Review

Retrovirus-Based Vectors—from Gene Therapy to Genetic Vaccines

Retroviruses have been studied extensively for almost 100 years (Figure 1, see Supplementary Text for a brief retrospective of milestone events). This work has created a vast knowledge base of relevance to the development of the retroviral vectors, which initiated the development of gene therapy.

Figure 1.

Historical timeline of retrovirology. The timeline depicts milestone events in the almost 100-year history of retroviruses. The first 80 years were largely devoted to fundamental studies of the retrovirus' structure and function and work that laid the ground for the development of retroviral vectors. Abbreviations: retroviral DNA, R-DNA; gene therapy, GT.

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Gene therapy involves the introduction of genetic material into a cell or tissue in order to (i) replace or repair a missing or defective gene to treat a genetic disorder, (ii) regulate the expression of genes for therapeutic purposes, or (iii) supplement a gene such as (a) a tumor suppressor or cytotoxin-activating gene to treat a malignancy or (b) an antigen-encoding or immunostimulatory gene to prevent or cure an infectious disease. Permanent modification of the host cell genome is the generally preferred outcome in the former case, whereas temporary regulation or complementation may suffice in the latter cases. In all cases, efficient and tissue-specific gene transfer as well as means to control the level and duration of gene expression are of paramount importance for efficacy and safety.

Retroviruses have several distinct inherent advantages with respect to both their gene and protein make-up, which constitute keys to their success in gene therapy. These include (i) well studied genomes that are amenable to genetic engineering, (ii) sufficiently small genomes to allow their incorporation into and expression from various heterologous vectors, (iii) exclusive ability to transform their single-stranded RNA genome into double-stranded DNA that stably integrates into and permanently modifies the host cell genome, (iv) relatively low immunogenicity compared to some other viral vectors, and (v) the unique ability to incorporate envelope proteins from a wide range of heterologous viruses, providing a means for specific cell targeting or foreign antigen display. These features are of differing interest depending on the intended application and this review focuses on the main developments of retroviral vectors for application in "classical" gene therapy, cancer gene therapy, or genetic vaccines.

Replication-defective retroviral vectors. The capacity of retroviruses to integrate into the host genome carries the risk of insertional mutagenesis and oncogene activation. Reducing this risk was a major goal of scientists designing the first retroviral vectors. To this end, replication-defective retroviral (RDR) vectors devoid of genes required for self-replication and thus incapable of cell-to-cell spread or general dissemination were developed.^{1, 2, 3} To decrease the potential emergence of replication-competent retroviral (RCR) vectors through inadvertent recombination events, overlapping sequences of the retrovirus genomes and those of the vector-producing cells were reduced to a minimum.^{4, 5, 6} Generally, RDR vector genomes consist of retrovirus genome cis sequences driving the expression of a therapeutic gene and include the packaging signal sequence necessary for their encapsidation (Figure 2). RDR vectors are produced in vitro by transient transfection or stable integration of RDR vector genomes in vector-producing cells containing stably integrated retrovirus genome trans sequences, encoding all of the structural elements necessary to assemble the vector particle (i.e., Gag, Pol, and Env), but lacking the packaging signal sequence necessary to encapsidate them (Figure 3). After infection of a target cell, the RDR vectors are able to integrate the genome sequence carrying the therapeutic gene, but are incapable of replication of secondary infection of adjacent cells. Indeed, the inability to replicate reduces the likelihood of unintended spread and mutagenic or oncogene-activating or tumor suppressor gene-inactivating insertion, and thus greatly improves the safety of these vectors. However, it also reduces their efficiency of gene delivery. Along these lines, these vectors have particular advantages and shortcomings depending on the application.

Figure 2.

Schematic representation of MoMLV-based retroviral vector genomes discussed in this review article. Wild-type (WT) MoMLV genomes consist of LTRs driving the expression of Gag and Env, which encode the structural elements of the vector particle, and Pol, which encodes the enzymatic elements protease, reverse transcriptase, and integrase. The genome also comprises the packaging signal (), necessary for its encapsidation. RDR vector genomes generally consist of retroviral LTRs driving the expression of a transgene (TG), and the retroviral packaging signal () necessary for its encapsidation. RDR vectors are produced in vitro through transient transfection of the (a) RDR vector genomes into packaging cells containing stably integrated genomes consisting of promoters (P) and (b, c) transcription termination sequences (T) of choice dictating the expression of the retrovirus' structural and enzymatic elements necessary to assemble the vector particle, but lacking packaging signal. RCR vector genomes generally consist of an intact retrovirus genome and, in addition, a cassette comprising an internal ribosome entry site (IRES) driving the expression of a transgene inserted immediately after the stop codon of the Env gene. s-RCR vector genomes generally consist of two transcomplementing RDR vectors, each transducing a transgene and either (d) Gag-Pol or (e) Env. retroVLP vector genomes generally consist of a promoter and a transcription termination sequence of choice driving the expression of the (f) retroviral Gag protein or, in addition, (g) a cassette comprising an internal ribosome entry site driving the expression of an envelope protein of choice inserted immediately after the stop codon of the Gag gene; both genomes lacking packaging signal. Epitopes of choice (Epi) may be inserted in Gag and/or Env. Env may express a retroviral or a non-retroviral glycoprotein. Of note, additional vector designs, including SIN vectors with modified LTRs,⁴⁷ exist but are not described in any detail in this review article. Also, more complex retrovirus species such as lentiviruses and foamy viruses have been developed as gene delivery vectors. Although these are quite promising, they are beyond the scope of this review.

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Figure 3.

Schematic representation of retroviral vectors resulting from transfection of the RDR, RCR, s-RCR, or retroVLP vector genomes discussed in this review article. Transfection of RDR vector genomes into packaging cells containing stably integrated packaging genomes results in release of infectious but replication-incompetent viral particles that carry two copies of the RDR vector genome. Transfection of RCR vector genomes results in release of infectious replication-competent viral particles that carry two copies of the RCR vector genome. Transfection of s-RCR vector genomes results in release, from co-infected cells, of infectious viral particles that carry two copies of either the same or one of each of the trans-complimentary s-RCR vector genomes. Upon co-infection of a cell, the inherently replication-defective but trans-complimentary s-RCR vectors can replicate and give rise to new trans-complimentary s-RCR vectors. Transfection of retroVLP vector genomes results in release of genome-free replication-defective viral particles that are capable of cell entry through the cognate receptors of the envelope glycoprotein. The envelope glycoprotein may be a heterologous one and the envelope and/or core protein may express foreign epitopes of choice. Of note, vector genomes that cannot be reverse transcribed or integrated into the host genome exist ⁴⁷ but are not described in any detail in this review article.

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The use of RDR vectors to deliver genes into hematopoietic stem cells ex vivo to cure two different forms of primary immunodeficiency, X-severe combined immunodeficiency, and adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID), long represented the only successes in human gene therapy clinical trials.^{7, 8} Several factors working in concert may explain why: (i) hematopoietic stem cells constitute optimal targets for retroviral vector-mediated gene transfer, as they can be manipulated with relative ease ex vivo, and transgenes can be expressed long-term in vivo by the stem cells' subsistence and generation of a large progeny of gene-modified mature cells, (ii) the relatively low immunogenicity of retroviral vectors and the immunodeficient status of the patients are likely to result in absent or minimal immune responses to vector components or transgene products, and (iii) the nature of the diseases causes a strong selective advantage for gene-corrected cells to proliferate or escape apoptosis. Unfortunately, selective advantage for growth may be linked not only to restored function induced by the transgene product, but also to inadvertent effects induced by the transgene insertion. This seems to be the case in the first X-severe combined immunodeficiency trial, where of the three treated children who developed leukemia-like clonal T-cell proliferation post-treatment, two died of complications.^{8, 9, 10} In all cases, the provirus had integrated within or near the LMO2 oncogene.¹¹ It thus appears that activation of the LMO2 gene is particularly advantageous to the growth or survival of T cells and, in fact, its translocation is among the most prevalent of the translocations seen in T-cell leukemias.¹² Purely coincidental integration in or near this gene is unlikely. To a certain extent, there is evidence for genome region-specific integration of retroviruses^{13, 14} and it was recently shown that in these cases the vector integration occurred at a common fragile site, known to correlate with chromosomal breakpoints in tumors, attracting a non-random number of murine leukemia virus (MLV) integrations.¹⁵ Finally, the inadvertent effects may have been induced by the transgene itself. Indeed, in a mouse model for X-severe combined immunodeficiency gene therapy, the corrective therapeutic gene itself could contribute to the genesis of T-cell lymphomas.¹⁶

In the third successful gene therapy clinical trial that was recently reported, two adults suffering from X-linked chronic granulomatous disease attained notable clinical improvement after infusion of genetically modified hematopoietic stem cells.¹⁷ Follow-up is ongoing to evaluate the long-term clinical outcome and malignant transformation, in line with lessons learned from the X-severe combined immunodeficiency trials. Altogether, RDR vectors deserve continued development. However, as we shall see below, certain diseases may benefit from retroviral vectors with quite different designs.

Novel retroviral vectors—transducing all or nothing. In essence, gene therapy for genetic disorders and gene therapy for malignancies have two opposite goals; the former aims to restore a particular cell compartment and the latter aims to oust it. In gene therapy for genetic disorders, gene transfer to a subset of the target cells may suffice for restoration, but care must be taken that no inadvertent growth advantage linked to the gene transfer occurs, as the goal is life-long persistence of correctly modified target cells and their progeny. To the contrary, in gene therapy for malignancies, gene transfer to most if not all target cells is a requisite, but less attention may be given to inadvertent growth advantage, as the goal is to kill all transduced cells. Thus, vector modifications aimed at efficacy or safety may be given somewhat different priorities, depending on their intended application.

As an example, in the largest clinical trial of RDR-mediated cancer gene therapy conducted to date, no more than 0.002% of the tumor cells were transduced in any examined patient,¹⁸ which probably explains why no such protocol has been therapeutically adequate. Thus, for this particular application, retroviral vectors retaining their capacity for replication might be justified. As another example, the use of moderately replicating vectors (live attenuated virus) in vaccines, i.e., attenuated human immunodeficiency virus (HIV) to prevent HIV infection, has provided promising results,¹⁹ but such vectors are considered far too dangerous for use in healthy subjects. For this particular application, retroviral vectors empty of any genome represent a safer alternative.

Likewise, many groups, including ours, have recently designed retroviral vectors that are either (i) fully replication competent for improved gene transfer, but which incorporate several alternative safety measures or (ii) completely devoid of any genome, but which incorporate foreign antigens of interest for specific immunogenicity. The remainder of this review will focus on the versatility of these two extremes of retroviral vector design.

Retroviral vectors for cancer gene therapy—opting for efficacy

RCR vectors. The use of replication-competent vectors has seen growing interest particularly in the field of cancer gene therapy, where replication-defective vectors have mostly failed. This is because each tumor cell infected with a replication-competent vector becomes a vector-producing cell that gives rise to more vectors and thus more efficient transduction. Indeed, the use of replication-competent viruses as oncolytic agents has scientific precedent. In the early 1900s, it was discovered that chance infection with cytolytic viruses could efficiently inhibit or even reverse tumor growth.²⁰ This discovery led to testing of the oncolytic power of various cytolytic viruses in the clinic. However, despite any initial tumor regression, the final result was often tumor recurrence resulting from this fact and the patients' anti-viral immune responses. Therefore, because of the contemporaneous advance of chemotherapeutic drugs, "oncolytic virotherapy" was largely abandoned. Since then, a better understanding of the molecular mechanisms involved in viral pathology and immunology, as well as improved means for precise manipulation of the gene or protein make-up of viruses, has allowed the engineering of replication-competent cytolytic viral vectors with more efficient oncolytic power.^{21, 22} Among them, RCR vectors, which are not inherently cytolytic, have been developed as oncolytic agents.²³

The first MLV-based RCR vectors were developed by insertion of either a transgene cassette into the U3 region of the 3' long terminal repeat (LTR), allowing for LTR-driven transgene expression,^{24, 25, 26} or an additional splice acceptor site and transgene cassette downstream of the env gene, allowing for transgene expression following alternative splicing.²⁷ However, these configurations led to genetic instability. Generally, most recent RCR vector genomes consist of an intact retrovirus genome including an internal ribosome entry site-transgene cassette inserted immediately after the stop codon of the env gene^{27, 28, 29} (Figure 2), thus comprising all elements necessary for replication and RCR vector production following cell transfection (Figure 3). In in vitro studies, a small number of such RCR vectors achieved a tremendous amplification of transduced cells, as compared to RDR vectors^{27, 30, 31} and in one study they proved genetically stable when propagated over seven serial cell-free passages.²⁹ However, murine tumor models have faster growth kinetics than many human tumors and, therefore, the efficacy of RCR vectors in murine models needs to be interpreted with caution. Also, although recent RCR designs show significant improvement over previous designs, the genetic instability of RCR vectors, i.e., the possible loss or incorporation of genetic sequences that may render the vectors inactive or give rise to chimeric vectors with unknown functions, must be kept in mind and deserves further study.

Beyond the hallmark stable integration of retroviruses, RCR vectors have two features of particular interest for oncotherapeutics. First, they can only integrate and replicate in dividing cells, providing an inherent means for targeting malignant cells that divide rather actively, while sparing surrounding normal cells that are largely quiescent. Indeed, in in vivo studies in mice, intratumoral injection of as little as 10⁴ infectious units of RCR vectors could spread and transmit a transgene throughout entire subcutaneous and intracranial tumor masses, whereas spread to normal tissues was undetectable.^{27, 32} Second, they are inherently non-cytolytic and their cytolytic power hinges on the transfer of suicide genes followed by administration of a non-toxic pro-drug that is converted intracellularly to a cytotoxic substance by the suicide gene encoded enzyme. This provides both a means for control, as tumor cell killing depends on pro-drug administration, and for safety, as the same pro-drug could stop inadvertent vector spread. In in vivo studies in mice, intratumoral injection of RCR vector supernatant transducing a suicide gene, followed by pro-drug administration, resulted in 100% survival over a 100-day follow-up period, compared to 0% survival of control groups receiving either vector or pro-drug alone.³² Furthermore, they achieved better therapeutic results than those reported using 10,000-fold higher levels of RDR vector supernatant.³³

It is thus clear that RCR vectors, as currently designed, are remarkably more efficient than RDR vectors to treat malignancies and, importantly, when administered to immunocompetent animals, their dissemination cannot be detected even by use of relatively stringent methods.³² Therefore, they may be considered in settings with dismal prognosis and lack of alternative effective therapies (e.g., advanced malignancies such as recurrent glioblastoma multiforme). However, both RCR and RDR vectors still suffer from the risk of inadvertent spread, and any one of the following steps implicated in retroviral gene transfer can impose unwanted side effects: vector production, transgene insertion and expression, target cell or tissue manipulation, and host immune responses.

Semi-replication-competent retroviral vectors. Aiming to make retroviral vectors worth considering in less extreme settings, we recently developed the so-called semi-replication-competent retroviral (s-RCR) vectors that are more efficient than RDR vectors and safer than RCR vectors. This alternative RCR vector design is based on two transcomplementing RDR vectors, each transducing a transgene and either gag-pol or env (Figure 2) that together, but not alone, hold all the genetic material necessary for replication and s-RCR vector production upon cell transfection (Figure 3). The s-RCR vector duo allows co-propagation of two different transgenes, which offers both a back-up therapeutic opportunity, should the effect of the first gene product wane owing to the development of drug resistance, and a means for vector replication shut off, if the transgene is a suicide gene. Furthermore, an s-RCR vector trio, i.e., mobilization of a third RDR vector including a third transgene, is also possible and further augments the system's versatility. With respect to efficacy, transgene propagation from s-RCR vectors was remarkably efficient both in vitro and in vivo, although not quite on par with RCR vectors.³⁴ On the other hand, with respect to safety, the s-RCR vector did not lead to detectable dissemination or leukemogenesis, as did the RCR vector, after direct intravenous vector injection and subsequent bone marrow transplantation in MLV-sensitive mice.³⁴

Beyond this approach, many others can be imagined. Most means currently explored to improve the integral safety of RDR vectors could be similarly applied to RCR vectors, including (i) physical targeting of cells of choice via modification of the envelope protein or expression from heterologous vectors,^{35, 36} (ii) transcriptional targeting via replacement of the viral enhancer/promoter elements with tissue-specific regulatory sequences,^{37, 38} (iii) transcriptional control via inducible/repressible regulatory sequences,^{39, 40} (iv) integrational targeting via homologous recombination,^{41, 42} and (v) self-inactivation or chromatin insulation via various cis-elements.⁴³ For reviews, see Anson,⁴⁴ Yi et al.,⁴⁵ Sinn et al.,⁴⁶ and Baum et al.⁴⁷

Although all of these designs serve to impede inadvertent vector dissemination within the treated patient, RCR vectors are inherently comparatively safe with respect to horizontal spread to the environment. Their transmission occurs mainly through exposure to blood or sexual fluids or cell–cell contact, which can be readily prevented. Transmission through aerosols, which can occur with other viral vectors, does not occur with retroviral vectors. Furthermore, transmission from an infected contact depends on the viral load.⁴⁸ In RCR-treated patients, the above-mentioned vector designs as well as the patient's antiretroviral immune responses and optional antiretroviral drugs should efficiently limit the viral load in blood and sexual fluids and thus prevent environmental dissemination. Finally, although retroviral endemics with the human retroviruses human T-cell lymphotropic virus (HTLV)-1 and -2 do exist, retroviral epidemics have only been seen with the more complex human retrovirus HIV.

Recently, germline transmission (endogenization), i.e., vertical spread, of retroviral vectors, as well as the potential interaction between retroviral vectors and human endogenous retroviruses (HERVs), (see Supplementary Text) have gained attention as safety concerns in the field of gene therapy. No endogenization of retroviral vectors or current transposition activity of HERVs has been documented so far. However, such events in our species cannot be completely excluded. Retroviral vectors and HERV elements could, owing to genetic instability, potentially give way to homologous recombination, gene conversion, or template switching, possibly generating adverse effects by altering the expression of normally active or inactive HERVs or by creating replication-competent chimeric provirus.^{49, 50} Indeed, creation of RCRs containing ERV sequences from producer cell lines has been described.^{51, 52} Unquestionably, further preclinical studies are needed to elucidate retroviral vectors' potential for endogenization and for interaction with HERVs. In the meantime, parameters such as vector design, dose, and route of administration, as well as the disease and population targeted, should be taken into account when assessing the risk of germline transmission.

Perspectives. The use of RCR vectors is already contemplated in clinical settings where the benefit/risk ratio can be deemed positive, such as advanced malignancies for which alternative treatment has failed. However, for broader application in gene therapy, retroviral vectors require additional fine-tuning with respect to both efficacy and safety. Continued improvements in their design and further understanding of the biology of the tissues to be treated, whether stem-, tumor-, or other cells, are certainly important steps toward their wider use in the clinic. Furthermore, their use as tools or therapeutics in other fields such as immunology and vaccinology will bring important know-how to take into account in future RCR vector designs.

Retroviral vectors for genetic vaccines—a virtual makeover

Background. The idea of rendering retroviral gene transfer vectors into retrovirus-based genetic vaccines has its roots in the fields of simian immunodeficiency virus (SIV) and HIV. Interest in live attenuated HIV vaccines was sparked by the observation that chronic infection of macaques with independently produced attenuated isolates of SIV prevented superinfection with pathogenic SIV.^{53, 54} Indeed, the attenuated SIV strains possessed many of the properties thought to be required for an efficacious HIV vaccine. Obviously, however, concerns about the safety of such vaccines—potential reversion to virulence, recombination with exogenous or endogenous retroviral elements, etc—called for integration of safety measures, before they could be considered in clinical trials. Here, the wealth of knowledge gathered over the years for engineering retroviral genomes and particles for gene therapy guided the development of two different kinds of HIV vaccine candidates: conditionally replicating vectors and genome-free particles. In turn, benefiting from the knowledge gained from these HIV vaccine candidates, simple retrovirus-based vaccines against a range of infectious diseases and even against cancer are now in development. Of note, such vaccines may be prophylactic, i.e., given before disease to prevent its establishment, or therapeutic, i.e., given after the establishment of disease to cure it.

Conditionally replicating retroviral vectors. For attenuated HIV vaccine candidates, replication competence and the degree of protection appear to be directly correlated.⁵⁵ Therefore, although further attenuation of vaccine vectors by alteration of viral genomic sequences implicated in replication may improve their safety, it may also reduce their immunostimulatory capacity. In attempts to circumvent this problem, HIV vaccine strains with conditional replication, relying on the insertion of a tetracycline-regulated system into the genome of selected HIV variants, were developed and resulted in strains with high replication capacity but dependence upon doxycycline for replication.⁵⁶ Adding to both safety and efficacy of attenuated HIV vaccine strains, this system is certainly promising. However, it has to be kept in mind that the tetracycline-regulated systems developed to date still suffer from some level of "leakiness", i.e., intrinsic activity refractory to doxycycline,⁴⁰ and will need further development before this type of vaccine candidates can be contemplated in the clinic. To get around this drawback, additional elements can be added to further condition viral activity. Along those lines, strains dependent both upon doxycycline and upon the peptide T20 for replication have been developed.⁵⁷ However, this system needs further study to eliminate the risk that the strain could evolve to become drug resistant.

Retrovirus-like particle vectors. In vaccinology, it has become widely accepted that vaccines based on attenuated forms of viruses are generally highly immunogenic and efficacious but, as indicated above, their potential reversion to virulence often poses too great a safety risk for use in healthy subjects. It has also become apparent that vaccines based on virus subunits, i.e., virus proteins delivered directly or via expression from viral or non-viral vectors that encode them, are quite safe but often induce inadequate immune responses for protection against infection.^{58, 59} From these observations, which apply to viruses in general and to viruses that cause chronic infections, such as HIV, in particular, it can be deduced that the particular form of a virus is of paramount importance for immunogenicity and that as much as possible of the viral genome should be deleted for vaccine safety. Genome-free, virus-like particles (VLPs) meet these criteria. Indeed, such VLPs, which have been derived from over 30 different viruses to date,⁶⁰ have demonstrated safe and efficient induction of humoral and cellular immune responses in animal studies,⁶¹ highly promising results in phase II and III clinical trials,^{62, 63, 64, 65, 66} and VLP-based vaccines against hepatitis B virus have already been marketed. Along with these developments in vaccinology, retrovirus-like particles (retroVLPs) have been developed as candidate vaccines against HIV.^{67, 68}

A number of groups have described the production of HIV-based retroVLPs. In their simplest form, they consist solely of Gag polyproteins which, requiring neither structural or enzymatic viral protein nor viral genome, suffice for assembly and budding of virions resembling their wild-type cognates.^{69, 70, 71} In more complex forms, both Gag and Env proteins are incorporated into the retroVLPs.^{72, 73} Above all, the latter type has proven to induce encouraging cellular and humoral immune responses, including neutralizing antibody responses, both in mouse⁷⁴ and in Rhesus macaque.⁷⁵ Indeed, the initial results were promising enough to motivate further studies to improve the overall immunogenicity and efficacy of this vaccine strategy.

For certain aspects of such studies, retroVLPs based on simple rather than complex retroviruses may offer some advantages. As with HIV-based retroVLPs, MLV-based retroVLPs can be designed in many ways. Cellular expression of the Gag protein suffices to generate particles^{76, 77} and additional expression of the Env glycoprotein allows generation of replication-defective particles that are capable of cell entry but lack the viral genome⁷⁸ (Figures 2 and 3). Whereas MLV-Gag readily forms VLPs when expressed in murine cells, HIV-Gag does not,⁷⁹ making the mouse model inappropriate for studies of the immunogenicity of HIV-based retroVLP expressed from genetic vectors. Beyond this technical difficulty, the use of HIV proteins in vaccines would render vaccinated people sero-positive for HIV. Finally, compared to HIV, the MLV genome is much less complex, rendering its genetic engineering more straightforward. As we shall see, MLV-based retroVLPs easily lend themselves to incorporation of foreign antigens, and they are small enough to be expressed from heterologous vectors, which are strong advantages in vaccinology.

Pseudotyped or antigen-displaying retroVLPs

The self-assembling core structures of many different viruses are amenable to pseudotyping, i.e., they can be adapted by recombinant technology to incorporate entire or truncated versions of an envelope protein from a heterologous virus of interest. The cores of retroviruses have proven exceptionally accommodating for this type of "make-up".⁷⁸ In vaccinology, pseudotyping serves two important functions: a means for ordered presentation of an antigenic determinant of interest and a means to enhance or target vector uptake by antigen-presenting cells (APCs).

The key to the success of VLPs as immunogens is thought to reside in their accurate mimicry of wild-type virus particles. The presentation of an antigen in a highly ordered, repetitive array, such as on many viruses and bacteria, normally provokes strong humoral responses, whereas the same antigen presented as a monomer usually is much less immunogenic.⁸⁰ RetroVLPs represent excellent platforms for the design of simple and safe, highly ordered immunogens. Indeed, MLV Env glycoproteins can be modified to display small or large epitopes⁸¹ or even entire proteins.⁸² Using this type of recombinant immunogen in the mouse, we recently showed that a dominant T-cell epitope displayed on the Env protein of a retroVLP induced significantly stronger cellular immune responses than the same antigen molecule administered as recombinant peptide formulated in Freund's adjuvant.⁸³ Furthermore, retroviral Gag core proteins can be modified to incorporate epitopes,⁸⁴ or be "pseudotyped" with glycoproteins from most retroviruses⁸⁵ and a large host of other families of enveloped viruses, including lymphocytic choriomeningitis virus, spleen necrosis virus, vesicular stomatitis virus, hepatitis C virus, yellow fever virus, West Nile virus, and influenza virus^{86, 87, 88, 89} (FL Cosset, personal communication), without harming their functionality. Obviously, this makes retroVLPs excellent vaccine candidates not only for retroviral diseases such as AIDS but also for a wide range of other viral diseases of clinical interest such as hepatitis C, yellow fever, West Nile fever, and influenza.

In gene therapy, in order to improve vector transfer efficacy or safety, retroviral vectors have been pseudotyped with numerous heterologous envelopes that confer improved vector stability or altered target cell specificity. For example, retroviral vectors pseudotyped with envelopes of the feline endogenous retrovirus, gibbon ape leukemia virus, or vesicular stomatitis virus-G, transduced hematopoietic stem cells^{90, 91, 92} and human primary T lymphocytes⁹³ more efficiently than did non-pseudotyped vectors.

Likewise, pseudotyping can be used in order to enhance vector uptake by APCs. Exogenous viral antigens are captured by professional APCs, by specific receptor-mediated mechanisms or nonspecific mechanisms such as phagocytosis, for subsequent processing via the major histocompatibility complex class I- and II-restricted pathways, which prime cytotoxic CD8⁺ T cells (CTLs) and helper CD4⁺ T cells, respectively.⁹⁴ Scientists found that efficient cytotoxic CD8⁺ T-cell priming depends on the entry of viral proteins into the cytosol and thus requires interaction between the virus envelope glycoproteins and their receptors as well as fusion of the virus and APC membranes. Indeed, retroVLPs, whether equipped with a retroviral envelope or pseudotyped with vesicular stomatitis virus-G, facilitated vector uptake by APCs and increased significantly the anti-Gag-specific cytotoxic CD8⁺ T-cell responses, compared to non-enveloped particles.^{95, 96}

Vectored retroVLPs

As described above, exogenous antigens, such as retroVLPs that mimic the natural form of viruses, are capable of inducing both cellular and humoral immune responses. However, it is important to keep in mind that exogenous antigens are generally processed via the major histocompatibility complex class II-restricted pathway that primes helper CD4⁺ T-cell responses and endogenously expressed antigens are generally processed via the major histocompatibility complex class I-restricted pathway that primes cytotoxic CD8⁺ T-cell responses, even though the inverse antigen/pathway pattern, so-called cross-presentation, also exists.⁹⁷ Obviously, this may have important implications for vaccine efficacy. Therefore, genetic vaccines that not only target APCs but also carry the genetic material necessary for expression of an antigen should bring the immune system into further play, by mimicking not only the natural form but also the natural expression of viral components, stimulating both the exogenous and endogenous antigen presentation pathways. Many recombinant vectors expressing non-particulate antigens have been constructed and tested.^{58, 98} Recently, however, since particulate antigens have proven to be more efficient immunogens than non-particulate ones, the idea of designing recombinant vectors that express VLPs has emerged. The genetic sequence necessary for expression of retroVLPs is small enough to be incorporated into a wide range of different vectors. To date, recombinant vectors based on DNA plasmids, adenovirus, poxvirus, rhabdovirus, and vesicular stomatitis virus have been used to generate functional retroVLPs.^{83, 99, 100, 101, 102, 103} Most of these express HIV-based retroVLPs, which are not well assembled in mice and are therefore not suitable for testing in this animal. To circumvent this problem, we recently constructed and tested the immunogenic properties of a plasmid expressing MLV-based retroVLPs, which are readily expressed in mice. Using this genetic vaccine vector, we showed that antigens presented on retroVLPs induced strong cellular immune responses and protected against MLV-induced leukemia, whereas the same antigens presented on matching viral proteins unable to assemble into VLPs, did not.⁸³ Similarly, retroVLPs expressed from a recombinant poxvirus vector showed enhanced cellular and humoral immune responses in mice compared to vectors expressing non-particulate matching antigens.¹⁰⁴ Furthermore, when modified vaccinia virus Ankara (MVA)-vectored retroVLPs were tested in macaques, vectors expressing a mix of mature and immature VLPs and intracellular protein aggregates elicited about twofold higher ELISPOT responses to Gag and Env than did vectors expressing immature VLPs alone, indicating again that the form of the antigen has a significant effect on elicited cellular and humoral immunity.¹⁰⁵ It must be kept in mind that the different vectors used and the antigens they expressed in the above-mentioned experiments have different capacities for APC targeting. However, taken together, these experiments and results show the versatility of retrovirus-based vectors both as tools to study the immunological aspects that are important to vaccines and as platforms for the design of vaccines against a wide variety of viruses.

Perspectives. The immunogenicity of co-expressed viral proteins probably depends upon their ability to assemble into a physical particle retaining wild-type ultrastructure with ordered antigenic display, as well as their ability to do so while retaining wild-type biophysical properties such as budding and capacity for receptor-mediated cell entry. This remains to be further investigated, and retroVLPs represent an excellent tool to this end. The fact that they can be easily pseudotyped or modified to display a foreign antigen without harming normal biophysical functions make them suitable for further studies of the role of the form, i.e., particulate versus non-particulate, and the function, i.e., the capacity for cell entry through their cognate receptors, of an immunogen for immunogenicity. Also, the fact that they are small enough to be incorporated into and expressed from a variety of viral or non-viral vectors makes them suitable for studies of the roles of expression, assembly, and budding for immunogenicity.

Furthermore, while genome-free retroVLPs represent safe and versatile vaccine candidates, RDR vectors transducing genes encoding select viral antigens such as immunodominant T-cell epitopes or immunostimulatory factors such as cytokines could be envisioned. Beyond infectious disease, retroVLPs displaying tumor-specific antigens or RDR vectors transducing immunostimulatory genes could be used in conjunction with RCR vectors to treat malignancies.

Finally, conditionally replicating vectors may offer some advantages. Studies with a series of SIV gene deletion mutants⁵⁵ suggest a direct relationship between replication competence and protection, i.e., viruses that replicate better protect better. On a more provocative note, integration of viral antigens into the host genome has been suggested to play a role in immunity against viral disease.^{106, 107} As we learn more about the bases for protective immunity, the retroviruses' capacity for integration might find a use not only in gene therapy but also in genetic vaccines.

Conclusion

Several aspects of simple retrovirus biology make them exceptionally apt as vectors for gene therapy or genetic vaccines. In clinical trials to date, RDR vectors have been used mainly in settings where alternative treatment has failed or does not exist, i.e., when matched bone marrow transplants are not available for patients with primary immunodeficiencies or when conventional surgery and chemotherapy has been unsuccessful or is predicted to have insufficient efficacy for patients with certain malignancies.

At this time of retroviral vector development, lessons learned from the use of RDR vectors and new insights emerging from the use of the two retroviral vector extremes discussed here—RCR vectors that contain largely intact retroviral genomes and retroVLP vectors that contain no genome at all—constitute valuable incremental steps toward the design of retroviral vectors with high integral efficacy and safety. As such, or in various hybrid forms, we may see their use moving from last resort to first line treatment in the clinic in the near future.

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