Review

Development of a novel AIDS vaccine: the HIV-1 transactivator of transcription protein vaccine 1.

Introduction

2.

The role of Tat in HIV-1 pathogenesis

3.

The protective role of the

Aurelio Cafaro, Antonella Tripiciano, Cecilia Sgadari, Stefania Bellino, Orietta Picconi, Olimpia Longo, Vittorio Francavilla, Stefano Butto`, Fausto Titti, Paolo Monini, Fabrizio Ensoli & Barbara Ensoli† †

Istituto Superiore di Sanit a, National AIDS Center, Rome, Italy

immune response to Tat:

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epidemiological evidence 4.

The Tat vaccine

5.

Conclusions

6.

Expert opinion

Introduction: 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. Areas covered: 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. Expert opinion: 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. Keywords: HAART intensification, HIV-1 eradication, HIV-1 therapeutic vaccine, neutralization Tat Expert Opin. Biol. Ther. [Early Online]

1.

Introduction

The introduction of effective antiretroviral drugs since the late 1990s has led to the establishment of combined antiretroviral therapies (cARTs) that have changed the scenario of HIV/AIDS. In particular, complete or almost complete suppression of virus replication is achieved in highly (95%) compliant patients and progression to AIDS is blocked. However, it is becoming increasingly apparent that the pace the epidemics expands overcomes the pace cART is provided to infected individuals, especially in developing countries where the infection hits the most [1,2]. Furthermore, evidence in successfully treated individuals of incomplete immune reconstitution, residual virus replication, inflammation and immune activation leading to increased frequency of non-AIDS-related pathologies and early senescence clearly indicates that additional interventions are needed to effectively cure the patients and eventually eradicate the infection. These are very challenging goals and have changed the way we look at HIV-1 infection. Prior to cART, the primary goal was to stop virus replication, and productively infected cells, mainly activated 10.1517/14712598.2015.1021328 © 2015 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. .

.

.

Vaccination with a biologically active HIV-1 Tat protein is safe and immunogenic in healthy and HIV-1 infected individuals (with B or C clade HIV-1). Tat vaccination in cART responders further restores lymphocyte populations and functional subsets and promotes proviral DNA decay. HIV-1 proviral DNA decay correlates with anti-Tat Ab-mediated neutralization of HIV-1 entry in DCs.

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This box summarizes key points contained in the article.

CD4 T cells and monocytes/macrophages, were the major targets. Upon the effective virus suppression achieved with cART, cells harboring the virus, the so-called reservoirs, have become the main targets. However, even cells hosting HIV-1 in an unintegrated form and for a short period of time may to some extent contribute to the reservoir maintenance and replenishment, making virtually all cells in the body potential reservoirs that need to be targeted, a very challenging objective at present. Instructing the host to deal with the infection through vaccination is conceivably the most feasible and cost-effective intervention to combat HIV-1. However, despite almost 30 years of efforts, an HIV vaccine is still lacking and only one of the four HIV vaccines tested in efficacy trials [3,4] has provided some evidence of low and transient (60% at 12 months but 31% at 42 months) protection from acquisition of infection in a lowrisk population [5]. New trials are being designed to reproduce and further improve this vaccine efficacy [6]. Moreover, oligomeric Envs are now available that resemble the native spikes more closely than the gp120 monomers or the truncated form of gp160 (HVTN 505) utilized so far [7,8], thus representing a better immunogen to vaccinate with and, hopefully, afford superior efficacy, as shown in macaques [6]. Trials evaluating vaccines aimed at inducing protective CD8+ T cell responses, alone (HVTN 502 and 503) or in association with anti-Env Abs (HVTN 505), have also been unsuccessful. However, novel approaches tested in the macaque model provide some encouraging results. In particular, strong control and apparent clearance of infection upon mucosal challenge with the pathogenic simian immunodeficiency virus (SIV) mac239 were obtained with a replication competent simian cytomegalovirus (CMV) vector engineered to express SIV Gag, Tat, Rev, Nef, Env and Pol [9]. While this strategy aimed at inducing and maintaining effector memory CD8 T cells in peripheral tissues, including the portal of HIV entry, mosaic [10] and conserved antigens [11,12] represent two additional promising approaches aimed at addressing the challenges of global HIV-1 diversity. However, all the above strategies face major difficulties (safety demonstration for the CMV vector in humans, better targeting of relevant epitopes, superior immunogenicity, durable immunity, identification of correlates of protection) prior to progress to 2

clinical evaluation. These types of vaccines are expected to contain infection (i.e., low to undetectable plasma viral load and CD4+ T-cell preservation), preventing progression to disease as well as virus transmission. Therefore, they may well be considered as therapeutic approaches, which are presently being pursued with renewed interest for a number of reasons (see Section 6). In this regard, the distinction between a preventative vaccine aimed at providing sterilizing immunity and a therapeutic one aimed at clearing the infection is fainting, as more acquisitions on the pathogenesis of the infection and response to treatment are available. In fact, evidence of ongoing residual virus replication or reactivation during suppressive highly active antiretroviral therapy (HAART) somewhat recapitulates and mimics the difficulties HIV encounters in the primary infection [13]. Thus, a vaccine blocking the mechanism(s) of virus transmission in primary infection should also be able to block ongoing replication in asymptomatic and naive to drug individuals as well as residual ongoing replication and/or reactivation in patients on effective cART, leading, respectively, to protection from infection and to virus eradication. Accordingly, for a number of reasons discussed below, the therapeutic setting may be particularly convenient to select effective preventative vaccine candidates. Although no therapeutic vaccine has been market approved, a growing number of vaccine candidates are being evaluated in Phase I/II clinical trials conducted in both naive and/or cART-treated patients (reviewed in [14]), a comprehensive list of vaccine candidates is available at [15]. With a notable exception [16], these vaccines have all proven safe and immunogenic, but not efficacious. Here, we review the rationale and the translational development of a therapeutic vaccine targeting the HIV-1 Tat protein, a key virulence HIV protein, providing a proof of concept of the validity to adopt a pathogenetic-driven approach to develop vaccine strategies against HIV/AIDS. Finally, we propose the therapeutic setting as the most feasible to speed up the testing and comparison of preventative vaccine candidates, challenging the current distinction between the two fields of preventative and therapeutic vaccines, which lies only in the use of the vaccine in uninfected versus infected subjects and not in the vaccine formulation. 2.

The role of Tat in HIV-1 pathogenesis

Tat is the transcription transactivator of HIV gene expression, which is essential for viral replication [17] and, therefore, for establishment of infection or virus reactivation [18,19]. In particular, Tat recruits host proteins to the RNA hairpin formed at the 5¢-end of nascent viral RNAs (TAR) to switch on transcription elongation. Upon virus entry into cells, Tat is expressed by proviral DNA prior to virus integration [20], and it is released extracellularly early during acute infection or virus reactivation [19,21-23] by a leaderless secretory pathway similar to that used by basic fibroblast growth factor or IL-1b to exit cells [21-23]. Upon release Tat binds heparan sulfate

Expert Opin. Biol. Ther. (2015) ()

Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

Anti-Env bn Ab (against high mannose determinants)

APC (DC, EC, Mo) High mannose chains

Block of Env-mediated HIV entry

RGD sequence

Tat Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Nyu Medical Center on 06/24/15 For personal use only.

DC-SIGN SIGN-R MR

Anti-Tat (RGD-blocking)

RGD-binding integrins avb3 a5b1 avb5

Tat/Env complex integrinmediated HIV entry

Block of Tat-mediated HIV entry

NO infection or transinfection

Figure 1. By binding Tat, HIV escapes anti-Env Abs and acquires the capability of using RGD binding integrins to enter cells, greatly expanding its spreading potential. Anti-Tat Abs effectively counteract this entry pathway. Reproduced with permission from [58]. APC: Antigen-presenting cell; DC: Dendritic cell; DC-SIGN: Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; DC-SIGN-R: DC-SIGN-related; EC: Endothelial cell; Mo: Monocyte/macrophage; MR: Mannose receptor; RGD: Arg-Gly-Asp motif; Tat: Transactivator of transcription.

proteoglycans of the extracellular matrix and is detected in tissues of infected individuals [21,24-26]. Extracellular, biologically active Tat exerts activities on both viral infection and immune activation that are key in acquisition of infection, as well as for virus reactivation and for HIV disease maintenance in HAART-treated individuals [19,20,22-25,27-37]. In particular, bioactive Tat has been shown to target cells expressing Arg-Gly-Asp(RGD)-binding integrin receptors such as dendritic cells (DCs), macrophages, activated endothelial cells (ECs) and lymphocytes via its RGD-binding site. However, extracellular Tat enters very selectively and efficiently only DCs and activated ECs [27,36,38]. In DCs, Tat promotes the maturation of DCs toward a T helper (Th)-1 polarizing phenotype, modulating further T cell responses [36-38] and activates the proteasome leading to increased antigen processing and presentation thus contributing to Th-1 cell activation [34,39,40]. Moreover, upon engagement of RGD-binding integrins on CD8 T cells Tat promotes the induction of short-living effectors [41,42], which undergo apoptosis upon encountering antigens in the presence of Tat [43]. Tat also suppresses T-cell activation by inducing in DCs the expression of indoleamine 2,3 dioxygenase [44], an important marker of disease progression in HIV-1-infected individuals [45]. Furthermore, Tat activates the expression of cytokines with key immunomodulatory effects and/or capable of activating HIV gene expression [37,46-51]. Moreover, extracellular Tat mimics chemokines [52] and induces HIV

co-receptor expression [53,54]. As a result, extracellular Tat facilitates R5-tropic viruses transmission to neighbor cells [55] even in the absence of cell activation [56], activates virus replication, rescues defective proviruses [22,28] and enhances virus infectivity [30]. Of note, the Tat protein is detected in highly purified virions [57], further supporting its key role in virus transmission and establishment of infection. In fact, it has been recently shown that Tat binds to Env trimers of different clades and forms a novel virus entry complex that favors -- via an integrin-mediated pathway -- virus entry and productive infection of DCs and efficient transmission to T cells (Figure 1). In the Tat/Env complex, the cysteine-rich region of Tat engages the CCR5-binding regions of Env, including the V3 loop and a region of Env partially overlapping the other Env CCR5-binding site, whereas the Tat RGD sequence remains free and directs the virus to the RGDbinding integrins present on the DC surface (Figure 1). As a result, HIV acquisition by relatively poor susceptible cell targets -- but important viral reservoirs, such as DCs, resting T cells and possibly other cell types not expressing the canonical HIV-1 receptors -- is greatly increased. Of importance, Tat also shields the bound Env oligomer from the attack of anti-Env neutralizing Abs (NAbs), as indicated by a novel neutralization assay that evaluates the capability of antiHIV-1 Abs to block the entry of oligomeric Env into DCs, in the presence or absence of Tat [58]. In fact, in the presence of Tat the capability of HIV-1+ sera (anti-Env-Ab-pos, anti-

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Percentage of dendritic cells internalizing Env

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–HIV sera

+HIV sera (anti-Env Ab positive/anti-Tat Ab negative)

+HIV sera (anti-Env Ab positive/anti-Tat Ab positive)

80 70 60 50 40 30 20 10 0 S IV- at at at 91 40 69 18 06 70 64 21 91 40 69 18 06 70 64 21 66 88 81 56 82 08 67 34 66 88 81 56 82 08 67 34 PB l H + T +T nti-T 2 2 2 8 8 2 2 8 2 2 2 8 8 2 2 8 2 2 2 2 2 8 2 2 2 2 2 2 2 8 2 2 o IVa o P lH Ab o m o –Tat +Tat –Tat +Tat + P at +T

Figure 2. Neutralization of Tat/Env complex entry in MDDCs by sera from HIV-infected individuals. Neutralization of trimeric Env deleted in the V2 domain entry in MDDCs by sera from HIV-infected subjects in the presence or absence of Tat in anti-Tat Ab-negative (n = 8) and anti-Tat Ab-positive (n = 8) subjects. The bars represent the percentage of entry of Env alone incubated in buffer (in blue) or with Tat (in red). The percentage of Env positive cells is shown. Data are expressed as the mean with standard deviation of experiments performed in duplicate. The codes of the anti-Tat Ab-negative or -positive sera are indicated at the bottom of the bars. Reproduced with permission from [58]. Tat: Transactivator of transcription.

Tat-Ab-neg) to block the entry of Env in DCs is abrogated; however, when anti-Tat Abs are present, the neutralization of Env entry is not only restored but further enhanced (Figure 2). Notably, this test has also the advantage to overcome the problem posed by the cART interference with neutralization of HIV-1 infection [59]. Thus, Tat may well be considered a virulence factor [60] that plays key roles any time the virus needs to establish or to reactivate infection, that is, at the acquisition of infection or under cART-mediated viral suppression, both of which are accompanied by the presence of unintegrated proviral DNA expressing regulatory gene products and RGD-containing Tat protein isoforms [56,61-64]. 3. The protective role of the immune response to Tat: epidemiological evidence

Consistently with the roles of Tat in HIV pathogenesis, the presence of anti-Tat immune responses correlates with low or no progression to AIDS. In fact, when present, cellular and humoral anti-Tat responses exert protective roles to control virus replication and to delay disease progression, both in 4

humans and monkeys [65-70]. Notably, anti-Tat Abs are infrequently induced in the course of natural infection, being found in ~ 20% of HIV-infected individuals in the asymptomatic phase, and are lost during progression [71-73]. In contrast, high Ab titers are produced against all viral products at all infection stages [74,75]. In particular, a cross-sectional and longitudinal study on 252 HIV-1 seroconverters, with a median follow-up time of 7.2 years, indicated that anti-Tat Abs are more frequently found in the asymptomatic stage of the infection and that the presence of anti-Tat Abs is predictive of a 60% slower progression to AIDS or immunodeficiency [69]. Progression was faster in persistently anti-Tat Ab-negative than in transiently anti-Tat Ab-positive subjects, whereas no progression was observed in individuals persistently anti-Tat-Ab positive [69]. Thus, the rationale of targeting Tat is based on experimental evidence indicating that: i) Tat is necessary for HIV gene expression, replication and cell-to-cell transmission; ii) antiTat Abs abrogate the Tat-mediated shielding of Env from the attack of anti-Env NAbs and block HIV infection of DCs and spreading to T lymphocytes (Figures 1 and 2) [58] and iii) epidemiological evidence indicating that immune

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Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

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Table 1. Reasons to use the native HIV-1 Tat protein as a vaccine candidate for HIV/AIDS. Key role in the virus life cycle (early expression and release by infected cells) and in AIDS pathogenesis Correlation in cross-sectional and longitudinal studies of the anti-Tat immune responses (humoral and cellular) with asymptomatic stage and non-progression to AIDS Conserved immunogenic sequences among HIV-1 clades Very efficiently taken up by dendritic cells inducing Th-1 polarization (adjuvant properties) Modifies hierarchy of CTL epitopes of heterologous antigens in favor of subdominant and cryptic epitopes (due to a modification of proteasome catalytic subunit composition) Preservation of the seronegative status in vaccinees* Use as both ‘preventative’ and ‘therapeutic’ vaccine See text for explanation and references. *Tat-vaccinated individuals will be distinguished from infected individuals as the routine tests for HIV diagnosis detect only Abs against structural HIV antigens and not against Tat. CTL: Cytotoxic T cell; Tat: Transactivator of transcription; Th-1: T helper 1.

Table 2. Rationale for therapeutic immunization of HIV-infected individuals. In HIV-infected Drug-Naive individuals Delay or block of progression to AIDS or cART therapy In HIV-infected individuals in need of therapy or cART-treated cART intensification to: Accelerate time-to-response to therapy Block or reduce virus transmission Solve unmet needs of ART (immune activation, immune defects, proviral DNA) Drug simplification or virus control despite low adherence to therapy cART: Combined antiretroviral therapy.

responses against Tat are associated with slow or no disease progression. Of importance, Tat is highly conserved among the different clades, especially in the first exon [76], assuring effective crossclade, possibly universal, coverage upon vaccination with a clade B (BH10) Tat protein (see below) (Table 1). Finally, because of the intrinsic immunomodulatory properties, biologically active Tat does not require adjuvants, a relevant advantage in terms of development, production and delivery costs. The therapeutic uses of Tat vaccination are to be confirmed in dedicated trials, and may range from HAART intensification to treatment interruption, depending on the effectiveness of the elicited anti-Tat immune response in controlling the infection (Table 2). 4.

The Tat vaccine

Preclinical testing Based on this evidence, preclinical studies in cynomolgus monkeys were undertaken. Immunization with the Tat 4.1

protein or tat DNA in cynomolgus macaques was found to be safe, elicited a broad and specific immune response and, most importantly, induced a long-term protection against infection with the highly pathogenic SHIV89.6P, a SIV carrying the HIV-1 tat gene, which rapidly causes AIDS and death in these monkeys [77-79]. In particular, in 9 out of 12 animals vaccinated with the Tat protein or DNA neither plasma viremia nor CD4+ T-cell decline were detected, even though a few proviral DNA copies were sporadically found in a few animals. In contrast, all controls became overtly infected, with high viral load and steep CD4+ T cell decline [77,79]. Both Tat protein and tat DNA elicited long-term specific memory T cells in protected monkeys, which did not show signs of systemic infection throughout a 104-week follow-up or even after two boosters with tetanus toxoid, a stimulus known to activate CD4 T cells and to increase virus replication. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died [80]. Tat-specific Abs, CD4+ and CD8+ T-cell responses were high and stable only in the animals controlling the infection, indicating that vaccination with Tat had induced long-term memory Tat-specific immune responses, and had controlled primary infection at its early stages, allowing a long-term containment of virus replication and spread in blood and tissues. To further investigate the breadth and durability of this protective immunity, four of the protected monkeys were rechallenged intravenously with a fivefold higher dose (50 MID50) of the same SHIV-89.6P. Although the vaccinated monkeys became overtly infected and underwent CD4+ T cell loss in the acute phase of infection, over time they regained control of infection, as indicated by the statistically significant and long-lasting reduction of viral replication and CD4+ T cell number restoration in comparison to control monkeys. This effect was associated with a strong anamnestic response to Tat, whereas responses to Gag and Env were nearly undetectable [81]. A retrospective analysis of 112 Mauritian cynomolgus macaques from different preclinical trials, vaccinated (n = 67) or not (n = 45) with Tat and challenged intravenously with the SHIV-89.6P, showed that vaccination induced a significant reduction of the rate of infection acquisition at 10 MID50 (p < 0.0001), and contained acute CD4+ T-cell loss at 15 MID50 (p = 0.0099). Of importance, vaccination also contained CD4+ T-cell depletion (p = 0.0391) during chronic infection, irrespective of the challenge dose [82]. Protection or containment of infection in cynomolgus macaques was also observed upon co-immunization with HIV-1 Tat and Env proteins. The rationale for this association was based on the evidence that HIV-1 Tat, which is necessary for HIV gene expression, replication and cell-to-cell transmission, appears also to be critical in the initial steps of virus acquisition. In this study, the macaques had been primed twice intranasally with HIV-1 Tat and Env, given together with the LT-K63 mucosal adjuvant, then boosted twice subcutaneously with Tat plus Env in Alum, and finally

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Plasma RNA (Eq/ml × 104)

A.

p = 0.0100

18 16 14 12 10

p = 0.0100 p = 0.0041

8 6 4 2 0

p = 0.0406

2

3

Proviral DNA (copies/mg genomic DNA) C.

4

5

2

3

4

5

Weeks after challenge

p = 0.0452

800

p = 0.0325

600 400

p = 0.0452 200 0 2

Proviral DNA (copies/mg genomic DNA)

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B.

3

4

5

2

3

4

5

Weeks after challenge

p = 0.0065

600 500 400 300 200 100 0 RT

LN

RT

LN

Figure 3. Virological outcome in Tat/Env-vaccinated or control cynomolgus macaques after intrarectal challenge with the SHIVSF162P4cy (70 MID50). Box plots of (A) viral RNA, (B) proviral DNA in blood at 2, 3, 4 and 5 weeks after challenge, respectively; and (C) proviral DNA at week 4 after challenge in rectal tissue (RT) and inguinal lymph nodes (LN). Statistical analysis was performed by the one-sided Wilcoxon rank sum test. Red: monkeys vaccinated with Tat/Env (n = 6); blue: control animals (n = 6). Vaccination with Tat and Env contained viral infection at the portal of entry (rectal challenge) in vaccinated but not in control animals, blocking dissemination to local lymph nodes (C panel). Reproduced with permission from [58]. Tat: Transactivator of transcription.

they were challenged intrarectally 14 weeks after the last boost with a high dose (70 MID50) of the R5-tropic SHIVSF162P4cy. No infection or a statistically significant reduction of viral load and proviral DNA was observed in the vaccinated monkeys when compared to controls [55]. Of importance, sera from vaccinated macaques neutralized the entry of the Tat/Env complex in DCs [58]. Strikingly, proviral DNA load in the inguinal lymph nodes was significantly lower in vaccinated monkeys when compared to controls, whereas it did not differ significantly in rectal biopsies (Figure 3) [58]. Thus, mucosal vaccination with the Env and 6

Tat protein combination induced anti-Tat NAbs and led to an effective containment of viral infection at the portal of entry, with block of virus dissemination from the rectum (site of virus inoculum) to lymph nodes following a very high rectal challenge dose. In a similar approach, rhesus macaques were primed mucosally with a replicating adenoviral vector carrying the HIV-1 tat transgene and an adenovirus expressing HIV-1 Env and boosted with the Tat and Env proteins. Following intravenous SHIV-89.6P high dose challenge, all macaques became infected. However, the Tat/Env vaccinated monkeys reduced

Expert Opin. Biol. Ther. (2015) ()

Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

Table 3. Clinical observational studies completed to date. Code (Clinicaltrials.gov identifier)

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ISS OBS T-003 (NCT01029548) ISS OBS T-002 (NCT01024556) OBS IFO ISS OBS T-004 (NCT01359800) All studies

Study

Country

No. of volunteers

Treatment-naive

Longitudinal

Italy

73

ARV-treated

Longitudinal

Italy

105

Treatment-naive ARV-treated and treatment-naive Total volunteers

Longitudinal Cross-sectional

Italy South Africa

29 531

chronic viremia by 4 logs when compared to controls (p < 0.0001) and maintained CD4+ T cells. Of note, control of infection correlated with Tat and Env binding Abs [83]. In a further approach, the sterilizing immunity or control of infection observed in rhesus macaques immunized with a multi-component vaccine -- (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) delivered either systemically or mucosally and challenged orally or intrarectally with the C clade r5-tropic SHIV-1157ip -- correlated only with anti-Tat Abs against the N-terminus of Tat, as determined by a novel biopanning strategy which, using recombinant phages encoding random peptide libraries, allows a complete and unbiased profiling of the antibody repertoire and identification of epitopes associated with vaccine protection [84]. In this regard, we recently found that, in addition to antiTat Abs against the N-terminus, also Abs directed to peptides spanning the glutamine-rich (aa 61--75) and the RGD-integrin-binding (aa 71--85) regions of Tat are associated with control of infection in cynomolgus macaques primed with the biologically active HIV-1 Tat protein delivered by anionic microspheres and boosted with Tat in Alum and then challenged intravenously with SHIV89.6Pcy [85]. These studies strongly suggest that the induction of antiTat Abs is key to achieve a protective immunity against HIV/AIDS, and support the concept that the combination of Tat with Env may generate a most effective, broad and cross-protective immunity against HIV acquisition. Based on these preclinical studies, Phase I preventative studies with the Tat alone and the Tat/Env combination were conducted (see below). In particular, a pilot preventative Phase I trial with the combined Env and Tat proteins has been recently completed in Italy and results are under analysis. Of note, in the therapeutic setting only immunization with Tat is required, as anti-Env Abs are present at all times and at high titers in infected individuals [75]. Observational studies Observational studies were conducted (Table 3) to: i) gain more insights into the role of anti-Tat Abs in the course of the natural infection (ISS OBS-T003, OBS IFO); ii) serve 4.2

Type

738

as a reference group for the ISS-T-002 vaccine trial and evaluate the occurrence and impact of seroconversion for anti-Tat Abs in successfully cART-treated individuals (ISS OBS T-002) and iii) acquire information on the characteristics of the epidemics in South Africa relevant to the design and conduction of the recently completed Tat vaccine trial ISS T-003 (ISS OBS T-004). A significant association between the presence of anti-Tat Abs and a slower disease progression was found in asymptomatic drug-naive HIV-infected adult volunteers enrolled in a prospective observational study (ISS OBS T-003, Table 1) aimed at evaluating the effects of anti-Tat Abs on the immunological, virological and clinical outcome of HIV-infected subjects. In particular, anti-Tat Ab-positive patients showed a remarkable preservation of CD4+ T cells and containment of viral load for the entire follow-up (3 years), and no individuals with high levels of anti-Tat Abs initiated HAART during follow-up [86]. Of note, the association of increasing anti-Env IgG titers with a lower risk of starting HAART occurred only in the presence of anti-Tat Abs, suggesting that anti-Tat and anti-Env Abs combined have increased HIV neutralizing effects by blocking the Tat/Env complex formation and virus entry, as shown earlier both in vitro and in vivo [58]. Thus, both anti-Tat and anti-Env Abs appear to be required to efficiently block HIV disease progression. In contrast, anti-Env or anti-Gag Ab titers had no significant effects on CD4+ T-cell counts and viral load in patients naive to therapy with or without anti-Tat Abs [86]. Thus, anti-Tat Abs appear to have a protective role and represent a predictive biomarker of slower progression to AIDS. Incidentally, this evidence provides some explanation to the repeated failure of preventative vaccines based solely on Env and indicates that Tat may represent an optimal target for preventative interventions in combination with oligomeric Env or possibly alone. Results from another observational study (OBS IFO) showed that response to therapy was significantly faster in anti-Tat Ab-positive than in the anti-Tat Ab-negative subjects (p < 0.0001, Log-Rank test). In addition, in anti-Tat Ab-positive subjects, reduction to undetectable levels was persistent. Cox model was used to estimate the relative hazards of

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A.

Anti-Tat Ab-

Viral load (log10 copies/ml)

6

Anti-Tat Ab+

5 p = 0.0305 4 3 2 p = 0.0044

1

p = 0.0116

0

p = 0.0022

3 6 9 Months from starting HAART

12

B. 1.00 Cumulative incidence of persistant viral load = 0

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0

0.75

C. 0.50 Parameter 0.25

Anti-Tat Ab+ vs anti-Tat Ab– CD4+ at month 0 Viral load at month 0

Hazard ratio 3.603 1.004 0.663

95% hazard ratio confidence limits 1.270 1.000 0.249

10.224 1.009 1.764

Pr > ChiSq 0.0160 0.0317 0.4105

0.00 0

3 6 9 12 Months since HAART initiation

No. at risk at baseline Anti-Tat Ab+ n = 10 (red) Anti-Tat Ab– n = 19 (blue)

Figure 4. Kinetics (A), Kaplan--Meier curves (B) and Cox Model (C) of time-to-response to cART in anti-Tat Ab-positive versus -negative. OBS IFO: HIV-infected subjects starting HAART (time to virologic response): anti-Tat Ab positive individuals (n = 10) have a significantly faster response to therapy when compared to Anti-Tat Ab-negative subjects (n = 19) (p < 0.0001, Log-Rank test). The probability (hazard ratio) to achieve the end point was > 3 times greater in anti-Tat Ab-positive when compared to anti-Tat Ab-negative subjects. cART: Combined antiretroviral therapy; Tat: Transactivator of transcription.

reaching the end point (persistent viral load = 0) for the antiTat Ab-positive and anti-Tat Ab-negative subjects, after adjustment for CD4+ and viral load at baseline (Figure 4A, B). The probability (hazard ratio) to achieve the end point was > 3 times greater in anti-Tat Ab-positive when compared to anti-Tat Ab-negative subjects (Figure 4C). Clinical development As the next step, preventative Phase I and therapeutic Phase I and II trials [87-90] were conducted and successfully completed (Table 4 and [91]). In all the trials the vaccine administered was a subunit vaccine made of recombinant biologically active HIV-1 B Clade (BH10) Tat protein (referred herein as ‘Tat’) 4.3

8

manufactured under GMP. The manufacturing process was specifically developed to prevent oxidation and to keep the protein in its native active form. In fact, only the biologically active Tat retains the native conformation, which is necessary to induce an effective antibody response against conformational epitopes that are key for Tat neutralization. Oxidation renders the protein biologically inactive and thus incapable of eliciting the proper antibody responses. The Tat vaccine was formulated in isotonic saline buffer with albumin and sucrose as excipients, and stored at -80 C until the administration. Stability studies of GMP lots indicate a shelf life of > 3 years. Here, only the therapeutic Tat vaccine, which is in an advanced phase of development, is discussed. In fact, the

Expert Opin. Biol. Ther. (2015) ()

Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

Table 4. The Tat vaccine clinical studies completed to date. Code (Clinicaltrials.gov identifier) ISS P-001 (NCT00529698)* ISS T-001 (NCT00505401) ISS T-002 (NCT00751595) ISS T-003 (NCT01513135) ISS P-002 (NCT01441193) Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Nyu Medical Center on 06/24/15 For personal use only.

All studies

Study

Type

Phase I preventive trial (Tat)

Randomized, controlled Phase I therapeutic trial (Tat) Randomized, controlled Phase II therapeutic trial (Tat) Randomized, Phase II therapeutic trial (Tat) Randomized, controlled Phase I preventive trial Randomized, (Tat + Env) Total volunteers (with placebo) Total vaccinated volunteers

Country

No. of volunteers

double blind, placebo

Italy

20

double blind, placebo

Italy

27

open label double blind placebo

Italy South Africa Italy

168 200

open label

11 426 314

Tat: Transactivator of transcription.

therapeutic setting has several advantages over the preventative approach. In particular, it: i) provides a rapid first proof-of-concept of efficacy of a vaccine design and biomarkers assessment; ii) requires a much smaller sample size; iii) can be conducted in developed countries; iv) is much less expensive and v) has a broad application with key potentials in the most affected populations. 4.4

Therapeutic trials Phase I trial with Tat in Italy (ISS T-001)

4.4.1

A Phase I therapeutic study (ISS T-001, ClinicalTrials.gov NCT00505401) was conducted in four clinical centers in Italy in HIV infected asymptomatic individuals, with CD4+ T cells/µl ‡ 400, viral load £ 50,000 copies/ml and CD4 nadir ‡ 250, independently from the anti-Tat serostatus at baseline. The study was designed as a randomized, double blind, placebo-controlled clinical trial in which the recombinant biologically active HIV-1 Tat protein was administered five times subacute with alum or intradermally without adjuvant at 7.5, 15 or 30 µg doses, respectively. The study included a treatment phase of 16 weeks, a post-immunization follow-up period up to week 48 (final analysis) and was followed up to 5 years after immunization. Twenty seven volunteers were enrolled in the study: 20 received the vaccine injections (11 for Arm A and 9 for Arm B, respectively) and seven received the placebo (4 for Arm A and 3 for Arm B, respectively). The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific Th-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat Abs, rarely seen in HIV-infected subjects. The data indicated the achievement of both the primary (safety) and secondary (immunogenicity) end points of the study [87,88,90]. In particular, administration of biologically active Tat did not increase plasma viremia levels, confirming former data obtained in infected macaques that bioactive Tat at the doses used does not promote viral replication. Conversely, the protein was immunogenic, with

anti-Tat Ab titers peaking between week 12 and week 24, to decrease thereafter during the follow-up period. The long-term assessment of the kinetics of anti-Tat Abs (48 -- 144 weeks) revealed that, though decreased over time, anti-Tat Abs generated by vaccination were still detectable in vaccinees 3 years from first immunization. The assessment of CD4+ T-cell counts was performed as a primary safety parameter. The statistical analysis of the data collected up to 48 weeks revealed a significant positive correlation in vaccinees between the levels of circulating CD4+ T cells and the anti-Tat IgG or IgA Ab titers, whereas no significant correlation was found for anti-Tat IgM titers. Of note, when the neutralization of Tat-mediated Env entry in DCs by sera from vaccinees or placebo who had remained naive to therapy for the entire follow-up (up to 5 years) was assessed retrospectively, vaccinees, but not placebo, were found to retain the neutralizing activity, despite the fact that anti-Tat Abs were barely detectable or undetectable (data not shown). Thus, the neutralization assay appears to be more sensitive than measuring binding Abs by ELISA. Nevertheless, sera from HIV-1-infected individuals negative for anti-Tat Abs by ELISA did not neutralize the Tat-mediated Env entry in DCs, confirming the low prevalence of anti-Tat Abs in the course of natural infection [58]. Phase II trial with Tat in Italy (ISS T-002) Based on the Phase I results an ‘exploratory’ multi-center, randomized, open-label Phase II therapeutic trial with the biologically active HIV-1 Tat protein (ISS T-002, Clinicaltrials.gov NCT00751595) to evaluate immunogenicity and safety was conducted and successfully completed in 2012 in Italy. The open-label design of the trial allowed us to explore immunological and virological biomarkers of disease as second-line testing, as foreseen in the Study Protocol, with the aim of including additional analyses deemed necessary to better understand mechanism(s) of action and to define the proper biomarkers for future trials. 4.4.2

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To partially overcome the limitation of the lack of a placebo group, an external reference group from a parallel observational study (OBS, see below) was used -- in accordance with the recommendations of regulatory guidelines (Note for Guidance on Choice of Control Group in Clinical Trials [CPMP/ ICH/364/96] and see below). To reduce the bias of the openlabel design, the OBS subjects were enrolled at the same clinical sites with the same inclusion criteria and the samples were analyzed simultaneously for the same parameters by the same centralized laboratory. All was done as for the trial subjects. This group was not used as a control of the pre-specified end points of the protocol (immunogenicity/safety), but served only to provide a side-by-side view of the immunological and virological disease biomarkers in anti-Tat Ab-negative subjects under effective ART without any direct comparison with the trial subjects, as they belong to two separate studies. Indeed, the open-label design of this ‘exploratory’ trial permitted verification of the rapid achievement of the study primary and secondary end points and to appreciate the immune reconstitution induced by vaccination [92]. This allowed the implementation of proper study protocol amendments such as inclusion of more immune compromised patients who, in fact, showed the most pronounced immunological benefit, as well as to increase the sample size of the study. Of note, this ‘exploratory’ approach also allowed the design and the definition of the proper laboratory testing for a placebocontrolled double-blinded Phase II study of the Tat vaccine (ISS T-003, Clinicaltrials.gov NCT01513135), which is now completed in 200 individuals on effective ART in South Africa (see below). The clinical study protocol submitted and approved by national competent authorities included 128 individuals with chronically suppressed plasma viremia (viral load < 50 copies/ml in the last 6 months prior to the screening) without a history of virologic rebound, CD4+ T-cell counts > 400 cells/µl, pre-ART CD4 nadir > 250 cells/µl. An interim analysis performed at 48 weeks in 87 subjects who completed the treatment phase [92] indicated that the Tat vaccine candidate did not raise any safety concern. Therefore, after approval by the Data Safety Monitoring Board (DSMB), the study was amended to include subjects with less restrictive inclusion/exclusion criteria (history of virologic rebound, CD4+ T-cell counts ‡ 200 cells/µl irrespective of pre-ART CD4 nadir, history of AIDS-related opportunistic or neoplastic diseases, HBV or HCV co-infections), and to increase the total number of subjects from 128 to 160, upon recalculation of the sample size. Upon study closure, an ‘ad hoc’ OBS of extended followup was initiated and volunteers were invited to enter the ISS T-002 Extended Follow-up study (ISS T-002 EF-UP, ClinicalTrials.gov NCT02118168), which is currently ongoing. Safety Immunization with Tat was safe and well tolerated without any notable dose-dependent relationship, as indicated by the 4.4.2.1

10

independent Data and Safety Monitoring Board after analyses of all adverse events (AEs). No relevant AE occurred during the study, and most of them were expected both in frequency and type, for HIV-infected subjects. The events were mostly local, related to the injection site and mild in severity. Thus, the AEs reported during the study indicated that the Tat vaccine may induce mild reactions at the injection site and systemic effects that are generally well tolerated. Immunogenicity Tat immunization induced anti-Tat Abs in most vaccinees (79%), with the highest frequency in the Tat 30 µg groups (89%), and particularly after three administrations (92%), when compared to the 7.5 µg arms (70%). In the Tat 30 µg groups, Abs persisted significantly longer, when compared to the Tat 7.5 µg groups. The 30 µg doses were also more effective at inducing anti-Tat Abs of different isotypes, and peak IgG titers. Tat immunization also increased the percentage of responders and intensity of anti-Tat cellular responses, including gIFN, IL-4, IL-2 production and CD4+, CD8+ T-cell proliferation, when compared to baseline without significant differences among the treatment groups [93]. Cellular responses to Tat were also present in OBS subjects [93]. Of utmost importance, a subgroup study conducted in 30 volunteers enrolled in the ISS T-002 study (anti-Tat Ab-negative for clade B at baseline as per protocol) and immunized with Tat 30 µg, 3, or Tat 7.5 µg, 5, showed that Tat immunization had induced cross-clade (clades C, D, A and AG) Abs. In fact, although none of the subjects (0/30) were anti-Tat Ab-positive for other clades at baseline, all of them 30/30 became anti-Tat Ab-positive for clade B after immunization, and 21/30 (70%) also for the other HIV Tat clades. 4.4.2.2

Effects of vaccination on disease biomarkers Published data of the interim analysis [92] as well as the results obtained at study completion [93] indicate that the therapeutic immunization with the Tat vaccine reversed a number of immune defects still present in patients under suppressive ART treatment (ART unmet needs), driving the immune system back toward homeostasis. In particular, peripheral blood CD4+ lymphocyte counts as well as B cell (including memory B cells) and NK cell numbers increased progressively over 3 years when compared to effective ART alone; the decrease of CD8+ T cell number seen in ART was reverted and, of key importance, the defect of central memory CD4+ and CD8+ T cells, which is known to persist despite a successful ART, was restored [92,93]. In addition to the reduction of effector memory CD8+ T cells, the inflammatory cytokine response (IL-6, TNF-a and Rantes) was homeostatically restored, in that it decreased in those with high cytokine serum levels and increased in those with low levels (data not shown). Functionally, the cellular immune responses against HIV (Env) and against different ‘recall’ antigens (Candida, CEF: CMV, EBV and Flu peptides) were improved when 4.4.2.3

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Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

compared to the reference group, whereas reactivation of Toxoplasma gondii, the cause of Toxoplasma encephalitis, was reduced [92,93]. In this regard, antibody IgG titers against T. gondii >150 IU/ml indicate reactivation of the pathogen and are known to be predictive of Toxoplasma encephalitis in HIV-infected subjects, which is resistant to ART [94]. Results in vaccinees showed that at baseline 36% (37/104) had anti-Toxoplasma IgG and of these 22% (8/37) had IgG values >150 IU/ml. In the OBS subjects, 37% (28/75) of subjects had anti-Toxoplasma IgG at baseline and of these 36% (10/28) had values >150 IU/ml. Anti-Toxoplasma IgG significantly decreased after 24 weeks from the first immunization in vaccinees (Tat 30 µg), whereas no relevant changes were observed in OBS subjects over time (data not shown). Of utmost relevance, immunization with Tat led to a statistically significant reduction of proviral DNA, especially when the vaccine was administered at the 30 µg dose, given 3 times and in conjunction with HIV protease inhibitors (PI) treatment, when compared to OBS subjects. In fact, although in the 30 µg, 3, group HIV DNA reduction progressed over the observation period of 3 years (144 weeks), and the predicted estimate of HIV-1 DNA decay was of 56% reduction after 3 years from vaccination, the decay was more pronounced under PI-based regimens for which the predicted estimate reached over 70% reduction after 3 years, with a half-life of 88 weeks [93]. Finally, these results have been confirmed in the ongoing extended follow-up of the trial (ISS T-002 EF-UP ClinicalTrials.gov Identifier: NCT02118168), with HIV-1 DNA becoming undetectable in the peripheral blood of about 20% of vaccinees evaluated over a period of time ranging from 144 to 304 weeks (median 200 weeks) since vaccination. 5.

Conclusions

Thus, results from the therapeutic trials indicate that the Tat vaccine is safe (to date over 300 people have received the vaccine, in either preventative or therapeutic trials) and immunogenic. Furthermore, the results from the Phase II trial showed a significant increment of CD4+ T cells, B cells and NK cells with an important increment of T and B cell functional (memory) subsets, in association with a reduction of immune activation, suggesting a return of immune functions to homeostatic levels. Furthermore, the Tat vaccine promoted proviral DNA decay, thus supporting the use of Tat immunization to intensify ART [93]. In fact, virus reservoirs and their replenishment by cell-to-cell virus transmission are ARTresistant and associated with chronic immune activation and lymphocyte dysfunction, which represent the ART unmet needs. These data are consistent with the primary role played by Tat in HIV pathogenesis, including virus replication, transmission, immune dysregulation, disease onset and maintenance and open new avenues for an effective ART intensification and therapy simplification and provide the basis for a functional cure and future virus eradication strategies. In

fact, vaccination with the Tat vaccine is expected to amplify the spectrum of ART efficacy providing an effective intensification of drug therapy by counteracting AIDS progression and non-AIDS defining diseases (Table 2), thus reducing the overall economic burden of AIDS care. The overall benefit to patients and governments will be further enhanced if therapy simplification regimens are adopted in conjunction with the therapeutic Tat vaccine. No other current treatment has been shown to achieve these effects. Future studies will address whether the immunity provided by the Tat vaccine is effective only in patients fully adhering to cART (therapy intensification), or whether it affords virus control despite low adherence to cART, therapy simplification or even therapy interruption (Table 2). Phase III trials are being designed to confirm the efficacy of the Tat vaccine (Figure 5). 6.

Expert opinion

Overall, the results obtained with the Tat vaccine in monkeys and in humans indicate that Tat is critical in HIV-1 life cycle, as, when targeted either during natural infection or upon vaccination, it contains viral replication with no/low progression, whereas in vaccinated macaques protection appears to confine the virus at the portal of entry (Figure 3) [58] or grant sterilizing immunity [77,82]. Of note, data from the T-002 therapeutic trial indicate that targeting Tat in successfully cART-treated individuals reduces immune activation, as indicated by the homeostatic recovery and balancing of all lymphocyte populations and functional subsets, which we interpret as an immunological ‘reset and restart’, conceivably occurring upon removal of extracellular Tat from tissues. In this regard, it should be underscored that cART does not prevent HIV-1 gene expression [64] and data from many groups strongly indicate Tat as a major dysregulator of the immune system (as discussed above) per se (i.e., even in the absence of HIV-1 infection). Data from the same trial also indicate that in vaccinees, but not in individuals followed in the parallel OBS the proviral load significantly declines, especially after 3 years from the vaccination [93]. The delayed kinetics is consistent with the evidence that ~ 88% of the HIV-1 DNA does not appear to code for replication competent virus [95], and its decay corresponds to the half-life of the cell that harbors it, which in blood may be of years [96]. Although the exact mechanism by which anti-Tat immunity promotes proviral load reduction is unknown, data indicate a correlation with the anti-Tat Ab-mediated neutralization of HIV-1 entry in DCs, suggesting blockade of replenishment of the reservoir as a possible mode of action (Figure 1) [58]. In addition, killing of Tat expressing cells, presumably carriers of replication-competent proviruses [97] by both anti-Tat Abs and cytotoxic T cells (CTLs) has to be considered. However, it appears that anti-Tat Abs are a key requirement, as indicated by both the epidemiological evidence of protection from disease progression in individuals naive to therapy [88]

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A. Cafaro et al.

1995

2014

GMP Approval for Candidate Pre-clinical production and testing human use development studies

Phase I

Phase II

2018

Phase III

Regulatory filings to MCC in South Africa for marketing authorization

Therapeutic and preventive Tat vaccine

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Phase I trial completed in Italy (2003 – 2007)

Preventive Tat + Env vaccine Phase I trial completed in Italy (2012 – 2014)

Therapeutic Tat vaccine: Phase II trial completed in Italy (2008 – 2012) Phase II trial completed in South Africa (2012 – 2014)

Planned

Figure 5. Time line of vaccine development and current status and plan of the Tat vaccine. Tat: Transactivator of transcription.

and the results of proviral DNA decay in subjects on cART developing anti-Tat Abs upon vaccination [93]. Conversely, the contribution of cellular anti-Tat responses alone, which are present in most infected individuals, appears lower. Accordingly, Tat-based vaccines aimed at inducing cellular responses alone failed at demonstrating any therapeutic efficacy, despite good induction of cellular responses [98]. In acute infection studies, anti-Tat CTLs are readily induced and escaped [66,68], indicating both that Tat is essential to the virus and must escape very rapidly to CTL control and that it may afford mutations in the linear sequence corresponding to the CTL epitope without loosing its biological activities. This may also explain the lack of efficacy of a therapeutic vaccine based on a single and presumably linear universal Tat B cell epitope [99]. In contrast, Tat is apparently unable to escape Abs indicating that structural changes affecting its conformation also impair its function. Of note, despite being reported as a very variable protein, Tat is highly conserved in the first 58 aa of exon 1 [76] and it is conceivable that most of the mutations found in the second exon do not translate into a functionally dead protein. Moreover, coevolving mutations in functionally distinct domains appear to be compensatory and to maintain Tat functions [100]. This is in substantial agreement with crystallography data indicating that, with a few constraints in the first exon, Tat is a poorly structured 12

protein capable of standing mutations at several sites without loosing vital functions [101,102] while maintaining the capability of interacting with an extraordinary high number of putative ligands [103]. Overall, the results with the Tat vaccine reinforce the concept that new paradigms must be applied to develop efficacious preventative/therapeutic intervention strategies against HIV/AIDS. In fact, empirical vaccines as well as vaccine based on structural knowledge have not been successful. In particular, it should be kept in mind that structures do not always translate in immunogenicity and immunogenicity does not always translate in efficacy. A ‘pathogenesis-driven’ approach should be aimed at targeting key viral products responsible for virus transmission, activation and maintenance of virus reservoirs. Furthermore, it should take advantage of the epidemiological evidence of spontaneous control of infection to identify immunological correlates of protection to exploit in vaccine development. Novel immunological platforms have been proposed to permit a systematic comparison of different vaccine candidates to select those to advance further [104]. However, in the absence of known or surrogate immune correlates of protection these strategies might be risky, and valuable candidates discarded. In this regard, when compared to preventative vaccines, therapeutic trials are more cost effective as they may provide, even in small Phase I/II trials, a first proof-of-concept

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Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

of efficacy and better define specific end points and laboratory biomarkers to be assessed, before advancing to preventative trials (Table 2). In addition, an effective therapeutic vaccine may offer a promising alternative strategy to the recurrent failure of preventive HIV vaccines, based on the consideration that it can reduce HIV replication and transmission to healthy individuals. Furthermore, a therapeutic vaccine may be worth to develop even if not fully effective, as it may be used in association with antiretroviral drugs. Thus, the therapeutic setting provides the unique opportunity to evaluate vaccine efficacy in a more rapid and convenient manner, hopefully speeding up the identification and development of an effective vaccine candidate. In this regard, novel biomarkers (in addition to viral load and CD4 T-cell counts) should be taken into consideration in trial design in order to assess the achievement of efficacy end points (i.e., assessment of functional T and B cell subsets, cellular and biochemical immune activation biomarkers, proviral DNA in reservoirs, cell-to-cell virus transmission and virus neutralization). This approach imposes new challenges to the scientific community, vaccine developers and regulatory bodies, which require new paradigms and a new ‘way ahead’. Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Acknowledgments The authors thank MT Maggiorella, L Sernicola, MR Pavone Cossut, B Collacchi, S Moretti, F Ferrantelli, A Scoglio, MJ Ruiz Alvarez, C Ariola, M Campagna, F Stivali (National AIDS Center, Istituto Superiore di Sanita, and Pathology and Microbiology, San Gallicano Hospital, ‘Istituti Fisioterapici Ospitalieri’, Rome, Italy) for laboratory support; and F Cammisa and G Fornari Luswergh (National AIDS Center, Istituto Superiore di Sanita, Rome, Italy) for support to study management and editorial assistance, respectively; S De Menna, A Biondi, S Tobelli and F Fedeli (National AIDS Center, Istituto Superiore di Sanita, Rome, Italy) for administrative support.

Declaration of interest This paper is part of a supplemental issue, sponsored by SciClone. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents, received or pending, or royalties.

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Development of a novel AIDS vaccine: the HIV-1 Tat protein vaccine

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Affiliation Aurelio Cafaro1, Antonella Tripiciano1, Cecilia Sgadari1, Stefania Bellino1, Orietta Picconi1, Olimpia Longo1, Vittorio Francavilla1, Stefano Butto`1, Fausto Titti1, Paolo Monini1, Fabrizio Ensoli2 & Barbara Ensoli†1 † Author for correspondence 1 Istituto Superiore di Sanita, National AIDS Center, Rome, Italy E-mail: [email protected] 2 San Gallicano Institute, ‘Istituti Fisioterapici Ospitalieri’, Pathology and Microbiology, Rome, Italy

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Development of a novel AIDS vaccine: the HIV-1 transactivator of transcription protein vaccine.

Classical approaches aimed at targeting the HIV-1 envelope as well as other structural viral proteins have largely failed. The HIV-1 transactivator of...
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