Immune responses to replication-defective HSV-1 type ... - Nature

Gene Therapy (2003) 10, 941–945 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt

REVIEW

Immune responses to replication-defective HSV-1 type vectors within the CNS: implications for gene therapy WJ Bowers1,4, JA Olschowka2 and HJ Federoff1,3,4 1

Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA; 2Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA; 3Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA; and 4the Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA

Herpes simplex virus (HSV) is a naturally occurring doublestranded DNA virus that has been adapted into an efficient vector for in vivo gene transfer. HSV-based vectors exhibit wide tropism, large transgene size capacity, and moderately prolonged transgene expression profiles. Clinical implementation of HSV vector-based gene therapy for prevention and/ or amelioration of human diseases eventually will be realized, but inherently this goal presents a series of significant challenges, one of which relates to issues of immune system involvement. Few experimental reports have

detailed HSV vector-engendered immune responses and subsequent resolution events primarily within the confines of the central nervous system. Herein, we describe the immunobiology of HSV and its derived vector platforms, thus providing an initiation point from where to propose requisite experimental investigation and potential approaches to prevent and/or counter adverse antivector immune responses. Gene Therapy (2003) 10, 941–945. doi:10.1038/sj.gt.3302047

Introduction

ment of HSV-based gene transfer vectors for diseases of the central nervous system (CNS).5,6 Viewed, albeit mistakenly, as a relatively immunopriviledged site, the brain appeared to be a highly compatible target for HSV vector-mediated gene transfer. However, as the numbers of CNS studies involving viral vectors (including HSVbased types) augmented, the effects of CNS immune responses on the safety and efficiency of gene transfer became increasingly apparent. These effects range from immune cell-mediated removal of transduced target cells and inflammation to detrimental influences on transgene expression duration. It has therefore become imperative to detail the profiles of HSV vector-elicited CNS immune responses. In the following discourse, we will outline the manner in which wild-type HSV stimulates the host immunological responses and contrast these mechanisms with those that are most applicable for the two major types of HSV-derived vectors: the recombinant and the amplicon. Understanding how the host’s immune system responds to each vector platform will likely provide insights into devising HSV-based vectors that exhibit safer and more effective in vivo profiles.

Herpes simplex virus type 1 (HSV-1) is a naturally neurotropic DNA virus proficient in establishing latent infection within neurons, but moreover possesses the ability to infect a wide range of cell/tissue types. The cellular receptors (or appropriate species-specific homologues) responsible for virion docking and uptake have been cloned, including the herpesvirus entry mediator A (HveA; formerly HVEM) and nectin-1 (formerly HveC), and, not surprisingly, have been shown to be nearly ubiquitously expressed.1–4 Intracellular transport of the viral genome to the nucleus leads to a highly coordinated cascade of viral gene expression. Based upon cellular/ molecular signals that gauge the status of the host cell environment, HSV infection can progress via two distinct paths: one that involves active viral gene expression to produce new virions (lytic phase) or another that involves an abolition of a majority of viral gene expression (latent phase). Its double-stranded 150-kb DNA genome encodes for approximately 80 polypeptides that play a role in establishing the two phases. Mutational analyses of the HSV-1 genome have determined that a number of the viral open reading frames (ORFs) are not essential to viral propagation. This finding combined with the inherent ability of this virus to gain entry into neurons initially led to the developCorrespondence: Dr HJ Federoff, Department of Neurology, Box 645, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642, USA

Wild-type HSV and the immune system Up to 90% of human adults possess circulating antibodies against herpes simplex virus.7 These are generated against the viral envelope-bound glycoproteins, predominantly glycoproteins B, C, and D (gB, gC, and gD).8 These antibodies bind to viral particles, and if in

Immune responses to HSV vectors within CNS WJ Bowers et al

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sufficiently high circulating levels, have the potential to inhibit viral adhesion and entry and participate in inducing antibody-dependent, cell-mediated cytotoxicity.9 Despite existing humoral immunity, however, wild-type HSV is proficient in evading the immune system.10 In fact, the natural HSV life cycle typically involves long periods of latency, indicating that the virus necessitates a series of elaborate and highly efficient mechanisms to avoid detection and elimination by immune cells.11 The unique ability of HSV to successfully avoid immune surveillance is also evident from numerous, unsuccessful investigations into the development of anti-HSV vaccines.12

Immune system evasion mechanisms utilized by wild-type HSV One mechanism by which HSV avoids immune detection is via inhibition of the complement cascades and in essence, antibody-dependent cell lysis. Although HSV-1 glycoproteins E (gE) and I (gI) are considered nonessential for viral propagation, these constituents of the viral envelope as part of a complex appear to serve an important role in immune evasion. The gE–gI complex has been shown to bind the Fc domain of IgG.13 It has been hypothesized that anti-HSV antibodies via their Fab domains recognize gB, gC, and/or gD but that simultaneous Fc binding by the gE–gI complex may result in a masking effect, termed ‘antibody bipolar bridging.’14 This would lead to an inability of the bound IgG to activate the classical complement cascade and would result in failure to induce phagocyte function.15,16 Glycoprotein C of HSV-1 acts via a different mechanism in that it possesses the capacity to bind the C3 factor resulting in ‘short-circuiting’ of the complement pathway.17 In addition, wild-type HSV evades the host immune system by interfering with MHC class I cytotoxic Tlymphocyte (CTL) recognition of the infected cell. One manner by which HSV achieves this is by extinguishing global protein synthesis. The tegument of the HSV virion harbors several viral proteins that act to initiate viral gene expression and/or to establish a favorable environment for viral propagation within infected cells. One of these proteins, designated virion host shutoff (vhs), acts as an mRNase to disrupt polysomes and degrade host cell mRNA. During the process of global protein synthesis shutdown, MHC class I gene expression is diminished early during infection, inherently leading to a decreased ability of circulating CTLs to recognize and destroy HSV-infected cells. The vhs protein is a highly regulated viral gene product since if levels are excessively high, vhs can be a source of cellular toxicity because of its global mRNase activity.18,19 Another clever mechanism employed by HSV to evade MHC Class I CTL surveillance relates to the ability of this virus to inhibit antigenic peptide transport to the endoplasmic reticulum (ER). This mechanism was initially proposed because the occurrence of CTLs specific to wild-type HSV is relatively low, and that later during the infection process, MHC class I is retained in the ER in a misfolded conformation.18,19,20 An HSVderived immediate-early gene product, designated ICP47, mediates this process by interrupting the function Gene Therapy

of transporter associated with antigen presentation (TAP) through a high-affinity interaction that ultimately results in diminished translocation of processed peptide into the ER.21 Working in concert, the ICP47 and vhs proteins are responsible for why anti-HSV MHC Class I CTL activity is rarely substantial in infected hosts. It is important to note that anti-HSV MHC Class II T cells have been reported in a majority of infected individuals, but their role in controlling HSV-1 infections is not fully elucidated.22 Latency plays an indirect role in immune evasion. During this phase of the life cycle, low-level transcription of a restricted number of viral sequences called the ‘latency-associated transcripts’ (LATs) occurs.23,24 The function of these transcripts has been a point of contention within the HSV field, arguments that range from the LATs acting as antisense RNAs to prevent exit from latency to these transcripts encoding proteins important for viral reactivation.25 Regardless of how the LATs function, the state of latency is a time of minimal viral gene expression, thus diminishing the likelihood that viral peptides are presented via MHC I molecules to circulating immune cells.

HSV-1-derived vector platforms These highly evolved and intricate mechanisms of immune system evasion appear to be an indication that host immune responses would not impose a major burden on the in vivo employment of HSV-based gene transfer vectors. However, depending upon the iteration, HSV-based vectors have not only lost many of these virally encoded immunomodulatory activities, but can produce cellular toxicity at the site of administration that lead to significant inflammatory responses. In addition, delivery of viral vectors to the CNS is typically performed by stereotactic surgery resulting in a temporary breach of the blood–brain barrier (BBB) and a potential influx of immune effectors from the periphery. Two disparate HSV-1-based delivery platforms capable of gene transfer have been developed: recombinant and amplicon vectors.26–32 Each vector demonstrates efficient gene delivery to a variety of tissues and cell types, including cells resident in the CNS. However, because of the inherent qualities specific to each of the platforms, recombinant and amplicon vectors are engaged differently by the host immune system. The following will detail the two HSV vector types and the information that is available regarding the immune responses elicited by them in the setting of the CNS.

Recombinant HSV vectors Recombinant HSV vectors comprise a wild-type HSV genome rendered replication defective via disruption/ deletion of an indispensable viral gene(s). Typically, the immediate-early gene loci, which encode for potent transactivation proteins that initiate the viral lytic cycle, are targeted for insertion of therapeutic transcription units via homologous recombination.33,34 Following construction, recombinant vectors are packaged into infectious virions using an engineered eucaryotic cell line that supplies the absent viral gene product(s) in


trans.26,27 The genome of recombinant vectors, at present, can accommodate approximately 30 kb of genetic material. Recombinant vectors are also attractive gene transfer vehicles for CNS disorders because they can be propagated to relatively high titers (108–109 PFU/ml). In addition, the threat of insertional mutagenesis is greatly diminished as the vector genome persists episomally within postmitotic cell nuclei. Immune responses arising from infusion of recombinant HSV vectors can arise from any of the following sources: viral particle components, copurified packaging cell debris, low-level de novo viral gene product expression, and transgene expression itself. What appears to be a major source of immune response elicitation is related to de novo expression of the intact viral transcription units. Recombinant HSV vectors, although replicationdefective, harbor virtually intact HSV genomes. These contain ORFs that are expressed at low levels even in the absence of immediate-early gene products, augmenting the potential for antigen processing and subsequent MHC Class I presentation.35,36 Detailed assessment of immune responses elicited against HSV recombinant vectors within the brain have been lacking. However, immunological insights may be gleaned from studies employing the amplicon vector platform, which have received more investigational attention.

HSV amplicon vectors The HSV-1 amplicon is a uniquely designed eucaryotic expression plasmid that harbors two nonprotein encoding virus-derived elements: an HSV origin of DNA replication (OriS) and the cleavage/packaging sequence (‘a’ sequence).37–45 Both cis sequences are specifically recognized by HSV proteins to promote the replication and incorporation of the vector genome into viable viral particles, respectively. This highly versatile plasmid can be readily manipulated to contain desired promoters, enhancers, and transgenes of substantial size (B130 kb).46 Heterologous transcription units either singly or in combination can be cloned into the amplicon plasmid using conventional molecular cloning techniques, and the resultant construct is packaged into enveloped viral particles for subsequent transduction of cells or tissues. Amplicon plasmids are dependent upon helper virus function to provide the replication machinery and structural proteins necessary for packaging amplicon vector DNA into viral particles. An engineered replication-defective HSV derivative that lacks an essential viral regulatory gene has conventionally provided helper packaging function. These helper viruses are similar to the recombinant HSV vectors discussed above in that they retain a majority of the HSV genome. The final product of helper virus-based packaging contains a mixture of varying ratios of helper and amplicon virions. The titers obtained from helper virus-based amplicon packaging range from 108 to 109 expressing virus particles/ml. Recently, helper virus-free amplicon packaging methods have been developed by providing a packaging-deficient helper virus genome via a set of five overlapping cosmids or a bacterial artificial chromosome.47–50 This packaging strategy requires the cotransfection of eucaryotic cells that are receptive to HSV

propagation (ie, BHK, Vero) with packaging-incompetent HSV genomic DNA, amplicon DNA, and any accessory HSV genes shown to enhance amplicon titers.51 Crude vector lysates are then purified by a series of ultracentrifugation steps and titered by expression or transduction-based methodologies.52 The titers obtained from helper virus-free amplicon packaging typically range from 107 to 108 expressing virus particles/ml. The lack of contaminating helper virus in these stocks, and thus loss of immunosuppressive proteins like ICP47, has made the HSV amplicon a powerful delivery platform for infectious disease and cancer vaccines.53–56 Similar to recombinant HSV vectors, immune responses arising from infusion of HSV amplicon stocks can arise from several sources and such responses are dependent upon the packaging system employed. Immune response-eliciting sources include viral particle components, copurified packaging cell debris, low-level de novo viral gene product expression (helper virus-based packaging only), and the expressed transgene. Early generation helper virus-based packaging methods lead to vector stocks that contain substantive levels of contaminating helper virus. Owing to the identical physical properties of amplicon and helper virus particles, preferential purification of amplicon particles is not possible. The replication-defective helper virus, comparable to the recombinant HSV vectors described above, expresses viral proteins at low levels within transduced cells. These viral proteins exhibit cytotoxic activity and can potentially undergo antigenic processing and subsequent immune presentation. Wood et al57 were the first investigators to examine the immune responses elicited against early iterations of packaged HSV amplicon stocks. For their studies they utilized an amplicon expressing the reporter gene product b-galactosidase and packaged using a recombinant HSV helper virus (tsK), which possessed a temperature-sensitive mutation in the ICP4 gene locus.57 Although tsK is replication-defective at the nonpermissive temperature of 391C, viral gene product-associated cytotoxicity remains observable. Administration of these amplicon stocks induced a vigorous inflammatory response. Elevated MHC Class I expression and microglial activation was evident by 2 days postinfection, which was followed by MHC Class II cell recruitment, Tcell activation, and macrophage influx at 4 days following delivery of helper virus-contaminated amplicon stocks. In a more recent study, our laboratories described the innate responses elicited upon stereotactic delivery of HSV amplicon vectors packaged via two different methods.58 C57Bl/6 mice were injected with sterile saline, b-galactosidase-expressing amplicon (HSVlac) packaged by a conventional helper virus-based technique, or a helper virus-free HSVlac preparation. Animals were killed at 1 or 5 days post-transduction and analyzed by immunocytochemistry and quantitative RT-PCR for various chemokine, cytokine, and adhesion molecule gene transcripts. All injections induced inflammation with BBB opening on day 1 that was similarly enhanced following all treatments. By day 5, mRNA levels for the proinflammatory cytokines (IL-1b, TNF-a, IFN-g), chemokines (MCP-1, IP-10) and an adhesion molecule (ICAM-1) had fully resolved in saline-injected mice and to near baseline levels in mice receiving helper virus-free

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HSVlac. In contrast, mice injected with helper viruspackaged amplicon stocks elicited elevated inflammatory molecule expression and immune cell infiltration even at day 5. In aggregate, these studies demonstrated that helper virus-free amplicon preparations exhibit a safer innate immune response profile, presumably because of the absence of helper virus gene expression, and provide support for future amplicon-based CNS gene transfer strategies. What remains to be assessed experimentally in greater detail is the role that virion structural components, packaging cell-derived debris, and transgene product appear to play in the activation of the innate immune response by helper virus-free amplicon stocks. In addition, the role of pre-existing HSV immunity in modulating host responses to braindelivered HSV vectors remains essentially unstudied.

Future directions While helper virus-free HSV amplicon stocks appear to exhibit the more favorable immunological profile when delivered to the CNS, there exist a number of modifications that can be made to the HSV recombinant and amplicon platforms to improve their inherent immune response-eliciting potentials. Enhanced methods of virion purification need to be developed to minimize packaging cell line-derived contaminants. This will be vital for future clinical implementation of either platform. Additionally, if the role of the transgene product in elicitation of immune responses is found to be substantive, it will likely become necessary to utilize speciesspecific transgenes to take advantage of the state of antigenic tolerance that exists in the transduce host organism. However, because of the high levels of transgene expression mediated by HSV vectors this path should be traveled carefully, as there exists the potential to break tolerance and induce an autoimmune-like disorder. A third approach that could be employed using HSV vectors involves the use of immunomodulation. Owing to the large transgene capacity of HSV vectors, it is possible to include transcription units that encode anti-inflammatory molecules or even cytokines that transiently shape the immune response to a favorable state. The viral envelope glycoproteins that exist on the exterior of vector-harbored virions represent another target for minimizing anti-HSV immune responses. Mapping of dominant epitopes within these viral glycoproteins provides the information necessary to initiate stepwise modification in order to ‘dampen’ these epitopes without compromising transduction efficiency. The further development of these approaches and countless others will undoubtedly advance the utility and safety of both HSV vector platforms, and will likely have broader applicability to other gene delivery systems.

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