New challenges in therapeutic vaccines against HIV infection.

Expert Review of Vaccines

ISSN: 1476-0584 (Print) 1744-8395 (Online) Journal homepage: http://www.tandfonline.com/loi/ierv20

New challenges in therapeutic vaccines against HIV infection Lorna Leal, Constanza Lucero, Josep M Gatell, Teresa Gallart, Montserrat Plana & Felipe García To cite this article: Lorna Leal, Constanza Lucero, Josep M Gatell, Teresa Gallart, Montserrat Plana & Felipe García (2017) New challenges in therapeutic vaccines against HIV infection, Expert Review of Vaccines, 16:6, 587-600, DOI: 10.1080/14760584.2017.1322513 To link to this article: http://dx.doi.org/10.1080/14760584.2017.1322513

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Date: 16 October 2017, At: 04:29

EXPERT REVIEW OF VACCINES, 2017 VOL. 16, NO. 6, 587–600 https://doi.org/10.1080/14760584.2017.1322513

REVIEW

New challenges in therapeutic vaccines against HIV infection Lorna Leala, Constanza Luceroa, Josep M Gatella, Teresa Gallartb, Montserrat Planab and Felipe García

a

Downloaded by [La Trobe University] at 04:29 16 October 2017

a Infectious Diseases Unit, HIVACAT, Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain; bRetrovirology and Viral Immunopathology Laboratories, HIVACAT, Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain

ABSTRACT

ARTICLE HISTORY

Introduction: There is a growing interest in developing curative strategies for HIV infection. Therapeutic vaccines are one of the most promising approaches. We will review the current knowledge and the new challenges in this research field. Areas covered: PubMed and ClinicalTrial.gov databases were searched to review the progress and prospects for clinical development of immunotherapies aimed to cure HIV infection. Dendritic cells (DC)-based vaccines have yielded the best results in the field. However, major immune-virologic barriers may hamper current vaccine strategies. We will focus on some new challenges as the antigen presentation by DCs, CTL escape mutations, B cell follicle sanctuary, host immune environment (inflammation, immune activation, tolerance), latent reservoir and the lack of surrogate markers of response. Finally, we will review the rationale for designing new therapeutic vaccine candidates to be used alone or in combination with other strategies to improve their effectiveness. Expert commentary: In the next future, the combination of DCs targeting candidates, inserts to redirect responses to unmutated parts of the virus, adjuvants to redirect responses to sanctuaries or improve the balance between activation/tolerance (IL-15, anti-PD1 antibodies) and latency reversing agents could be necessary to finally achieve the remission of HIV-1 infection.

Received 14 November 2016 Accepted 20 April 2017

1. Introduction Currently, over 36 million people worldwide are infected with HIV, most of them living in developing countries. In 2013, 1.5 million people died of AIDS-related illnesses. Combined antiretroviral therapy (cART) has proven to be highly effective to prevent clinical progression and death [1]. In addition, recent evidence from clinical trials has confirmed the powerful impact that ARV drugs can have on the AIDS pandemic as part of an effective option for HIV prevention [2,3]. As a consequence of these preventive and therapeutic efforts, the global incidence of HIV infection has stabilized and has begun to decline in many countries. Despite these successes, current cART has a number of public health, economical and clinical limitations. First, the current European Guidelines for treatment of HIV-infected adults in Europe 2016 (EACS: http://www.eacsociety.org/guide lines/eacs-guidelines/eacs-guidelines.html) agrees that all HIVinfected patients should be treated, irrespective of the clinical stage and as early after infection as possible to reduce morbidity and mortality in patients and to reduce sexual transmission [4]. UNAIDS has proposed to curb the epidemic and reduce the number of new infections from 1.4 million in 2014 to half a million in 2020. To reach this aim, UNAIDS recommends that 90% of people living with HIV should know their HIV status, 90% of people with diagnosed HIV infection should receive sustained antiretroviral therapy and 90% of people receiving antiretroviral therapy should have viral suppression (http://www.unaids.org/en/resources/docu ments/2014/90-90-90). However, poor engagement in care CONTACT Felipe García

[email protected]

Therapeutic vaccine; immunotherapy; HIV-1; functional cure; reservoir

for HIV-infected individuals will substantially limit the effectiveness of these test-and-treat strategies [5]. Although 80% of HIV-infected individuals in USA and Europe know that they are HIV infected, only 30% and 50%, respectively, are virally suppressed (and with low probability of sexual transmission) [5,6]. Therefore, it is not clear how to implement this proposal and this problem has consequences. As no effective preventive HIV vaccine is currently available [7] and preventive strategies have failed [3], still too many people are acquiring HIV infection (142,000 new HIV infections diagnosed in Europe in 2014, the highest number ever) (http://www.euro.who.int/en/mediacentre/sections/press-releases/2015/11/highest-number-ofnew-hiv-cases-in-europe-ever). Currently, the number of new infections still surpasses the number of new patients on treatment [8]. It is estimated that for every HIV-infected person who starts antiretroviral therapy, two individuals are newly infected with HIV; this is clearly unsustainable (UN SecretaryGeneral. Uniting for universal access: toward zero new HIV infections, zero discrimination and zero AIDS-related deaths. United Nations 2011). Second, although access to treatment has significantly improved for patients in developing countries over the last years, only 41% of the individuals in need of treatment (15 millions) receive standard cART. The progressively higher cost of the medication (the current calculated yearly cost of treating all HIV-infected patients in Western and Central Europe is approximately 7 billion €), remain as an important issue to implement the UNAIDS strategy.

Infectious Diseases Unit, Hospital Clínic, Villarroel, 170, 08036 Barcelona, Spain

© 2017 Informa UK Limited, trading as Taylor & Francis Group

KEYWORDS


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Finally, the treatment by itself is unable to cure or eradicate the infection [9]. Indeed, it only suppresses the HIV productive replication cycle in infected cells. This cycle involves the integration of HIV DNA into the genome of a host cell, and the generation of new viral particles that are released by budding from the cell. A small proportion of cells survives the cell-lytic consequences of infection, and goes on to form a latent reservoir of cells that have HIV DNA integrated into host-cell chromosomes [9]. This reservoir can rekindle a raging infection if cART is interrupted [10,11]. Consequently, the patients necessitate cART throughout their lives. Risk of viral resistance development and adverse effects in the medium-long term are important limitations for lifelong adherence to antiretroviral treatment and for widespread use mainly in developing countries, but also in the developed world. Moreover, standard cART do not fully restore health or a normal immune status in HIV-infected individuals [12], and patients still experience comorbidities, such as increased cardiovascular disease, bone disorders, and cognitive impairment [13]. These concerns highlight the urgency by which new therapeutic strategies must be developed and tested. A safe, affordable, and scalable cure could address both the individual and public health limitations that are associated with lifelong cART. The scientific community has acknowledged this position and there is a growing interest in developing curative strategies to tackle HIV [14,15], and therapeutic vaccines are one of the most promising approaches. Irrespective of the complexity of the procedure, the cost and feasibility of a strategy limited in time would be more affordable than cART for life. There are a number of excellent reviews about therapeutic vaccines and immunotherapies in recent years [16–20]. Here, we will update the current knowledge in this research field and will review the new challenges associated with this strategy.

2. The functional cure of HIV infection: immune system and control of viral replication In an Opinion article of the International AIDS Society about the functional cure [14], the authors argue that two broadly defined approaches for curing HIV infection were considered by the group: first, the elimination of all HIV-infected cells (a sterilizing cure); and, second, the generation of effective host immunity to HIV that would result in lifelong control of the virus in the absence of therapy, despite not achieving the complete eradication of HIV (a functional cure). Intensification of cART [21], stem cell therapy [22], gene therapy [23], cell therapy [24,25], immune-based therapies (therapeutic vaccines [16,26,27], broadly neutralizing antibodies (bNAbs) [28–30], passive immune therapy [31], structured treatment interruptions [32], immunosuppressors [33,34], cytokines [35–38], checkpoint inhibitors [39]) and activation of HIV-1 from latent reservoir [40–42] are different strategies to improve the immune system or to target HIV-1 reservoirs that have been or are currently being assessed to achieve an HIV cure [43]. Some of these strategies are not effective [21,31–36], have potential safety problems [22–24,39], are technically very complicated [22], and/or are in a very preliminary stage of development [24]. Based on the data available [20], immune-based therapies in combination with other approaches appear to be one the best options to obtain a functional cure. In fact, the International AIDS Society global scientific strategy toward an HIV cure [15]

argues that the development of immune-based strategies that control viral replication would be very relevant for defining the path to a functional cure. Therefore, T cell vaccines that would be able to enhance HIV-specific immunity would remain a priority. Such strategies might also limit the replenishment of viral reservoirs that may occur through ongoing replication in treated subjects (particularly in tissues) and hence could help to achieve a sterilizing cure. A good natural model of functional cure is found in a small proportion of HIV-infected patients that show a lack of clinical progression associated with strict control of viral replication in the absence of any treatment (they have referred as elite controllers). This so-called ‘functional cure’ has been linked with low level of viral reservoir and potent HIV-specific immune responses observed in these patients, including CD8+ T cell-mediated viral suppression [44]. The main limitation to achieve these characteristics in chronic HIV-infected patients is the inability of cART to eliminate the persistent reservoir of latently infected cells or improve the HIV specific immune response [45,46]. This situation leads to a rapid reemergence of viremia and disease progression if cART is interrupted [11,32]. The objective of a therapeutic vaccine in ‘noncontroller patients’ would be to induce or augment immune responses against HIV infection using a planned exposure to HIV viral antigens in a strong immunogenic context. The main question to be answered is whether the immune system has the capacity to clear or control HIV without cART. A major limitation is that the types of immunological responses able to control viral replication are not accurately known. Some data suggest that strong T cell-mediated immunity to HIV can indeed limit virus replication and protect against CD4 depletion and disease progression in chronic HIVinfected patients. Direct data on the critical role of the CTL response in the control of viral replication have been obtained both in the infection model with macaques devoid of CD8 + T lymphocytes [47,48], and in the immunodeficient murine model [49]. Recently, Cartwright et al. [50] have shown that depletion of CD8+ lymphocytes in SIV-infected treated macaques resulted in increased plasma viremia in all animals and that repopulation of CD8 + T cells was associated with prompt reestablishment of virus control. These data suggest that CD8+ lymphocytes are required for maintaining viral suppression even in SIV-infected macaques treated with cART. There is also clear evidence that a specific helper T response against HIV is crucial in obtaining an optimal specific CTL response, which can control viral replication both in humans and in animal models [51,52]. This concept is consistent with reported data on other chronic viral infections [53].

3. Types of therapeutic vaccines In the last 30 years, important efforts have been performed to develop a successful vaccine. Initially, ‘classical approaches’ such as whole inactivated virus (REMUNE) [54] or recombinant protein (gp120) [55] were tested as therapeutic vaccines. In general, the capacity of these early vaccines, as well as those based on peptides [56] or DNA vectors [57], to increase the HIVspecific responses and control viral load were very limited. New approaches have been used in recent years, based on more innovative vectors such as recombinant viral vectors or

EXPERT REVIEW OF VACCINES

autologous dendritic cells (DCs). Regretfully, although most viral vector vaccines were able to induce HIV-specific immune responses in clinical trials, they showed very limited efficacy to control viral replication [58]. Finally, DC-based vaccines, on the other hand, have yielded the best results in this field [26]. The safety profile has been excellent with only minor local side effects reported in some clinical trials. No severe side effects or

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induction of autoimmunity have been reported. See Table 1 for a comprehensive explanation of different types of vaccine tested in HIV-1-infected patients. Despite these initial poor results, some innovative work has permitted to improve the immunogenicity of some candidates and obtain partial virological responses [26,68,76,79,86]. A promising candidate is a potent cytomegalovirus (CMV) vector

Table 1. Therapeutic vaccine types used in clinical trials. Description Whole-inactivated virus/Replication defective virus - HIV-1 immunogen (REMUNE) [59]


- HIVAX [60] Proteins/subunits - gp120 [55,61,62] - gp160 [63–67] - Tat [68–72] Peptides -HIV-v [73] -C4-V3 [74] - VAC3S [75] - VACC-4X [76] - F4/AS01B [56] - Afo-18 [77] DNA - multiantigen (MAG) DNA vaccine [78]

®

- GTU -Multi-HIVB [79] - pTHr.HIVA [57] - VRC-HIVDNA009-00-VP [80] -

VRC-HIVDNA016-00-VP [81] EP HIV-1090 [82] Dermavir [83] PENNVAX-B [84]

- APL 400–047 and APL 400–003 [85] - pGA2/JS7 DNA [86]

- A 1:1 mixture of whole, inactivated (with beta-propiolactone and 60Cobalt irradiation) HIV-1 virus particles and incomplete Freund’s adjuvant (IFA). - Replication-defective HIV-1 attenuated by multiple deletions of the pol, vif, env, nef genes and pseudotyped with Vesicular Stomatitis Virus G protein (VSV-G) - Purified MNrgp120 protein absorbed to aluminum hydroxide adjuvant - Recombinant HIV-1 IIIB gp160 (rgp160 IIIB) - Recombinant Tat vaccine [68–70], synthetic protein of 101 amino acid residues (Tat Oyi)[71], Tat fragments (TUTI-16) [72] - Sterile equimolar admixture of four multi-epitope polypeptides VPR (30 aa), VIF (40 aa), REV (27 aa), NEF (43 aa) - Synthetic peptide vaccine representing 4 epitopes from HIV gp120, including an HLA B7-restricted CTL epitope - A highly conserved motif of HIV gp41 envelope protein (called 3S) where interacts with CD4 + T cells to induce NKp44L - Conserved domains on the HIV-1 core protein p24Gag. - Recombinant fusion protein (F4) containing 4 HIV-1 clade B antigens (p24, reverse transcriptase, Nef, and p17) combined with the AS01B adjuvant system - 15 HLA-supertype-restricted subdominant and conserved CD8 T cell epitopes and three CD4 T-helper cell epitopes - 2 pDNA expression vectors: a single promoter expression vector encoding an HIV-1 clade B Gag/Pol fusion and a dual promoter expression vector encoding an HIV-1 clade B Nef/Tat/Vif fusion and a clade B primary isolate (6101) Envelope gp160. It is codelivered with a third expression plasmid encoding the p35 and p40 subunits of human interleukin-12 (IL-12). - Containing genes nef, rev and tat, parts of the structural proteins coded by gag and a stretch of selected T helper (Th) and CTL epitopes coded for pol and env. E2, a transcriptional regulator from bovine papilloma virus (BPV) was added - Encodes an immunogen, HIVA, comprising HIV-1 clade A p24/p17 fused to a string of cytotoxic T-cell epitopes - 4 plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope constructs from HIV-1 subtypes A, B and C - Encodes for clade B Gag, Pol, and Nef and clade A, B, and C Env - 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 - 15 HIV antigens in a synthetic pDNA nanomedicine formulation - Three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation - APL 400–047 is a gag/pol plasmid, APL-400–003 plasmid encodes a modified env gene and a rev gene from an HIV-1MN isolate under the control of the human cytomegalovirus promoter - Encodes for Gag–Pol–Env

Viral vectors - ALVAC (Canary pox) *vCP1452 [87–91]

- Expresses the gene products of the HIV-1MNstrain gp120 and the LAI strain including the anchoring region of gp41, p55 gag polyprotein, the protease, and reverse transcriptase and Nef CTL epitopes - Expresses for gp120 (MN strain) and a part of the anchoring transmembrane region of gp41 (LAI strain); for the p55 *vCP1433 [92] polyprotein, expressed by gag (LAI strain); for a portion of pol encoding the protease; and for genes expressing cytotoxic T lymphocyte peptides from pol and nef - MVA (Modified vaccinia Ankara) - Expresses Bx08 monomeric gp120 and the fused IIIB Gag-Pol-Nef (GPN) polyprotein of clade B *MVA-B [93] - Expresses nef *MVA-nef [94] - Expresses Gag-Pol and Env *MVA62B [86] - Delivers a combination of HIV-1 gag/pol with the cytokine human interferon-gamma gene - Fowlpox [95] -Adenovirus5(VRC-HIVADV014-00-VP) [81] - Encodes clade B Gag, Pol and clade A, B, and C Env (adenoV5) - See Table 3 for HIVconsv - ChAd-MVA.HIVconsv [96]

DC-based vaccines - peptides [97–101] - whole inactivated virus [26,102] - ALVAC-HIV vCP1452 [103] - mRNA [104–107] - Apoptotic bodies [108] RNA - iHIVARNA.01 [109]

- Env, gag, pol [97]; gag [98]; env, gag, pol [99]; env, gag, pol, vpu, vif [100]; Lipopeptides gag, pol, nef, CD40L [101] - Chemically inactivated virus (AT-2) [102], heat inactivated virus [26] - Expresses the gene products of the HIV-1MNstrain gp120 and the LAI strain including the anchoring region of gp41, p55 gag polyprotein, the protease, and reverse transcriptase and Nef CTL epitopes - Autologous RNA encoding HIV Gag, Nef, Rev, and Vpr [104]; encoding Tat, Rev and Nef [105]; encoding Gag and a chimeric Tat-Rev-Nef [106]; encoding Gag and Nef [107] - Autologous HIV-1–infected, apoptotic CD4 + T cells - Encoding activation signals (TriMix: CD40L+CD70+caTLRA4) combined with HIV antigenic sequences (HTI sequence: comprised of 16 joined fragments from Gag, Pol, Vif and Nef)


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reengineered to stimulate sustained responses to novel, nonimmunodominant epitopes that has shown promise in nonhuman primate models [110]. Hansen et al. found that 50% of rhesus macaques vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors manifested durable, aviremic control of infection [110,111]. These are some of the best results in the field of preventive HIV vaccines and clinical trials in humans are expected; whether this vaccine is also useful as a therapeutic vaccine, i.e. to control viremia when administered to infected animals remains to be tested. In any case, it seems that these RhCMV/SIV were able to induce broadly targeted MHC-E–restricted CD8 + T cell responses [112]. It is possible that these types of responses could be responsible of the impressive control of viral load observed in these animals. In addition, these MHC-E-restricted CD8 + T cell responses have the potential to exploit pathogen immuneevasion adaptations. If it could be replicated in humans, it would open the door to attack pathogens with novel immune responses that they are not adapted to evade. Recently, it has been proposed the direct administration of mRNA targeting DCs as an alternative to the classic antigen forms mentioned above. Compared with other gene-based approaches such as recombinant viruses or plasmids, mRNA is expected to have a much better safety profile because it is only transiently present in the cell, and is a naturally and quickly degraded molecule, in contrast to plasmid DNA, which could permanently modify the cells and carries the potential risk of insertional mutagenesis. For this reason, mRNA-based therapies have been classified as nongene therapy by the American (Food and Drug Administration) and German (Paul Ehrlich Institute) authorities. Lyophilized mRNA can be stored at elevated temperatures without loss of activity and is not less stable than other conventional vaccines that require a cold chain to retain efficacy. In addition, compared with proteins, mRNA is easier to produce and to store regardless of the encoded antigen. In contrast to peptides, mRNA-based vaccination offers the advantage of not being restricted to a defined HLA allele of individuals and is thus broadly applicable to all patients, irrespective of their HLA haplotype. Promising preliminary results have been reported in other infectious diseases and cancer with this candidate. Indeed, an mRNA influenza vaccine candidate has demonstrated similar efficacy to licensed vaccines in animal models [113] and direct administration of mRNA has entered clinical testing in cancer [114–116]. Our group has recently performed the first in-humans clinical trial with intranodal naked mRNA encoding activation signals (TriMix: CD40L+CD70+caTLRA4) combined with HIV antigenic sequences (HTI sequence: comprising 16 joined fragments from Gag, Pol, Vif and Nef) (NCT 02413645) that has shown promising results in the in vitro and in vivo preclinical tests [109].

4. Therapeutic vaccine clinical trials A number of reviews have detailed the therapeutic vaccine clinical trials performed in the last years [16–20]. Following a search in clinicaltrials.gov, we can conclude that 93 therapeutic vaccine clinical trials have been performed and 17 are currently ongoing. Different strategies and candidates have

been used in these clinical trials [whole inactivated virus (n = 7), HIV proteins (n = 19), HIV peptides (n = 17), DNA (n = 11), recombinant viral vectors (n = 22), DC-based vaccines (n = 17)]. Although many candidates have been immunogenic, the ability to control viral replication after interruption of cART have been limited [16]. We detail below the results of 5 representative clinical trials that used HIV proteins, HIV peptides, DNA, recombinant viral vectors or DC-based vaccines. Ensoli et al. [68–70] have proposed that as Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during cART, a Tat-based vaccine could be a good candidate to be used as a therapeutic vaccine. A phase II multicenter, randomized, open-label, therapeutic trial (ISS T-002) conducted in 11 clinical centers in Italy on 168 HIV-positive subjects under cART demonstrated that the vaccine was safe and well tolerated and induced anti-Tat Abs in most patients [70]. A significant reduction of blood proviral DNA was seen after week 72 in patients receiving Tat 30 μg given 3 times. This decay was significantly associated with anti-Tat Abs. A second 48-week randomized, double-blinded, placebo-controlled trial (ISS T-003) was performed to evaluate immunogenicity and safety of B-clade Tat (30 μg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART [68]. Immunization was safe and well tolerated and induced durable, high titers of anti-Tat B-clade antibodies in most vaccinees. Tat vaccination increased CD4 + T-cell numbers, particularly when baseline levels were still low after years of therapy, and this had a positive correlation with HIV neutralization. The authors propose that their vaccine candidate could improve the T cell subset homeostasis not completely recovered in HIV-1treated patients. Although immunogenicity and efficacy of some of the peptide-based immunogens used have been poor, some studies have yielded promising results. A multinational doubleblind, randomized phase II clinical trial assessed the efficacy, safety, and immunogenicity of Vacc-4x a peptide-based HIV-1 therapeutic vaccine targeting conserved domains on p24Gag [76]. While no differences were observed between vaccine and placebo groups in the proportion of patients who reinitiated cART (primary end point) or in CD4 T cell counts, a significant difference in viral load was noted for the Vacc-4x group. A new clinical trial combining this vaccine with a latency reversing agent (Romidepsin) (see below) have recently been reported with promising results [117]. Regarding DNA-based vaccines, Vardas et al. [79] performed a placebo-controlled phase II clinical trial with a recombinant plasmid DNA, GTU-multi-HIVB, containing 6 different genes derived from an HIV-1 subtype B isolate, in 63 untreated HIV-1-infected patients. The vaccine was safe and both a significant decline in viral load and increases in CD4 + T cell counts were observed in the vaccine group compared to placebo. A prime-boost strategy with DNA/adenovirus 5 was tested in a double-blind study in 17 HIV-infected individuals [81]. The vaccine was able to induce significantly stronger HIV-specific T cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire. However, no changes in single-copy viral load or the frequency of latent infection were observed.



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Finally, DC-based vaccines have yielded the best results in this field [27]. Our group has recently reported the results of a blinded placebo controlled clinical trial which suggest that HIV-1-specific immune responses elicited by therapeutic DC vaccines (made up of DCs pulsed with heat inactivated autologous whole virus) could significantly reduce the plasma viral load (pVL) set-point (mean peak drop of pVL of −1.2 log10 copies/ml) after cART interruption in most chronically HIV-1infected patients treated in early stages [26]. At weeks 12 and 24 after cART interruption, a decrease of plasma viral load setpoint ≥1 log was observed in 12 of 22 (55%) versus 1 of 11 (9%) and in 7 of 20 (35%) versus 0 of 10 (0%) patients in the DC-HIV-1 and DC-control groups, respectively. This significant decrease in plasma viral load observed in immunized recipients was associated with a consistent increase in HIV-1-specific T cell responses. These findings are some of the best, most solid data demonstrating a benefit of therapeutic vaccination. Although not yet a functional cure, these results support future studies optimizing a therapeutic vaccine to control HIV-1 replication in infected patients and emphasize the importance of targeting DCs to improve antigen presentation and specific immune response. A number of potential strategies to improve the outcomes of MD-DC-based therapeutic vaccines should be taken in account in the next generation of these candidates. Different adjuvants could be used combined with the immunogen to improve antigen presentation [118]. Although it is known that MD-DC migrate to regional lymph nodes after acquiring the antigen, it remains uncertain whether MD-DC generated in vitro exhibit an equivalent migratory capacity. In fact, it seems that less than 10% of intradermally/subcutaneously injected MD-DC migrate to lymph node [119]. New routes of injection (i.e. intranodally), new strategies to favor migration or in vivo DC targeting immunogens could be tested to improve their effectiveness. Table 2 summarizes the therapeutic vaccine clinical trials that are currently ongoing. New candidates (VSV, mosaic DNA/ adenovirus 26) will be tested in 131 primary-infected and 969 chronic-infected patients in the next few years.

that could be of high avidity, broad polyfunctionality, and superior in vitro virus-suppressive capacity. Vaccination depends on the uptake of immunogen by DCs and its potential to induce the optimal maturation of DCs. There is evidence that some HIV-1 vaccines by themselves are insufficient to fully harness the stimulatory potential of DCs. It has been suggested that targeting in vivo DCs by costimulatory molecules improve the effectiveness of the vaccines [120,121]. This type of strategy has already been tested in humans with a vaccine co-expressing immune activator molecules. A clinical trial testing a recombinant fowlpox virus vector coexpressing HIV-1 Gag/Pol and human interferon-gamma has been reported [95,122]. Although the results were modest, showing only a trend toward lower rates of HIV replication following cessation of ART of patients who received the vaccination as compared to placebo, this type of design should be taken into account to plan new candidates able to co-express stimulatory molecules. Immunomodulation by mRNA-based vaccines has also been proposed [120,123]. It has been suggested that naked mRNA or other type of vaccines are insufficient to fully harness the stimulatory potential of DCs. Therefore, Van lint et al. [120] have designed mRNAs encoding a mixture of antigen presenting cell (APC) activation molecules, including CD40L, a constitutively active variant of Toll-like receptor (TLR) 4 and CD70 (referred as TriMix). The aim is to induce DC maturation with CD40L and TLR4 and to support activated T cell survival and proliferation with CD70. DCs in vitro or in vivo modified with TriMix mRNA have been shown to be significantly more potent and immunogenic than unmodified DC [120]. In addition, mRNA encoding TriMix in combination with HIV antigens confers a T cell stimulatory capacity to DCs and enhances their ability to stimulate antigen-specific immunity in vitro and in vivo in animal models as compared with mRNA expressing HIV antigens alone [109]. This candidate (iHIVARNA.01, NCT02888756) has already been tested in a phase I clinical trial in HIV-1-infected patients in the iHIVARNA project in a dose-escalating dose. Preliminary results are expected for the end of 2016.

5. Major obstacles to achieve a functional cure

5.2. CTL escape mutations

Despite the relevance of HIV-specific immune responses both in acute and chronic stages of the infection, these responses are not able to control viral replication to undetectable level and avoid the progression of the infection to AIDS in most of the patients. This means that major immune-virologic obstacles can cause resistance to current vaccine strategies. The antigen presentation by dendritic cells (DCs), CTL escape mutations, B cell follicle sanctuary, host immune environment (inflammation, immune activation, tolerance), latent reservoir and the lack of surrogate markers of response are some of the major drawbacks that should be addressed by rationally designing therapeutic vaccine candidates alone or in combination with other strategies to improve the effectiveness of the different immunotherapies.

The vast majority (98%) of latent viruses in chronic HIV-1-infected patients carry CTL escape mutations that render infected cells insensitive to CTLs directed at standard (canonical) epitopes [124]. It is likely that most of the therapeutic vaccines preferentially expand preexisting clones which are exhausted and target escape variants. There is specific interest in approaches that stimulate responses against novel, nondominant epitopes [125–127]. New therapeutic vaccine candidates should re-direct the responses toward vulnerable sites and away from irrelevant epitopes. It has been demonstrated in animal model of cancer [128] and HIV-1 [124] and in humans in cancer [129] that a personalized mRNA [128], peptide [124], or dendritic cell [129] -based vaccine, produced by the prioritization of unmutated epitopes, can redirect the CTL responses to these epitopes and in HIV-1 infection eliminate target cells infected with autologous virus derived from the latent reservoir. Table 3 shows 4 different approaches that are currently being assessed in animal models and clinical trials in humans.

5.1. Antigen presentation by DCs A correct antigen presentation by DCs is of paramount importance for a vaccine to induce robust and broad T cell responses

- iHIVARNA.01 NCT02888756

RNA - iHIVARNA.01 NCT02413645a

- N = 38, chronic, undetectable viremia on cART, CD4 > 600/mm3

- N = 33, cART during PHI, CD4 > 450/mm3

- N = 27, cART during PHI (Fiebig I-IV)


- Immunogenicity, reservoir

- Safety, immunogenicity, reservoir, time to viral load rebound after cART discontinuation

- Safety, immunogenicity, reservoir

- N = 21, chronic, undetectable viremia on cART, CD4 > 450/mm3 - N = 70, chronic, undetectable viremia on cART, CD4 > 450/mm3


- N = 12, PHI or chronic, undetectable viremia on cART, CD4 > 350/mm3

- Phase I, dose-escalation injections of iHIVARNA.01 at weeks 0, 2 and 4 - Phase II, double-blind, placebo controlled, multicenter study

- Single-center, national clinical trial, phase I, randomized, prospective, placebocontrolled, partially masked, parallel group - Phase I/II, single-site, pilot study, 2 arms: 6 patients cART initiated during PHI and 6 chronic - phase I study, one arm

- Dose-escalation (2 × 109 VP, 2 × 1010VP or 2 × 1011VP) injections of Adenovirus vector vaccine (Ad5-gag) at weeks 0 and 4 - Pilot phase I/IIa study, double blind placebo controlled study - Phase I Randomized, Double-Blind, PlaceboControlled Study - Phase I/II is a randomized, double-blind and placebo controlled study, dose-escalation (5 × 106 TU, 5 × 107TU or 5 × 108VP) injections of THV01-1

- Pilot phase I, roll-over of ChAd-MVA. HIVconsv_BCN01 study - Two-arm, open-label randomized study

- N = 105 (70 vaccine), chronic, undetectable viremia on - Multi-center double blind randomized cART, CD4 > 600/mm3 versus placebo phase II - N = 30, chronic, undetectable viremia on cART, - Randomized Phase I/II, transcutaneous vs. 3 CD4 > 200/mm electroporation

- Safety, viral load set-point at week 12, time to rebound, - N = 32, chronic, undetectable viremia on cART, reservoir, immunogenicity CD4 > 450/mm3

- Safety, viral load set-point after cART discontinuation, reservoir, immunogenicity - Safety, reservoir, viral load rebound after cART discontinuation - Safety, immunogenicity

- Safety, immunogenicity

- Dose escalating, 3 injections of placebo, Tat Oyi 11, 33 or 99 microg

- Multi-center double-blind randomized Phase III

Design

- N = 15/N = 90, chronic, undetectable viremia on cART, - Dose escalating, every 4 weeks for 3 months followed by 3 maintenance CD4 200–500/mm3 vaccinations every 12 weeks after the third initial vaccination, dose VAC-3S 16, 32 and 64 µg/ml


- N = 500, on CART, chronic

Patients

- Safety, reservoir, immunogenicity, time to rebound after - N = 15, cART during PHI, CD4 > 500/mm3 cART discontinuation, proportion below 2000 copies/ml - Reservoir, immunogenicity and markers of inflammation - N = 52, PHI starting cART

- AGS-004, autologous mRNA + vorinostat NCT02707900 - Safety, reservoir

- AGS-004, autologous mRNA NCT02042248a

DC-based vaccines - Autologous whole inactivated virus + Pegylated interferon NCT02767193

- HIV-MAG pDNA vaccine prime (IL-12 adjuvant) rVSV HIV gag boost NCT01859325 - THV01-1: lentiviral vector encoding an HIV antigen NCT02054286

- Ad26.Mos.HIV and MVA-Mosaic NCT02919306

- ChAdV63.HIVconsv prime MVA.HIVconsv boost vaccines + vorinostat NCT02336074 - Adenovirus 5 gag NCT02762045

Viral vectors - MVA.HIVconsv + romidepsin NCT02616874

® ®

Clinical trials completed, not yet reported.

a

- Safety, anti-3S ab, markers of progression to AIDS, reservoir

- Optimal dose, safety, viremia undetectable after cART interruption

- Changes in HIV viral load at Week 52

End points

DNA - GTU -Multi-HIVB + Lipo5 (lipopeptide) NCT01492985 - Plasma HIV-RNA after stopping antiviral treatment, immunogenicity, reservoir - GTU -Multi-HIVB NCT 02457689a - Safety, immunogenicity

Peptides - VAC3S NCT02390466/NCT02041247

Proteins/subunits - Tat Oyi NCT01793818

Characteristics. ClinicalTrials.gov code Whole-inactivated virus/Replication defective virus - HIV-1 immunogen (REMUNE)+ AMPLIVAX 1.0 NCT02366026

Table 2. Ongoing therapeutic vaccine clinical trials.


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Table 3. Innovative antigenic targets proposed in different therapeutic vaccine candidates to redirect the response to unmutated/non dominant epitopes. Candidate HIVACAT-T immunogen (HTI) [125,127] HIVconsv [130,131] p24CE1 and 2 [132,133]


Bivalent HIV-1 mosaic antigens [134,135]

Insert 16 segments (between 11 to 78 aa) linked to each other by single, dual or triple alanine amino acids linkers. The final polypeptide sequence included the regions p17, p24, p2p7p1p6, protease, RT, integrase, Vif, nef 14 most conserved subprotein domains of HIV-1 irrespective of known CD8 + T-cell epitopes. For each region, the consensus aa sequence alternates between the four major clades A, B, C, and D, including Gag (A, C, D), Pol (A, B, C, D), Vif (D) and Env (C, D) Sequences of at least 8 aminoacids in length, in which all aminoacids were conserved in at least 98% of all sequences and correlate epitope recognition with clinical viral load. Two plasmids, each with 7 conserved segments from 12–24 AA in length, separated by 2–4 AA spacers and differing only by the single toggle AA. Polyvalent “mosaic” proteins assembled from natural sequences by in silico recombination and optimized to provide maximal coverage of potential T cell epitopes for a given valency: single clade C Gag, Pol, and Env natural sequences (C.IN.-.70177 Gag, C.ZA.04.04ZASK208B1 Pol, C.SN.90.90SE_364 Env). clade B antigens (B.CAM-1 Gag, B.IIIB Pol, B.Con Env)

Mothe et al. [125] proposed a rational design for the selection of the HIV antigens based on the viral targets of protective HIV-1-specific T cell responses from three large cohorts of HIV-infected individuals [125]. They studied 950 untreated HIV1- clade-B or -C-infected individuals for responses to sets of 410 overlapping peptides (OLPs) spanning the entire HIV-1 proteome. For each OLP, a ‘protective ratio’ (PR) was calculated as the ratio of median viral loads (VL) between OLP nonresponders and responders. Of the 410 overlapping peptides, 26 OLPs were identified as beneficial as the group of individuals who reacted to these OLPs had a significantly reduced median viral load compared to the group of OLP nonresponder. These beneficial OLPs were located in HIV Gag protein (n = 10), Pol (n = 12), Vif (n = 3) and Nef (n = 1) proteins of the virus. This approach resulted in the design of 16 segments (between 11 and 78 aa each) that were linked to each other by single, dual or triple alanine amino acids linkers to ensure optimal processing and to avoid premature epitope digestion. These regions formed the basis of the HIVACAT T cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8 + T cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. Mice immunized with mRNA TriMiX also encoding HTI [109] and mice and rhesus macaques immunized with DNA/MVA expressing HTI [127] showed broad and balanced T cell responses to several segments within Gag, Pol, and Vif. These data demonstrate that it is possible to redirect responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T cell responses toward variable and less protective viral determinants. The mRNA iHIVARNA.01 candidate encodes HTI and as mentioned above preliminary results in humans will be available at the end of 2016. Two groups have hypothesized that T cell vaccines targeting the most conserved regions of the HIV-1 proteome will induce more efficient immune response than whole protein-based T cell vaccines [130,132]. They argue that these conserved regions are common to most variants and bear fitness costs when mutated. Letourneau et al [131] designated HIVconsv, by assembling the 14 most conserved regions of the HIV-1 proteome into one chimeric protein. They inserted the gene coding for the HIVconsv protein in MVA, chimpadenovirus and DNA. A recent clinical trial has shown

Vectors mRNA TriMIX.HTI DNA.HTI MVA.HTI ChAd.HTI ChAd.HIVconsv MVA.HIVconsv p24CE DNA

Adenovirus 26 MVA

the safety and immunogenicity of ChAd.HIVconsv and MVA. HIVconsv therapeutic vaccines in a cohort of early treated HIV-1infected individuals [96]. Twenty-four individuals identified with recent HIV infection received an intramuscular ChAdV63.HIVconsv prime MVA.HIVconsv boost immunization after 6 months under cART. No unspecific expansion of T cells targeting HIV-1 regions outside HIVconsv insert was noted, allowing for an optimal focusing of T cell responses on conserved regions and demonstrating the ability of this candidate to shift preexisting immune responses toward conserved, vaccine-encoded regions of HIV [96]. The other group that has focused in designing an immunogen by selecting conserved parts of the HIV genome selected seven highly conserved elements (CE) identified in HIV-1 p24gag [133]. These authors have demonstrated in mice [133] and macaques [132,136] that this novel strategy is able to significantly increase CTL responses to subdominant highly conserved Gag epitopes and avoid decoy epitopes. Finally, Barouch et al. [134] have proposed to divert immune response by designing a ‘mosaic’ candidate able to induce HIV-1 immune responses against all possible HIV-1 variants. They have demonstrated the immunogenicity and protective efficacy of this candidate expressed in adenovirus 26 or 35 and MVA in animal models in the preventive arena [134,135]. The safety, immunogenicity, and efficacy as a therapeutic vaccination to control viral replication after cART discontinuation are currently being assessed in an Ad26.Mos.HIV and MVA-Mosaic prime-boost clinical trial in HIV-1-infected adults who initiated antiretroviral treatment during acute HIV infection (NCT02919306). Results are expected for 2019.

5.3. B cell follicle sanctuary B cell follicle sanctuary permits persistent productive virus infection and CTL responses are excluded from this sanctuary [137,138]. Recently, it has been identified a specialized group of CTL that expressed the chemokine receptor CXCR5 and selectively entered B cell follicles and eradicated infected TFH cells [139–141]. This population of virus-specific CD8 + T cells proliferates after blockade of the PD-1 inhibitory pathway in mice chronically infected with lymphocytic choriomeningitis virus (LCMV) [140]. In addition, it has been shown that a heterodimeric IL-15 treatment in animal models enhances the number, activation and cytotolytic potential of CD8 cells in lymph nodes and

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germinal centers [142,143]. These findings provide new ideas to attempt to redirect CTL responses induced by therapeutic vaccine toward HIV sanctuaries by using IL-15 or optimizing PD-1directed immunotherapy in chronic HIV-1 infection. Potential toxicity associated with these therapies should be carefully assessed since it could limit their applicability in HIV infection [144,145]. A potential safer alternative would be the expression of the soluble extracellular part of IL-15 or PD-1/PD-L1 in DCs during antigen presentation. Expression, secretion and binding of PD-1/PD-L1 soluble molecules after mRNA electroporation in DCs has been demonstrated [118], opening the door to design vaccines able to encode these molecules and acting at local level, avoiding the potential systemic side effects related to these products.


5.4. Host immune environment Host immune environment (inflammation, immune activation, tolerance) could be an obstacle to generate an effective immunity. In fact, it has been reported that some therapeutic vaccines tilt the balance between activation and regulation of the response of HIV-specific CD8 + T cells toward regulation, what could partially explain the failure of these vaccines to control the virus upon analytical treatment interruption [146,147]. Adjuvant strategies to break the balance between activation/tolerance favoring the induction of effective responses should be a research priority of the next therapeutic vaccine candidates. PD-1 is a coinhibitory receptor that plays a major role in exhaustion, dysfunctional state of effector cells caused by antigen persistence. Exhausted T cells present defects in effector function including impaired proliferation, cytotoxic capacity and cytokine production. These defects can be partially restored by blocking the interaction between PD-1 and its ligand programmed death ligand-1 (PD-L1), which notably reduces viral loads, improves immune responses and reduces hyperimmune activation and microbial translocation in SIVinfected macaques [148,149]. The use of this kind of products has been a revolution in the cancer field. Usually, they are used in combination with other therapies, including other immunotherapies as anti-CTLA-4 antibodies [150]. A first prospective study of a PD1:PD-L1 axis inhibitor in HIV-infected participants on ART has recently been reported [144]. Despite that moderate improvements in immunological responses were observed, the clinical trial had to be discontinued for safety reasons. As it is mentioned above, the delivery of these molecules at local level could help to solve these toxicity problems. It is likely that if safety problems are solved, this type of therapy should be combined with other kinds of immunotherapy to avoid the increases in tolerance observed in some therapeutic vaccine clinical trials [146,147].

5.5. Latent reservoir It is not clear if immune-based therapies (therapeutic vaccines and bNAbs) by themselves can target and decrease viral reservoir. Although is a priority end point in the ongoing clinical trials (see Table 2), in the past years few therapeutic vaccine clinical trials have tested for the effect of immunizations on the resting CD4 + T-cell reservoir in patients receiving

cART [70,93,117,151–154]. A combination of whole-inactivated virus (REMUNE) plus a virus vector vaccine (ALVAC) showed no effect in the viral reservoir in a clinical trial [154]. Li et al. [151] studied the relationship of HIV-1 reservoir characteristics with immune status and viral rebound kinetics in HIV-infected patients receiving a therapeutic rAd5 HIV-1 gag vaccine (ACTG A5197). They reported that at study entry, cell-associated HIV-1 RNA and DNA levels were inversely correlated with the numbers of HIV-1-specific CD4+ interferon-gammaproducing cells. However, although the vaccination induced HIV-specific CD4+ activity, this did not significantly affect levels of viral reservoir. In other clinical trial, HIV-pox-based vaccinations in infected young adults on effective cART resulted in a modest, but measurable and statistically significant transient decrease in the frequencies of latently infected CD4 + T cells detected 40 weeks following the first vaccine dose [152]. HIV-1 Tat immunization had a significant impact on immune restoration that was followed by a significant and progressive HIV-1 DNA decay in blood, which was associated with the presence of both anti-Tat IgM and IgG Abs and Env neutralization [70]. Finally, our group vaccinated 24 patients with a dendritic cell-based therapeutic vaccine and, although we did not find any change of viral reservoir during the vaccination period (4 weeks), an inverse correlation was observed between viral reservoir (as measured by total and integrated HIV-1 DNA) and HIV-specific CD8 T cells after vaccination and before cART interruption. Moreover, a significant delayed replenishment of integrated HIV-1 DNA after cART interruption associated with HIV-1-specific responses was observed [153]. As therapeutic vaccines have had limited efficacy, it has been proposed combining therapeutic vaccine with latencyreversing agents (LRA), a strategy termed ‘shock & kill’ [155]. Three clinical trials are currently ongoing testing this approach (see Table 2), combining new innovative vaccine candidates (ChAdV63.HIVconsv and/or MVA.HIVconsv, DC-based vaccines) with romidepsin or vorinostat. The rationale basis is to use therapeutic vaccines to induce robust and broad T cell responses able to suppress HIV replication [26] and LRA [41,42] to induce virus expression in latently infected cells. To our knowledge, only 2 clinical trials assessing the effectiveness of this strategy have been reported. Our group has performed a clinical trial combining a therapeutic vaccine (MVA-B) and low dose of disulfiram that is considered as a potential candidate to effectively reawaken the latent viral reservoir [156]. The size of the latent reservoir did not decrease after the intervention, although we observed that pVL rebounded faster in patients taking disulfiram, which may indicate that an undetectable and/or transient increase of virus reactivation could have been induced in vivo [93]. Recently, Leth et al. [117] have reported the results of a single-arm, phase 1B/2A trial studying the safety and the effect on the HIV-1 reservoir of combining Vacc-4x, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin. They found a mean reduction of 40% in total HIV-1 DNA, and 38% in infectious units per million (IUPM), while integrated HIV-1 DNA remained unchanged. However, these changes did not have an effect in viral load rebound dynamics after cART discontinuation.


These data, although modest, are a proof of concept demonstrating the feasibility of the shock & kill strategy and open the door to the optimization of these strategies to improve the results with the final aim of changing the dynamics of viral load rebound after cART interruption.


5.6. Surrogate markers of response The International AIDS Society convened a group of international experts to develop a scientific strategy for research toward an HIV cure [14,15]. Seven key scientific priorities for HIV cure research were outlined. Overall, the authors recommend that studies on viral reservoir, HIV persistence, immune activation, mechanisms that control HIV replication and immunological strategies to enhance the capacity of the host immune response to control active viral replication are urgently needed. In addition, they recommend that assays need to be developed and validated such that they can be used to monitor the impact of a potential therapy on the HIV reservoir and control of viral replication. The immune correlates of viral control in HIV infection are not extensively known. Therefore, allowing participants to interrupt their antiretroviral treatment is still crucial to assess the efficacy of any investigational cure strategy in controlling HIV replication [157–159]. However, there are concerns about the discontinuation of cART. Safety [11], reservoir replenishment [160], risk of developing resistance [161], and risk of HIV transmission are some of the issues that should be taken in account before discontinuation. Some of these issues could be adequately addressed to minimize the risk in patients. In fact, a 3–4 months’ interruption of cART has been demonstrated to be safe in patients with a high nadir CD4 T cell count [162], the reservoir drop to pre-discontinuation levels in adults [163] and the risk of resistance mutations seems to be restricted to NNRTI. Although it seems clear that cART interruption is necessary to assess the response to any immunotherapy, it is not clear which virologic outcome measures should be assessed. Different virologic outcome vary in different cART interruption trials hindering the comparison between studies. Time to rebound, set point and VL at a predefined time following ATI, time to reach set point, time to reach a certain threshold, peak, time to peak and area under the curve are some of the virologic measures evaluated in different studies [157]. Monitored antiretroviral pause (MAP) restarting ART at the moment of viral rebound have been proposed as safer strategies to evaluate the efficacy of interventions aimed at reducing the HIV reservoir [157]. However, it seems that time to viral load rebound does not correlate with viral load set-point or magnitude of the viral rebound [59,87,159]. Some authors have suggested that the information obtained with MAP could be partial and that is probably not an appropriate outcome measure for assessing the effectiveness of HIV therapeutic vaccines. New studies are needed to compare the different virologic outcomes after cART interruption to determine the most relevant measure to assess the efficacy of interventions aimed at achieving a functional cure. In any case, data of responses of the proof-of-concept clinical trial might be useful to investigate other correlates of response, comparing vaccinated patients vs. controls or responders vs.

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nonresponders. Moreover, it would be necessary to compare immune responses between virologic responder patients (as defined as patients with undetectable level of viral load after 12 weeks of interruption of cART or patients with a significant drop of viral load >1 log10) with baseline values before any cART. Responses would be measured on different levels: CD8/CD4+ T cell responses (ELISPOT and ICS), reduction in the cultivability and fitness of HIV, induction or enhancement of the CD8+ T cell suppressive capacity, effect on reservoir as measured by qVOA, proviral DNA and the intracellular viral RNA (unspliced and multiple spliced viral RNA and microcultures), ultrasensitive RNA, assessment of viral CTL escape, and mRNA expression profiling.

6. Expert commentary Therapeutic vaccines and immunotherapies have demonstrated their effectiveness to induce immune responses, and partially change reservoir and control viral replication after cART interruption. Given this proof of concept, it is necessary to continue investigating new strategies and candidates to obtain the final aim of any HIV therapy, the functional cure of the infection. This objective has shown, however, hard to achieve. No therapeutic vaccine or immunotherapy has obtained a permanent remission of viral load in the patients. Whole inactivated virus, proteins, peptides, DNA, recombinant viral vectors, and DC-based vaccines have extensively been tested in more than 90 clinical trials. As the results have been poor, there are a growing interest in assessing new candidates or strategies to improve the magnitude and the quality of the immune responses. Major immune-virologic obstacles can cause resistance to current strategies. Poor antigen vaccine presentation by DCs, vaccine inducing responses that target escape mutations, CTL responses that do not migrate to sanctuaries (i.e. B cell follicle), an adverse host immune environment (inflammation, immune activation, tolerance), the persistence of a hard to target latent reservoir and the lack of surrogate markers of response are some of these problems. As it can be seen in Table 2 (ongoing clinical trials), the field is moving to in vivo targeting DCs (i.e. TriMix), in order to support the activation and antigen presentation (interferon gamma, pegylated interferon) and avoid tolerance (anti-PD1 or antiPDL1 antibodies). In addition, new rationally designed antigens with the final aim of redirecting the response to more vulnerable, subdominant sites of the virus are being tested. Mothe et al [125] propose a rational design for the selection of the HIV antigens. The selection of viral targets of protective HIV-1-specific T cell responses from large cohorts of HIV infected individuals, the most conserved regions of the HIV-1 proteome or a ‘mosaic’ candidate able to induce HIV-1 immune responses against all possible HIV-1 variants are some of the new strategies that are currently being tested. Some of them have already demonstrated their ability to shift preexisting immune responses toward conserved, vaccine-encoded regions of HIV [96]. Whether these results are associated with a control of viral load after cART interruption remains to be elucidated. A step further in this research field has been to combine therapeutic vaccines with mobilizers of the reservoir. Two clinical trials have been reported and 3 more are ongoing, combining different vaccine candidates (MVA-B, VACC-4x, ChAdV63.

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HIVconsv, MVA.HIVconsv and DCs-based vaccines) with LRA (disulfiram, romidepsin, and vorinostat). Preliminary results suggest that these approaches could target viral reservoir, but that there are not potent enough to avoid the viral load rebound after cART discontinuation. In the next future, the combination of DCs targeting candidates, inserts to redirect responses to unmutated parts of the virus, adjuvants to redirect responses to sanctuaries or improve the balance between activation/tolerance (IL-15, anti-PD1 antibodies) and LRA could be necessary to finally achieve the remission of HIV-1 infection.


7. Five-year view Stem cell therapy, gene therapy, cell therapy, immune-based therapies (therapeutic vaccines, bNAbs, checkpoint inhibitors) and activation of HIV-1 from latent reservoir are different strategies to improve the immune system or to target HIV-1 reservoirs that will be taken in account in a near future to achieve an HIV cure. It is possible that a combination of some of these strategies (i.e. therapeutic vaccines, bNAbs, checkpoint inhibitors, LRA, and cell therapy) and other innovative therapeutic vaccine candidates could be necessary to succeed. In the next 5 years, we will know the safety and the feasibility of implementation of some very complex approaches (stem cell therapy, gene therapy, cell therapy). In addition, we will have learnt about the effectiveness of new candidates able to redirect immune responses to more vulnerable sites of the virus (ChAdV63.HIVconsv, MVA. HIVconsv, Ad26.Mos.HIV, MVA-Mosaic, iHIVARNA.01), new vectors (mRNA iHIVARNA, nanoparticles), adjuvants (IL-15, antiPD-1), LRA (romidepsin, vorinostat, TLR7/9) or a combination of these strategies to achieve the functional cure of HIV-1 infection. The remission of the virus even in a low proportion of patients (20%) would be a proof of concept that the control of viral load without cART is possible. It will open other questions as the durability of this remission or the ability of these strategies to control other important problems in HIV infection as immune activation/ inflammation that are the reason of many of the morbidity and mortality currently observed in these patients.

Key issues ● cART is a major achievement in the fight against HIV infection with excellent public health and clinical results. ● Major problems are the fact that cART is a medication for life and the difficulty of achieving a universal availability of cART to all infected individuals. ● Therapeutic vaccines and immunotherapies are some of the most promising strategies to take into account to achieve a functional cure of HIV infection ● Although the therapeutic vaccines have demonstrated their effectiveness to induce immune responses and partially control viral load after cART interruption, the results have been modest ● New candidates and approaches are currently being tested with a rationally design of antigenic inserts, able to target DCs and reservoir

● Combined strategies taken in account major immune-virologic obstacles to achieve the cure should be evaluated in the future.

Funding This manuscript was funded by the European Commission [grant numbers: H2020-SC1-2016-RTD 731626, H2020-PHC-2015- RTD 681032, FP7HEALTH-2013-INNOVATION-1 602570-2 ERA-Net HIVERA JTC 2014 HIVNANOVA], the Foundation for AIDS Research US, amfAR [grant number: 108821-55-RGRL], Ministerio de Economía y competitividad, Spain [grant numbers: SAF2015-66193-R, FIS PI15/00641, FIS PI15/00480, RIS: Spanish AIDS Research Network].

Declaration of interest 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.

ORCID Felipe García

http://orcid.org/0000-0001-7658-5832

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