HIV-derived vectors for therapy and vaccination against HIV

Vaccine 30 (2012) 2499–2509

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Review

HIV-derived vectors for therapy and vaccination against HIV F. Di Nunzio, T. Félix, N.J. Arhel, S. Nisole, P. Charneau, A-S. Beignon ∗ Molecular Virology and Vaccinology Unit, Virology Department, Institut Pasteur & CNRS URA 3015, 25-28 rue du Dr. Roux, 75015 Paris, France

a r t i c l e

i n f o

Article history: Received 6 December 2011 Received in revised form 26 January 2012 Accepted 31 January 2012 Available online 13 February 2012 Keywords: AIDS/HIV vaccine Lentiviral vectors Pre-clinical studies

a b s t r a c t Despite being at the origin of one of the world’s most devastating public health concerns, the Human Immunodeficiency Virus (HIV) has properties that can be harnessed for therapeutic purposes. Indeed, the ability of HIV to efficiently deliver its genome into the nuclear compartment makes it an ideal vector for gene delivery into target cells. The design of so-called HIV-derived vectors, or more generally lentiviral vectors (LVs), consists in keeping only the parts of the virus that ensure efficient nuclear delivery while entirely removing all coding sequences that contribute towards the replication and pathogenesis of the virus: as a result, the vector genome is composed of less than 10% of the original virus genome and exclusively cis-active sequences. Proteins required for the formation of the lentivector particles and for the early steps of viral replication (including Gag- and Pol-derived proteins) are provided in trans. HIVderived vectors are thus non-replicative virus shells that deliver genes of interest into target cells with high efficiency. Undoubtedly, there is a hopeful twist of fate in our fight against AIDS, which consists in using these vectors to achieve gene therapy and vaccination against HIV itself. This review summarises the current generation of LVs with a special focus on vaccine applications against AIDS. Preclinical data are very encouraging and efforts are ongoing to optimise these vectors, to increase their safety and improve their immunogenicity. © 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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How HIV-1 causes AIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2500 Milestones in the development of lentiviral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2500 2.1. Principle of a lentiviral vector as gene transfer vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2500 2.2. Different generations of lentiviral vectors and safety issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2500 Using HIV-derived vectors for gene therapy against HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2502 Using HIV-derived vectors for vaccination against HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2502 4.1. Lentiviral vectors as vaccine vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2503 4.2. Lentiviral vectors as HIV/AIDS vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2503 4.3. Live-attenuated virus, single-cycle virus and virus-like particles as HIV/AIDS vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2504 How to improve lentiviral vectors for gene therapy and vaccination? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2504 5.1. Strategies to make lentiviral vectors safer/safe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2504 5.2. Strategies to optimise gene transfer in myeloid cells by overcoming retroviral restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2505 5.3. Non-human primate animal models to assess lentiviral vectors vaccine potential against AIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2506 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2506 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2506 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2506 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2506

∗ Corresponding author. Tel.: +33 1 44 38 95 85; fax: +33 1 40 61 34 65. E-mail address: [email protected] (A.-S. Beignon). 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2012.01.089

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1. How HIV-1 causes AIDS HIV-1 is the causative agent of an ever-increasing pandemic, the Acquired Immunodeficiency Syndrome (AIDS). It is a lentivirus that is transmitted between humans through unprotected sexual intercourse (in over 90% of cases worldwide), contact with contaminated blood products, or mother-to-child transmission. Following transmission, HIV infects essentially activated CD4+ T lymphocytes, macrophages and dendritic cells (DCs), and is rapidly disseminated through cell-to-cell transmissions. Furthermore, HIV infection leads to integration of the viral DNA into the host chromosome, which persists for the life of the infected cell, can be transmitted to daughter cells, and is sufficient to generate new virions. This results in viral reservoirs that are difficult to eradicate and may be reactivated upon removal of therapeutic pressure. HIV-1 infections are characterised by a chronic course of disease, with persistent viral replication, gradual destruction of CD4+ T cells and chronic activation of the immune system leading to immune exhaustion [1,2]. Severe immunodeficiency occurs in late stages of disease and gives rise to opportunistic infections or development of tumours. Highly active antiretroviral therapy (HAART) constitutes an effective approach for the treatment of AIDS and also prevents HIV transmission by reducing viral loads [3]. However, eradication of HIV in infected patients is currently not possible, thus fuelling efforts to develop alternative treatment or prevention approaches. The development of lentiviral vectors (LVs), typically based on the HIV-1 genome, for either gene therapy or vaccination against AIDS constitutes a promising approach in the field. 2. Milestones in the development of lentiviral vectors 2.1. Principle of a lentiviral vector as gene transfer vector Many viruses, such as retroviruses, exploit the cellular machinery to deliver their genome to the nucleus and ensure spreading infection. They have thus evolved into highly efficient gene transfer vectors that can in turn be exploited for the delivery of therapeutic genes. The first retroviral vectors were derived from retroviruses of the oncovirus sub-family such as the Moloney Murine Leukaemia Virus derived vector (MoMLV) following pioneering work by the Temin, Miller and Mulligan laboratories in the 1980s [4]. The approach consisted in removing all retroviral sequences (gag, pol and env) from the vector DNA and maintaining only cis-active sequences (acting as nucleic acids) required for vector particle formation (␺ sequence for vector RNA encapsidation), efficient reverse transcription (primer binding site (PBS) and polypurine tract (PPT) sequences for initiation of minus and plus strand synthesis, respectively) and integration (inverted repeats att and IR for MoMLV and HIV, respectively, at the tips of the long terminal repeats (LTRs) for integrase binding). The structural and enzymatic retroviral proteins (Gag and Pol) required for vector particle production and efficient transduction, as well as the envelope required for vector particle entry, are provided in trans (as proteins, not as genes) by transient or stable packaging systems. The first LV, described in 1991 by the Sodroski laboratory [5], applied the same design strategy of retroviral vectors to HIV-1 (Fig. 1a and b), i.e. removing all viral coding sequences while providing viral structural and enzymatic proteins in trans, together with an envelope in a separate expression vector. Originally, this vector was designed to retain the CD4 cell tropism of the parental virus by keeping the autologous HIV-1 envelope. However, the HIV1 envelope is poorly fusogenic and highly unstable as a result of the non-covalent association between the surface (SU, gp120) and transmembrane (TM, gp41) Env proteins; gp120 is easily shed from the particle, thus preventing the concentration of HIV-1 vector particles by ultracentrifugation without major loss of vector titre.

Meanwhile, a method for generating high titre MoMLV-based retroviral vector by the use of the Vesicular Stomatitis Virus G glycoprotein (VSV-G) was described [6]. The exceptional properties of the VSV-G envelope protein for gene transfer rapidly made this envelope the gold standard of MoMLV vector pseudotypes. Indeed, since the VSV-G protein consists in a single transmembrane polypeptide, it was found to be remarkably stable, allowing concentration of vector particles with minimal titre loss. Furthermore, the unique ubiquitous tropism of the VSV-G protein allowed the use of this retroviral vector system with virtually any cell type from any species as target cell. The pseudotyping of HIV-1 derived vector particles by the VSV-G envelope, with an otherwise identical vector construction strategy to HIV-1 vectors described in 1991, greatly increased gene transfer efficiency and versatility of usage of HIV-1 derived vectors [7]. A key feature of lentiviral replication, of central interest for lentivirus-derived gene transfer vectors, is their ability to efficiently integrate and replicate in non-dividing target cells. In contrast, all other retroviruses are strictly dependent on cell division to replicate. The complementation approach used for “classical” MoMLV-derived vectors, where the entire coding genome was deleted, was with hindsight sub-optimal when applied to HIV-1, since central cis-active sequences key to HIV-1 DNA nuclear import [8] but not present in MoMLV, the central polypurine tract/central termination sequence (cPPT/CTS), were also deleted. Reinsertion of these sequences in vector DNA led to the formation of a DNA Flap as in HIV DNA and stimulated LV gene transfer efficiency in both dividing and non-dividing cells [8]. Vector DNA nuclear import efficiency was complemented to that of wild-type parental HIV-1 virus, indicating that all necessary cis and trans viral determinants for nuclear import were now present in DNA Flap-containing LVs. LVs derived from other lentiviruses, such as HIV-2, Simian Immunodeficiency Virus (SIV), or non-primate lentiviruses such as Equine Infectious Anaemia Virus (EIAV), Bovine Immunodeficiency Virus (BIV), Caprine Arthritis and Encephalitis Virus (CAEV) and Feline Immunodeficiency Virus (FIV) [9–12] use similar design strategies. The rationale for their development was based on safety issues, i.e. to avoid the formation of replication competent lentiviruses (see below). However, the physiopathology associated with interspecies transmission of non-human lentiviruses is highly unpredictable and restriction mechanisms might decrease the transduction efficiency of human cells by those vectors. Thus HIV-1 derived LVs represent nowadays the vast majority of LVbased clinical developments.

2.2. Different generations of lentiviral vectors and safety issues Although the use of HIV-1 derived LVs is sometimes met with scepticism as a result of their derivation from a parental deadly virus, they actually offer better safety features when compared to classical MoMLV retroviral vectors, which present two main potential safety problems: (1) the generation of replication-competent retrovirus (RCR) during vector production, a mechanism called generation of “helper virus” in case of MoMLV vectors, and (2) potential tumourigenic events due to integration of the retroviral vector DNA within the chromatin of transduced cells, a mechanism referred to as insertional mutagenesis. The formation of helper virus within MoMLV vector stocks is problematic. Retroviral recombination during reverse transcription between the vector RNA and passively encapsidated gag/pol and env RNA sequences within the vector particle can occasionally lead to the formation of a wild-type replication competent virus, whose physiopathology is the induction of leukaemia in mice. This is a point of particular concern when the amphotropic MoMLV envelope (able to fuse with human cells) is used, or when the vector

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Fig. 1. Design of HIV-1 derived vectors. (a) HIV-1 NL4-3 and SIVmac251 genomes. Schematic viral genome representations (not to scale) showing genes coding for structural/enzymatic viral proteins (dark blue), regulatory proteins (light blue), accessory proteins (red), cis-acting sequences (yellow), and promoter sequences (grey). (b) Comparison of live attenuated SIV, single cycle SIV and lentiviral vectors derived from HIV-1, either integrative (ILV) or non-integrative (NILV), evaluated or to be evaluated as candidate AIDS vaccines in NHP models. A single example for each category is represented here, with same colour coding as in a. a Infected/transduced cells and their daughter cells (for NILV, cell divisions dilute episomal DNA) are expected to be cleared by the vaccine-induced effectors T cells. b Vaccine-induced protection evaluated in NHP models, mostly against viral replication and disease progression (after single high dose viral challenge with the homologous virus). c Cis-acting sequences preclude full codon-optimisation. d Low replication implies potential mutations and recombination and possible reversion to fully infectious variants. e sc viruses achieve a single round replication since virions released by the first infected cells are non-replicative. SIVmac239FS− IN Nefsff is one example of scSIV. It is produced by co-transfection of two plasmid DNA constructs, an SIV genome that is deficient for Pol expression as a result of a combination of mutations in the gag-pol frameshift site and in pol, and a Gag-Pol complementation plasmid. It is thus limited to one round of proteolytic maturation, reverse transcription, and proviral integration [70]. Nefsff refers to a premature stop codon followed by 2 single-nucleotide deletions which were introduced into the nef gene to eliminate residues essential for MHC class I downregulation. In addition, for a RRE/Rev independent mRNA export, a cis-acting RNA element, the CTE from Mason-Pfizer monkey virus, was used. f Non-replicative, no release of new vector progeny. g The potential risk of insertional mutagenesis by enhancer proximity effect is obviated when using an enhancer-less promoter. h Up to nuclear import of vector DNA. i Not yet assessed. j No risk related to HIV integrase dependent-integration. Abbreviations: LAV, live attenuated virus; sc virus, single cycle virus; coR, HIV co-receptors (CCR5 and/or CXCR4); Ag, antigen; LTR, long terminal repeat; VSV-G, vesicular stomatitis virus glycoprotein; MV H, measles virus haemagglutinin; FS, frameshift; GP fusion, gag-pol fusion; RRE, Rev responsive element, an RNA element encoded within the env region which is necessary for Rev function; i.e. promoting the nuclear export of viral mRNA; CTE, constitutive transport element; , encapsidation signal; cPPT/CTS, central polypurine tract/central termination sequence responsible for the formation of the DNA Flap structure during reverse-transcription; which is a determinant of HIV-1 nuclear import; pTRIP, transfer vector plasmid used for lentiviral vector production containing the cPPT/CTS sequence in contrast to previous generation without cPPT/CTS called pHR’; p8.74, encapsidation plasmid (EnvVprVifVpuNef); WPRE, Woodchuck hepatitis virus post-transcriptional response element to enhance mRNA nuclear export on a Rev/RRE independent fashion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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stock is destined for a clinical trial, thus imposing a careful assessment of the lack of helper virus within the vector stock. In contrast, and despite extensive production and thorough testing of HIV-1 derived vectors, the presence of replicative HIV1 in LV stocks has never been described. In fact, the design of LV makes the formation of replication-competent lentiviruses (RCL) unlikely. First, genetic constructs using PCR and synthetic DNA has allowed to minimise or even entirely avoid homologous sequences between the different plasmids necessary for LV production and thus strongly decrease the potential of recombination “jumps” of reverse transcriptase leading to recombination. Second, lentiviruses are complex retroviruses whose genome contains numerous additional genes when compared to the canonical gagpol-env genome structure of simple retroviruses. The accessory and regulatory genes vif, vpr, vpu and nef are not required for efficient gene transfer by LV and are therefore entirely absent from various LV constructs [13], however they are all required for efficient replication of HIV-1 in vivo. Third, LV technology involves the use of heterologous envelopes, most frequently the VSV-G glycoprotein. Taken together, these points make it very unlikely to generate a wild-type infectious HIV-1 by recombination. Insertional mutagenesis after integration of retroviral vectors has become a major safety concern after the description of several leukaemia cases in a two SCID-X1 gene therapy trials [14,15]. Clonal expansion leading to leukaemia resulted from an enhancer proximity effect from a strong viral enhancer included within the MoMLV retroviral vector, which was found to trans-activate a proximal LMO2 proto-oncogene in all leukaemic children tested [14]. With the exception of an oncogenic potential within the encoded transgene itself, numerous subsequent studies showed that enhancer proximity is to-date the only described mechanism of tumourigenesis after retroviral gene transfer in animal models. It is therefore the nature of the integrated retroviral sequence and not the integration per se that constitutes a potential safety problem. Therefore, each LV construct should undergo a careful risk assessment of their insertional transformation potential. The safety issues refer to the presence or not of transcriptional enhancer sequences within the vector promoter but also to potential side effects linked to the over-expression of the encoded transgene. The use of a LV entirely devoid of transcription enhancer sequences would be a major and obvious safety improvement. A LV lacking enhancer sequences no longer activates the LMO2 proto-oncogene in human T cells [16]. However, leukaemia induction can still occur in corrected SCID-X1 mice [17], emphasising the need of a careful analysis of tumour read-outs in gene therapy pre-clinical studies. In the case of LVs, the deletion of enhancer and promoter elements comprised within the U3 region of the LTR (in so-called “self-inactivating” vectors, SIN) offer the dual advantage of increasing safety by removing the HIV-1 enhancer [18] and allowing a stronger expression from the internal promoter, most probably by removing poorly described silencing sequences comprised within the NRE region (Negative Regulatory Element) of the HIV U3 region [19].

or cellular factors (e.g. entry receptors) [20]. Targeting of CCR5 is particularly appealing, since its deletion protects against HIV-1 infection and is not associated with significant immunological dysfunction. Indeed, a 32/32 allogeneic stem cell transplantation in an HIV-1 positive leukaemia patient, initially reported to generate long-term control of viral replication in the absence of antiretroviral therapy [21], has since been announced as the first potential cure for HIV infection by reporting reconstitution of CD4+ T cells and virus eradication [22]. Alternatively, gene therapy approaches may stimulate the immune system to productively recognise virus and control replication (immunotherapy) for instance by genetically engineered cells to produce neutralising Abs, or to express a recombinant specific T cell receptor (TCR) or a chimeric antigen receptor (CAR). Ideally, gene therapy against HIV involves gene transfer in haematopoietic stem cells (HSCs). Firstly, this is predicted to generate resistance against HIV in all the haematopoietic cell lineages, which include all HIV-target cells: macrophages, CD4+ T lymphocytes, DCs, microglial cells [23], as well as HSCs themselves, also reported as potential targets for HIV infection [24]. Interestingly, CD4+ T cells expressing a transferred gene conferring HIV resistance, either following direct transduction or derived from transduced HSCs, are likely to be selected due to HIV-mediated killing of non-resistant CD4+ T cells. Secondly, HSCs are capable of self-renewal, which would ensure treatment endurance and thus minimise viral loads and reduce latent reservoirs on the long-term. Pre-clinical trials in mice and non-human primates (NHPs) provide encouraging proofs of concepts for the use of LVs for gene therapy against HIV/AIDS. LVs are effective for generating RNA interference targeting CCR5 expression in vivo following delivery of shRNAs in HSCs of rhesus macaques [25] and can be designed to efficiently generate neutralising antibodies [26,27] or specific T cell responses in vivo [28–30]. The Wiley database on gene therapy trials worldwide indicates that roughly a quarter of gene therapy trials use retroviral or lentiviral vectors. LVs, which are more recent compared to MoMLV-derived vectors, currently constitute only 3% of all gene therapy trials, however this proportion is sure to increase considering the increasing body of literature reporting the use of LVs for gene transfer protocols. LVs have been successfully used in gene therapy clinical trials, such as for treatment of adrenoleukodystrophy and ␤-thalassaemia [31,32]. These have shown that gene transfer and transplantation are well tolerated without significant adverse effects and demonstrated clear therapeutic benefit. There are currently 9 gene therapy clinical trials that use LVs for therapy against HIV/AIDS [20]. These include the delivery of antisense RNA against Env in CD4+ T cells (VIRxSYS) [33], RNAi against tat/rev with TAR decoy and CCR5 ribozyme in CD34+ HSCs [34], and autologous T cells modified through expression of high affinity Gag-specific TCRs in HLA-A*02 patients with HIV.

4. Using HIV-derived vectors for vaccination against HIV 3. Using HIV-derived vectors for gene therapy against HIV The principle of gene therapy against HIV is to deliver genetic information coding for an RNA or protein able to interfere with HIV replication. This can be achieved either at the level of infected cells, through the delivery of therapeutic genes targeting viral or host cellular factors, or through the manipulation of immunity to enhance immune responses against the virus. Approaches that seek to induce resistance against HIV infection in target cells typically use RNA based decoys, ribozymes or shRNAs to block viral (e.g. tat, rev/RRE (rev-response element))

In the past few years, two candidate AIDS vaccines have been tested for efficacy in large phase II/III clinical trials. In the STEP trial, a human adenovirus serotype 5 (AdHu5)-derived vector expressing HIV-1 Gag/Nef/Pol failed to protect against HIV infection despite the induction of HIV-specific T cell responses [35,36]. In the RV144 trial, a combination of a canarypox vector and a gp120 subunit vaccine showed a relatively modest protection from HIV acquisition despite the induction of weak CD8+ T cell responses and non-neutralising antibodies [37]. In this context, it is essential that additional candidate AIDS vaccines, including novel vector platforms, be evaluated for immunogenicity and efficacy.

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Fig. 2. Delivery of vaccine Ag by HIV-1 derived vectors. Conventional MHC class I presentation occurs in DCs transduced by LVs (bold arrows): (1) receptor binding of vector particles; (2) receptor-mediated endocytosis of vector particles; (3) fusion with the endosomal membrane via a low pH-induced rearrangement and cytoplasmic release of the encapsidated vector RNA; (4) DNA synthesis through reverse transcription (RT) and transport to the nuclear pore. RT is inhibited by host restriction factors such as TRIM5␣ and SAMHD1; (5) completion of RT including DNA Flap formation and uncoating; (6) translocation of the pre-integration complex through the nuclear pore and vector DNA integration into host cell chromatin and/or circularisation into 1- or 2-LTR circles (exclusively circularisation when using NILV); (7) transcription of integrated and circular vector DNA driven by the internal promoter (P); (8) mRNA export to the cytosol, (9) translation into protein (endogeneous Ag in green); (10) degradation by the proteasome into antigenic peptides; (11) transport of peptides to the lumen of the endoplasmic reticulum (ER) by TAP; (12) peptide binding to MHC class I, transport of MHC class I/peptide complexes through the Golgi apparatus to the cell surface and conventional presentation to CD8+ T cells. In addition to conventional MHC class I presentation of endogeneous peptides after gene transfer, DCs undergo maturation. They also present exogeneous Ag (derived from incoming vector particles, in red) on MHC class II to CD4+ T cells and on MHC class I to CD8+ T cells (referred to cross-presentation) (thin arrows): (13) innate immune recognition of dsRNA, ssRNA and dsDNA by endosomal TLR3/7/9 resulting in the secretion of pro-inflammatory cytokines and maturation; (14) acidification of endocytic vesicles, activation of proteases and degradation of proteins provided in trans (VSV-G, and Gag/Pol-derived proteins) into peptides; (15) fusion of the vesicles containing antigenic peptides with vesicles containing newly synthesised MHC class II from the ER. Binding of peptides to MHC class II. (16) Transport of the MHC class II/peptide complexes to cell surface through the Golgi apparatus and presentation of exogeneous Ag to CD4+ T cells; (17) proteasome degradation of cytosolic exogeneous Ag followed by loading on MHC class I molecules in the ER; (18) cross-presentation to CD8+ T cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

4.1. Lentiviral vectors as vaccine vectors The use of LVs as vaccine vectors is more recent compared to their use for gene therapy purposes discussed in Section 3. It has boomed after the demonstration of their capacity to efficiently transduce DCs. In vitro, LV transduction of human and murine DCs or their precursors results in the persistent and non-toxic expression of reporter genes [38,39]. DCs can also be transduced in vivo as shown in mice after direct LV injection [40]. LVs are not only powerful Ag expression vectors, but they also induce phenotypic and functional maturation of DCs through the activation of innate receptors, such as TLR3 and TLR7 (toll-like receptors) [41–43]. Once transduced, maturing DCs migrate to draining lymph nodes where they directly prime Ag-specific CD8+ T cells after a prolonged endogenous presentation of the Ag [44–46] (Fig. 2). This contrasts with other viral vectors that rely on cross-priming to induce immunity [47], for which Ag presentation is more transient. B cells, macrophages, and non-APCs are also transduced in vivo after injection of LV particles [48,49] and likely participate in the establishment of immune memory. Immune responses induced by LVs are characterised by a high frequency of Ag-specific CD8+ T cells, even after a single

injection, and by a long-term immune memory [50,51]. The potency and durability of T cell responses elicited by LVs are likely due to the capacity of LVs to ensure a long-lasting Ag presentation in vivo when compared to other viral vectors [44]. Protective efficacy, both in the prophylactic and therapeutic settings, has been reported after LV particles injections in several mouse models of viral infections or cancer [40,44,49,52]. It should be pointed out that pre-existing Abs specific to the VSV-G often used to pseudotype LV particles are extremely rare in humans [53] and that, additionally, it is possible to pseudotype LV particles with non-cross reactive VSV-G serotypes [54] to allow iterative injections in case of prime–boost immunisations and thus circumvent the presence of anti-VSV-G Abs induced after the first immunisation. The low pre-existing immunity to vaccine vectors is advantageous, since Abs can otherwise neutralise the vaccine and blunt its efficacy.

4.2. Lentiviral vectors as HIV/AIDS vaccines The development of LVs as HIV/AIDS vaccines is in its early stages in comparison to other viral vectors. Nevertheless, preclinical studies performed in mice have provided encouraging

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results, demonstrating the remarkable ability of LVs to induce HIV/SIV specific cellular immunity [55–58]. We performed the first pilot study in NHPs and demonstrated that LVs can confer protection against viral replication and CD4 depletion in Mauritius cynomolgus macaques infected with SIVmac251 [54]. After two injections of LV encoding SIVmac Gag, animals mounted strong specific cellular immunity. After intrarectal challenge with a high dose of virus, plasma viral loads were reduced at the peak of acute infection (a mean of over 2 log10 reduction) and blood CD28+CD95+ memory CD4+ T cells were fully preserved during the acute phase, in contrast to non-vaccinated monkeys. We are currently evaluating the protective efficacy of a new generation candidate LV AIDS vaccine, optimised both for its safety and immunogenicity in rhesus macaques. Ultimately, such protective efficacy studies will expedite the advancement of LVbased AIDS vaccines into human efficacy trials, should they provide strong protection in NHP models. It is notable that, although LVs only transfer the gene coding for the protein of interest, i.e. HIV Ags in the case of an AIDS vaccine, the physical vector particles also serve as an additional source of HIV-1 class I and class II epitopes provided in trans because they are composed of HIV-1 Gag- and Pol-derived proteins [54,59] (Fig. 2). 4.3. Live-attenuated virus, single-cycle virus and virus-like particles as HIV/AIDS vaccines It is not uncommon to confuse LVs with other HIV/SIV-derived AIDS vaccine approaches. Live-attenuated virus (LAV), single cycle (sc) virus or HIV/SIV derived virus like particles (VLPs) obviously share some properties with LVs but LVs also have some unique features. Live-attenuated virus (LAVs) have been evaluated in macaques using SIV that is deleted for some accessory genes and hence replicates less [60]. For instance, SIVmac2393 is deficient in nef, vpr, and the negative regulatory elements of the LTR. LAVs certainly represent the best approach to-date in terms of protective efficacy [61,62] even against heterologous challenge [63,64]. However, for safety reasons they are not expected to advance to clinical trials, except if it is possible in the future to completely turn off replication (by using conditionally replicating LAVs for instance) [65]. Attenuated HIV-1 variants cause chronic infection and their replication, although low, may give rise to pathogenic variants over time because of the high mutation and recombination rates of HIV-1. Moreover, attenuation is relative to the host. Among the individuals from the Sydney Blood Bank cohort who were infected with an attenuated HIV-1 variant with deletions in the nef/LTR region via contaminated blood products from a common donor, some were long-term non-progressors but others were progressors [66]. While the infection of adult macaques with LAV is non pathogenic, it can lead to AIDS in newborns [67]. Still, LAVs are the subject of intensive research for the rationale design of an HIV/AIDS vaccine, since they represent a very helpful tool to identify immune correlates of protection [68]. Single cycle (sc) viruses have been developed with the aim to uncouple viral expression levels from the replicative capacity, while maintaining viral proteins as far as possible in their native conformation. After experimental injection in monkeys, target cells are productively infected. They express almost all of the viral gene products as in the case of LAV, but the released virions are noninfectious. Various approaches to produce scSIV, pseudotyped or not with VSV-G in order to increase cellular tropism, have been described [69–73]. Mutations of viral genes reduce viral replication and infectivity as well as the ability of the virus to manipulate the host immune system. After immunisation with scSIV, peak and set-point viral loads are typically decreased as compared to unvaccinated animals but not as much as with LAV [62].

SIV/HIV virus-like particles (VLPs) are essentially viral structural shells devoid of genome or regulatory and accessory proteins. The expression of HIV-1 Gag alone is sufficient for assembly of “immature VLPs” (made from unprocessed Pr55 Gag). The expression of both Gag- and Pol-derived proteins results in “protease processed mature” VLP, whereas additional Env expression leads to “enveloped fusion competent mature” VLP [74]. Pseudotyping VLPs with VSV-G ensures a larger tropism compared to HIV-1 Env [75]. Plasmid DNA such as the component of DermaVir can express the thirteen full-length HIV proteins, which self-assemble into a complex non-infectious (genome-free) VLP [76]. When used as a subunit or DNA vaccine, they can elicit Ab and T cells responses, but are poorly immunogenic and do not provide strong protection except when used as part of a heterologous prime/boost regimen [77,78]. In addition to the fact that LAV and sc virus are limited to AIDS vaccines while LVs can also be used to transfer genes not related to HIV/AIDS (gene therapy for genetic disease, cancer vaccine, etc.), the main difference between LVs, LAVs and sc viruses is obviously their replication capacity, which determines the persistence of Ag expression. LVs are non-replicative although they are integrative. LVs, LAVs and sc viruses also differ by the intensity and quality of Ag expression they trigger, which depend on the envelope (HIV env versus VSV-G versus DC-targeting), the internal promoter (HIV LTR versus a heterologous internal promoter) as well as on the number of encoded viral Ag, their form (wild-type versus codon-optimised) and ratio (the ratio of Gag/Gag-Pol is 20:1 in wild-type retroviruses) (Fig. 1b). In the case of LAVs, there is an inverse relationship between the degree of protection and the level of attenuation [79]. This is not the case for LVs where Ag expression does not require viral replication. A head-to-head comparison of these vaccine platforms in large and detailed pre-clinical trials in NHPs is desirable to fully and objectively judge their relative potentials. 5. How to improve lentiviral vectors for gene therapy and vaccination? There are two critical milestones for the clinical translation of LVs as gene therapy against HIV and as AIDS vaccine: safety and efficacy. In terms of protective efficacy, a LV AIDS vaccine may be optimised in a number of ways, on Ag design in particular, for instance to increase viral diversity coverage with mosaic Ag [80], to stimulate both B and T cells, or to induce antibody production by inclusion of Env [81]. Clearly, before we can move into clinical trials with LV AIDS vaccine, additional NHP studies will be required to document LV vaccine-induced immunity (including mucosal immunity) and to evaluate whether a LV AIDS vaccine can afford protection not only against viral replication (after a single high dose challenge) but also against infection acquisition (after repeated low dose challenge), against heterologous challenge, or in a therapeutic setting in already infected animals. Whether a LV AIDS vaccine would stand alone or whether a heterologous prime/boost strategy would be more potent will also need to be evaluated in monkeys. In addition, the development of robust and safe largescale GMP (good manufacturing practice) production of LVs is of paramount importance for their implementation in clinical trials [82,83]. 5.1. Strategies to make lentiviral vectors safer/safe The safety concerns associated with the use of LVs for gene therapy or vaccination are not the same because they have fundamentally different requirements for the duration of transgene expression. Contrary to the delivery of a therapeutic protein in gene therapy, permanent Ag expression in vaccination approaches is not

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only superfluous but would actually be detrimental and result in T cell exhaustion. In fact, prolonged Ag expression does not occur following injection of LVs since transduced cells are spontaneously and rapidly cleared by the effector T cells they have primed [84] and by innate immunity (type I IFN) [85]. In this context, the risk of insertional tumourigenesis is low. If insertional mutagenesis were to occur in a transduced cell, then the resulting transformed cells, which express the Ag, would be eliminated in a short time frame in an Ag specific manner. Still, strategies are currently under development to reduce or abrogate the risk of insertional oncogenesis when using LVs for vaccination. One obvious approach to improve the safety of LVs is to use an internal promoter devoid of enhancer activity, since insertional oncogenesis is mainly mediated by enhancer proximity, as discussed in Section 2.2. The safety of a LV with a hPGK promoter (devoid of enhancer activity) has been demonstrated in a tumourprone murine model, in contrast to a retroviral vector and despite a higher integration load and transgene expression [86]. To compensate for the lower transgene expression expected with a cellular enhancer-less promoter, compared with the strong ieCMV promoter notorious for its cross-talk with other promoters, it might be necessary to improve design, for instance by co-expressing the Ag together with immunostimulatory elements [87,88]. Transductional targeting, which consists in modifying vector tropism to transduce cells of interest only, is another approach. Potent specific gene transfer into quiescent T and B cells, for instance, is possible using Measles virus glycoprotein-pseudotyped LV [89]. Alternatively, targeting of DCs has been achieved using a modified Sindbis virus envelope glycoprotein (SVGmu) engineered to be DC-SIGN-specific [90]. LV-SVGmu pseudotypes were reported to induce a higher frequency of specific CD8+ T cells compared with VSV-G and SVGwt [59] and to elicit low levels of anti-vector NAbs [91]. Similarly, LVs pseudotyped with a measles virus haemagglutinin (MV H), engineered so it is unable to bind its natural receptors but able to target MHC II cells, were reported to be immunogenic, but less so than with VSV-G [92]. Another reported means to increase safety/efficacy is through transcriptional targeting, for instance through the use of an APCspecific promoter. The dectin-2 promoter was reported to trigger strong immune responses when compared with the spleen focus forming virus (SFFV) promoter [93]. However, a note of caution should be made regarding this approach since non-APCs also integrate the vector genome but do not express the Ag. Cells with silent vector proviral DNA will not be eliminated by vaccine-induced specific immunity, leading to undesirable long-term persistence of vector DNA in the vaccinated host, which could constitute a safety concern. In addition, targeting APCs does not always result in improved immunogenicity, as shown recently with the HLA-DRa [94] or DC-STAMP [95] promoters, which resulted in the induction of T cell tolerance. Transcriptional targeting may nevertheless be useful to limit immunity against a therapeutic protein and maintain its expression in the case of gene therapy. An additional strategy is based on the use of chromatin insulators, such as the prototypic insulator derived from DNase hypersensitive site 4 of the locus control region of the chicken ␤globin gene cluster (cHS4). It can be inserted into the proximal end of the 3 LTR from which it is copied into the 5 LTR, resulting in a LV flanked by insulators. cHS4 insulators were used in a recent gene therapy trial. A patient with severe ␤-thalassaemia became transfusions independent after transplant with autologous bone-marrow haematopoietic stem cells transduced ex vivo with a LV encoding a functional ␤-globin. However, one haematopoietic cell clone appeared to be dominant. Vector integration near the HMGA2 gene resulted in its transcriptional activation in erythroid cells and despite the presence of a cHS4 chromatin insulator in the U3 region [32]. Scaffold/matrix attachment regions (S/MAR),

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which associate with the nuclear matrix and organise chromatin into independent loops, can also insulate transgenes from the genomic surroundings, and may increase the accessibility of the promoter region and boost transgene transcription [96]. However, in a recent systematic in vitro study, the benefit of the addition of a MAR sequence on transgene expression was not confirmed [97]. It should also be noted that insulators may also exert a potential genotoxic effect since they can modify the physiological transcriptional regulation of the cellular chromatin domain in which the vector integrates. In addition, vectors with such insulators are usually produced at lower titre thus limiting their potential use [98]. Finally, bicistronic LVs encoding the Ag together with a conditional suicide gene like the herpes simplex virus-thymidine kinase could be used to clear transduced cells in vivo after administration of ganciclovir as reported in a gene therapy pre-clinical study in cynomolgus monkeys [99]. However, killing by ganciclovir is dependent on cell division. Actually, the best alternative to improve the safety of LVs is likely to use non-integrative vectors (NILV). A defective integrase obviates the risk of insertional oncogenesis by eliminating the integration step. The D64V mutation, most frequently used for NILVs, targets the integrase catalytic domain, specifically blocking the DNA cleaving and joining reactions of the integration step. The defect of integration leads to the accumulation of doublestranded circular DNA in the nucleus, which are dead-end products of infections in the case of HIV-1 [100], but are competent for the transcription in the case of vector episomes. The transduction of cells with NILVs results in the efficient expression of the gene of interest. More precisely, NILVs mediate stable gene expression in non-dividing cells but transient gene expression in proliferating cells because of the dilution of episomes upon cell division [101,102]. Efficient and long-term expression was reported in rodent postmitotic tissues including brain tissues [103] and with evidence of a therapeutic effect in preclinical models of retinal degeneration [104]. NILVs were also shown to be immunogenic [105–107]. However, current NILVs do not compete with integrative LVs (ILVs) in terms of magnitude of the elicited T cell immune response. Higher doses and iterative immunisations of NILVs are typically required [108,109]. This difference may be related to the intensity of Ag expression, which may be insufficient with NILV compared to ILV, and/or to the critical involvement of some dividing cells in the induction of immunity by LVs since mitotic cells lose episomal but not integrated DNA. Since NILV might represent the future of LV prophylactic vaccines including AIDS vaccines, their optimisation and evaluation in NHP models for AIDS is critical. 5.2. Strategies to optimise gene transfer in myeloid cells by overcoming retroviral restrictions Although HIV-1 and HIV-2 share the capacity to replicate in non-dividing cells, only HIV-2 is able to efficiently infect myeloid cells such as monocytes, DCs and macrophages. This property is conferred by a virion-associated accessory protein called Vpx, which is exclusively encoded by HIV-2 and related simian immunodeficiency viruses (Fig. 1a). Since they lack Vpx, HIV-1-derived vectors transduce DCs inefficiently, but this can be significantly enhanced by delivering virus-like particles carrying HIV-2 Vpx in trans [110]. How HIV-2 Vpx helps HIV-1 infection in myeloid cells has remained elusive for quite some time, but the mystery is now partially solved. Vpx was first believed to be involved in the nuclear import of PICs in quiescent cells [111–113]. In 2007, Goujon et al. hypothesised that Vpx counteracts an anti-HIV proteasomedependent restriction activity in human DCs [114]. This restriction activity affects an early step of viral replication, since the

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synthesis of full-length reverse-transcripts is inhibited in the absence of Vpx [114,115]. Further studies showed that Vpx usurps the Cul4A-DDB1DCAF1 ubiquitin-ligase complex, presumably to inactivate a putative restriction factor expressed in cells of the monocytic lineage [116–118]. The existence of an anti-retroviral restriction factor counteracted by Vpx was further supported by Sharova et al., in a study where they showed that heterokaryons between macrophages, in which Vpx is necessary for viral infection, and COS cells, in which Vpx is dispensable, were resistant to infection by Vpx-deleted viruses [116]. Three years later, SAMHD1 was identified by two independent groups as the myeloid cell-specific restriction factor counteracted by Vpx [119,120]. As predicted, Vpx binds SAMHD1 and mediates its degradation by the proteasome through the recruitment of the Cul4A-DDB1DCAF1 complex. The biological functions of SAMHD1 (for SAM- and HD-domain containing protein 1) are largely unknown, although it is now established that it is a deoxynucleoside triphosphate triphosphohydrolase [121,122]. Understanding how this newly identified restriction factor inhibits HIV replication in the absence of Vpx will be the next breakthrough in the field. Why HIV-1 can replicate without the help of Vpx is another question that would be important to address. In the meantime, Vpx delivered in trans will be of great help for HIV-1-derived vectors to transduce myeloid cells [110]. SAMHD1 is a new member of a growing and probably incomplete list of retroviral restriction factors [123,124]. The first restriction factor affecting HIV replication, APOBEC3G, was identified in 2002 [125], followed by TRIM5␣ [126] and BST-2/Tetherin [127]. These factors inhibit various steps of viral replication and constitute an intrinsic barrier to retroviral infections, limiting cross-species retroviral infections [128]. In human cells, HIV is able to escape these intrinsic defences using accessory genes to promote their degradation. This is the case of APOBEC3G, counteracted by Vif [125] and BST-2/Tetherin targeted by HIV-1 Vpu [127]. On the contrary, no accessory protein is necessary for promoting the degradation of TRIM5␣, since the human protein does not recognise its target, the HIV-1 capsid core, whereas its NHP counterpart, such as rhesus macaque TRIM5␣ (rhTRIM5␣) does, and strongly inhibits HIV-1 replication [126]. 5.3. Non-human primate animal models to assess lentiviral vectors vaccine potential against AIDS Although restriction factors confer a relative resistance to crossspecies retroviral infections, they also pose a problem for AIDS research, since they prevent efficient HIV-1 replication in NHPs. Thus, animal models of AIDS are so far limited to HIV-related simian lentiviruses, which imperfectly mimic the human disease in macaques. Similarly, these blocks probably underestimate the efficacy of HIV vectors. To bypass these limitations, some groups have thought to develop lentiviruses capable of escaping both TRIM5␣ and APOBEC3 restriction in macaque cells. These are almost entirely derived from HIV-1, since they differ from the human virus only in CA and Vif, which have been replaced by those from SIV [129,130,131]. By avoiding CA- and Vif-based restrictions, these chimeric particles are able to efficiently infect macaque cells, raising hope for more relevant animal models of human AIDS and of LVs. In the case of HIV-derived vaccines, mutations within the cyclophilin A (CypA)-binding loop of the CA protein have also been tested. However, the transduction efficiency of these vectors cannot compete with HIV-1/human cells or SIV/Simian cells homologous systems, either in vitro or in vivo [132–134]. In fact, only HIV-derived vectors containing SIVmac239 CA are able to transduce rhesus macaque CD34+ haematopoietic cells as efficiently as SIVmac239-derived vector [131]. Further development of simianadapted HIV-1 may include R5-tropic viruses as well as viruses

that can also overcome the restriction imposed by macaque tetherin. Interestingly, contrary to T cells, rhesus macaque DCs do not restrict HIV-derived LVs, thus allowing their efficient transduction by non-simianised HIV-1 vectors [135]. But the ideal NHP model for AIDS does not exist, since no single model can fully reflect the complex natural HIV infection in humans. First, the choice of the macaque species greatly influences the course of infection. Genotype is also a major element to consider since macaques, such as humans, present an important genetic polymorphism. For instance, rhesus macaques with the Mamu-A*01, -B*08, -B*17 major histocompatibility complex (MHC) I allele display a strong control of SIV replication compared to macaques harbouring other alleles [136–138]. TRIM5 polymorphism is another example of genetic factors influencing retroviral infection, and thus the efficacy of transduction, hence the immunogenicity of a LV vaccine [136,139–141]. In addition, besides TRIM5␣ polymorphism per se, some macaques encode a TRIM5-CypA chimeric protein named TRIMCyp, an anti-retroviral protein with distinct restriction pattern than TRIM5␣ [142–145], further complicating the picture. These differences in TRIM5 genotypes may have dramatic effects on the transduction efficiency and the outcome of SIV infection in macaques. Taken together, these recent findings suggest that candidate HIV vaccines should ideally be tested in NHP cohorts excluding animals with MHC or TRIM5␣/TRIMCyp that allow them to control viral replication. At least, they should be genotyped for these factors and matched accordingly. However, since many host genetic factors also influence HIV replication and progression to AIDS in humans [146], the use of animals with defined genotypes may also constitute a bias, by underestimating the complexity and diversity of retrovirus-host interactions. 6. Concluding remarks Since 1991, several laboratories have focused their efforts on the design of LVs and their applications as gene transfer vectors. Twenty years later, 9 gene therapy clinical trials using LVs against HIV/AIDS are in progress. We expect that, in a close future, many other clinical strategies, including LV-based vaccines, will be evaluated in humans. Conflict of interest Pierre Charneau is the founder of theravectys, a company that develops a therapeutic AIDS vaccine using lentiviral vectors. Acknowledgements The authors thank the Institut Pasteur, the CNRS (Centre National de Recherche Scientifique), the ANRS (Agence Nationale de Recherche sur le SIDA et les hépatites virales), Sidaction, the ANR (Agence nationale de la Recherche) and the FRM (Fondation pour la Recherche Médicale) for financial supports. Apologies are extended to those colleagues whose studies could not be mentioned due to space limitation. References [1] Moir S, Chun TW, Fauci AS. Pathogenic mechanisms of HIV disease. Annu Rev Pathol 2011;6(February):223–48. [2] McCune JM. The dynamics of CD4+ T-cell depletion in HIV disease. Nature 2001;410(April (6831)):974–9. [3] Simon V, Ho DD, Abdool Karim Q. HIV/AIDS epidemiology, pathogenesis, prevention, and treatment. Lancet 2006;368(August (9534)):489–504. [4] Miller AD. Development and applications of retroviral vectors. In: Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1997.

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