1-LacZ) and immune responses were characterized in different anatomical districts. IVag immunization with HSV1-Tat enhanced both expansion and memory phases of HSV-specific immunodominant CD8 responses at systemic, but not local, level and induced short- and long-term protection against mucosal challenge. Conversely, ID immunization with HSV1-Tat favored HSV-subdominant CD8 responses, which protected mice only at early time points after immunization. IVag immunization, in particular with HSV1-Tat, compared to ID immunization, induced the differentiation of CD8(+) T lymphocytes into short-lived effector (SLEC) and effector memory (Tem) cells, generating more robust recall responses associated with increased control of virus replication. Notably, systemic SLEC and Tem contributed to generate protective local secondary responses, demonstrating their importance for mucosal control of HSV. Finally, IgG responses were observed mostly in IVag HSV1-Tat immunized animals, although seemed dispensable for protection, which occurred even in few IgG negative mice. Thus, HSV1 vectors expressing Tat induce protective anti-HSV1 immune responses.

Background The phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 μg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks. Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia

The use of the Tat protein of HIV in vaccines against AIDS showed promising results in primate and human studies. To characterize the impact of the administration route on the induction of humoral responses at systemic and mucosal levels, we compared intradermal, intramuscular and mucosal immunizations with Tat and a Tat-derived peptide. Mice were immunized with the Tat protein by different routes and the titer and isotype of anti-Tat antibodies were assessed in serum and mucosal lavages. Intramuscular and intradermal administrations showed comparable immunogenicity, while the mucosal administration was unable to induce IgM in serum and IgG at mucosal sites but showed superior immunogenicity in terms of IgA induction. Anti-Tat antibodies were also obtained upon vaccination with the immunodominant Tat 1–20 peptide which was, however, less immunogenic than the whole Tat protein.

Over the past 20 years most of the efforts in HIV vaccinedevelopment have focused on sterilizing immunity bytargeting the Envelope protein (Env). However, resultsfrom preclinical and clinical trials have been largelydisappointing [1–11]. Therefore, current vaccine strat-egies are not only aimed at preventing virus infection butalso at blocking virus replication and disease onset. Inparticular, the control of virus replication should provideprotection from disease development and reduce virustransmission, halting the HIV epidemic. This objectivemay be achieved by targeting virus regulatory genes,which are expressed early after infection, are essential forvirus replication and pathogenesis, and are moreconserved among HIV clades. This approach may beeffective for both preventive and therapeutic vaccinestrategies [12–68]. In this article we review thecharacteristics of Tat and why it was selected for use in avaccine.Wealsocitethelessonlearnedinthedevelopmentof this anti-Tat vaccine for use in human clinical trials.

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.

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.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin–avidin–biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat. Balb/c mice were then immunized with 10g of soluble Tat in complete Freund’s adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5g soluble Tat in incomplete Freund’s adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies. © 2002 Elsevier Science Ltd. All rights reserved.

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.

Many attempts have been made or are ongoing for HIV prevention and HIV cure. Many successes are in the list, particularly for HIV drugs, recently proposed also for prevention. However, no eradication of infection has been achieved so far with any drug. Further, a residual immune dysregulation associated to chronic immune activation and incomplete restoration of B and T cell subsets, together with HIV DNA persistence in reservoirs, are still unmet needs of the highly active antiretroviral therapy (HAART), causing novel “non-AIDS related” diseases that account for a higher risk of death even in virologically suppressed patients. These “ART unmet needs” represent a problem, which is expected to increase by ART roll out. Further, in countries such as South Africa, where 6 millions of individuals are infected, ART appears unable to contain the epidemics. Regretfully, all the attempts at developing a preventative vaccine have been largely disappointing. However, recent therapeutic immunization strategies have opened new avenues for HIV treatment, which might be exploitable also for preventative vaccine approaches. For example, immunization strategies aimed at targeting key viral products responsible of virus transmission, activation and maintenance of virus reservoirs may intensify drug efficacy and lead to a functional cure providing new perspectives also for prevention and future virus eradication strategies. However, this approach imposes new challenges to the scientific community, vaccine developers and regulatory bodies, such as the identification of novel immunological and virological biomarkers to assess efficacy endpoints, taking advantage from the natural history of infection and exploiting lessons from former trials. This review will focus first on recent advancement of therapeutic strategies, then on the progresses made in preventative approaches, discussing concepts and problems for the way ahead for the development of vaccines for HIV treatment and prevention

Background: Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination.Findings: Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization.Conclusions: Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy. © 2014 Bellino et al.; licensee BioMed Central Ltd

Objective HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. Methods CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Results Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Conclusion Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

Little is known about the effects of Major Histocompatibility Complex (MHC) haplotypes on immunity to primate lentiviruses involving both acquired and innate immune responses. We present statistical evidence of the influence of MHC polymorphism on antiviral immunity of Mauritian cynomolgus macaques (MCM) following simian/human immunodeficiency virus SHIVSF162P4cy infection, involving the production of pro- and anti-inflammatory cytokines and α-defensins, which may modulate acquired immune responses. During the acute phase of infection, IL-10 correlated positively with viral load and negatively with CD4+T cell counts. Furthermore, α-defensins production was directly correlated with plasma viral RNA, particularly at peak of viral load. When the effects of the MHC were analyzed, a significant association between lower anti-Env binding and neutralizing antibody levels with class IB M4 haplotype and with class IA, IB M4 haplotype, respectively, was observed in the post-acute phase. Lower antibody responses may have resulted into a poor control of infection thus explaining the previously reported lower CD4 T cell counts in these monkeys. Class II M3 haplotype displayed significantly lower acute and post-acute IL-10 levels. In addition, significantly lower levels of α-defensins were detected in class IA M3 haplotype monkeys than in non-M3 macaques, in the post-acute phase of infection. These data indicate that the MHC could contribute to the delicate balance of pro-inflammatory mechanisms, particularly with regard to the association between IL-10 and α-defensins in lentivirus infection. Our results show that host genetic background, virological and immunological parameters should be considered for the design and interpretation of HIV-1 vaccine efficacy studies.

Objective:The identification of still unrevealed mechanisms affecting the anti-HIV CD8(+) T-cell response in HIV-1 infection.Design:Starting from the observation that anti-Tat immunization is associated with improved CD8(+) T-cell immunity, we developed both in-vitro and ex-vivo assays to characterize the effects of extra-cellular Tat on the adaptive CD8(+) T-cell response.Methods:The effects of Tat on CD8(+) T-cell activation were assayed using CD8(+) T-cell clones specific for either cellular (MART-1) or viral (HIV-1 Nef) antigens, and HIV-1 Gag-specific CD8(+) T cells from HIV-1 patients.Results:The interaction between CD8(+) T lymphocytes and immobilized Tat, but not its soluble form, inhibits peptide-specific CD8(+) T-lymphocyte activation. The inhibition does not depend on Tat trans-activation activity, but on the interaction of the Tat RGD domain with 51 and v3 integrins. Impaired CD8(+) T-cell activation was also observed in cocultures of CD8(+) T cells with HIV-1-infected cells. Anti-Tat Abs abrogate the inhibitory effect, consistently with the evidence that extracellular Tat accumulates on the cell membrane of virus-producing cells. The Tat-induced inhibition of cell activation associates with increased apoptosis of CD8(+) T cells. Finally, the inhibition of cell activation also takes place in Gag-specific CD8(+) T lymphocytes from HIV-1-infected patients.Conclusion:Our results support the idea that CD8(+) T-cell apoptosis induced by surface-bound extracellular Tat can contribute to the dysregulation of the CD8(+) T-cell adaptive response against HIV as well as other pathogens present in AIDS patients.

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6P(cy243), vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4(+) T cell depletion rate during the acute phase of infection and higher ability to resume the CD4(+) T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.

Natural killer (NK) cell recognition and function in humans is regulated by multiple cell surface receptors. The "on" signal leading to NK cell triggering is primarily mediated by natural cytotoxicity receptors (NCR). Analysis of NK cells in primate animal models is of particular relevance because NK cells may play an essential role in host defenses against infections. We analyzed Macaca fascicularis PBMC and in vitro-derived NK cell populations and clones by cytofluorometry, using a wide panel of mAb, and by cytolytic activity assays. In addition, RT-PCR strategy and transient transfections were used to isolate M. fascicularis NCR. NCR-specific mAb reactivity (anti-NKp46 and anti-NKp30) was present on M. fascicularis PBMC and on NK cell cultures. Macaque NCR were functional in both redirected killing and in mAb-mediated masking assays. Cloning of macNKp46 and macNKp30 NCR homologous genes showed a high sequence similarity (86 % and 88 %, respectively) with their human counterparts. Attempts at identifying NKp44 surface reactivity and at cloning the macaque homologue were unsuccessful. NKp46 and NKp30 NCRs, but not NKp44, are highly conserved in M. fascicularis NK cells. This suggests the possibility of a staged appearance of the NCR during phylogenesis and provides a useful tool for the study of natural immunity correlates of protection in primate SIV/SHIV infection models.

Various simian immunodeficiency virus (SIV)sm/mac and simian/human immunodeficiency virus (SHIV) strains are used in different macaque species to study AIDS pathogenesis, as well as to evaluate candidate vaccine and anti-retroviral drugs efficacy. In this study we investigated the effect of route of infection, species of macaques and nature of virus stock on early plasma viral RNA load. We monitored the plasma RNA concentrations of 63 rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) infected with well-characterised virus stocks administered either by oral, rectal, vaginal or intravenous (i.v.) routes. In SIV(mac)-infected macaques, no significant difference in plasma RNA loads was observed between the rectal, oral and i.v. routes of infection. Cynomolgus macaques developed lower steady state SIV plasma RNA concentrations compared with rhesus macaques and no significant difference was observed between rectal and i.v. routes of infection. In SHIV(89.6p)-infected macaques, no difference between species or between route of infection was observed with this particular chimeric virus.

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.