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

WJ Bowers1,4, JA Olschowka2 and HJ Federoff1,3,4 1Department 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 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 (HSV) is a naturally occurring double- detailed HSV vector-engendered immune responses and stranded DNA virus that has been adapted into an efficient subsequent resolution events primarily within the confines of vector for in vivo gene transfer. HSV-based vectors exhibit the central nervous system. Herein, we describe the wide tropism, large transgene size capacity, and moderately immunobiology of HSV and its derived vector platforms, thus prolonged transgene expression profiles. Clinical implemen- providing an initiation point from where to propose requisite tation of HSV vector-based gene therapy for prevention and/ experimental investigation and potential approaches to or amelioration of human eventually will be prevent and/or counter adverse antivector immune re- realized, but inherently this goal presents a series of sponses. significant challenges, one of which relates to issues of Gene Therapy (2003) 10, 941–945. doi:10.1038/sj.gt.3302047 involvement. Few experimental reports have

Introduction ment of HSV-based gene transfer vectors for diseases of the central nervous system (CNS).5,6 Viewed, albeit virus type 1 (HSV-1) is a naturally mistakenly, as a relatively immunopriviledged site, the neurotropic DNA virus proficient in establishing latent brain appeared to be a highly compatible target for HSV within , but moreover possesses the vector-mediated gene transfer. However, as the numbers ability to infect a wide range of /tissue types. The of CNS studies involving viral vectors (including HSV- cellular receptors (or appropriate species-specific homo- based types) augmented, the effects of CNS immune logues) responsible for virion docking and uptake have responses on the safety and efficiency of gene transfer been cloned, including the herpesvirus entry mediator A became increasingly apparent. These effects range from (HveA; formerly HVEM) and nectin-1 (formerly HveC), immune cell-mediated removal of transduced target cells and, not surprisingly, have been shown to be nearly and inflammation to detrimental influences on transgene ubiquitously expressed.1–4 Intracellular transport of the expression duration. It has therefore become imperative viral to the nucleus leads to a highly coordinated to detail the profiles of HSV vector-elicited CNS immune cascade of viral gene expression. Based upon cellular/ responses. In the following discourse, we will outline the molecular signals that gauge the status of the host cell manner in which wild-type HSV stimulates the host environment, HSV infection can progress via two distinct immunological responses and contrast these mechanisms paths: one that involves active viral gene expression to with those that are most applicable for the two major produce new virions (lytic phase) or another that types of HSV-derived vectors: the recombinant and the involves an abolition of a majority of viral gene amplicon. Understanding how the host’s immune system expression (latent phase). Its double-stranded 150-kb responds to each vector platform will likely provide DNA genome encodes for approximately 80 polypep- insights into devising HSV-based vectors that exhibit tides that play a role in establishing the two phases. safer and more effective in vivo profiles. 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 Wild-type HSV and the immune system to gain entry into neurons initially led to the develop- Up to 90% of human adults possess circulating anti- bodies against .7 These are gener- Correspondence: Dr HJ Federoff, Department of Neurology, Box 645, ated against the -bound glycoproteins, University of Rochester School of Medicine and Dentistry, 601 Elmwood predominantly glycoproteins B, C, and D (gB, gC, and Ave., Rochester, NY 14642, USA gD).8 These antibodies bind to viral particles, and if in Immune responses to HSV vectors within CNS WJ Bowers et al 942 sufficiently high circulating levels, have the potential to of transporter associated with antigen presentation (TAP) inhibit viral adhesion and entry and participate in through a high-affinity interaction that ultimately results inducing antibody-dependent, cell-mediated cytotoxi- in diminished translocation of processed peptide into the city.9 Despite existing humoral immunity, however, ER.21 Working in concert, the ICP47 and vhs proteins are wild-type HSV is proficient in evading the immune responsible for why anti-HSV MHC Class I CTL activity system.10 In fact, the natural HSV cycle typically is rarely substantial in infected hosts. It is important to involves long periods of latency, indicating that the virus note that anti-HSV MHC Class II T cells have been necessitates a series of elaborate and highly efficient reported in a majority of infected individuals, but their mechanisms to avoid detection and elimination by role in controlling HSV-1 is not fully eluci- immune cells.11 The unique ability of HSV to successfully dated.22 avoid immune surveillance is also evident from numer- Latency plays an indirect role in immune evasion. ous, unsuccessful investigations into the development of During this phase of the life cycle, low-level anti-HSV vaccines.12 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 Immune system evasion mechanisms utilized contention within the HSV field, arguments that range by wild-type HSV from the LATs acting as antisense RNAs to prevent exit from latency to these transcripts encoding proteins One mechanism by which HSV avoids immune detection important for viral reactivation.25 Regardless of how is via inhibition of the complement cascades and in the LATs function, the state of latency is a time of essence, antibody-dependent cell lysis. Although HSV-1 minimal viral gene expression, thus diminishing the glycoproteins E (gE) and I (gI) are considered nones- likelihood that viral peptides are presented via MHC I sential for viral propagation, these constituents of the molecules to circulating immune cells. viral envelope as part of a complex appear to serve an important role in immune evasion. The gE–gI complex 13 has been shown to bind the Fc of IgG. It has HSV-1-derived vector platforms been hypothesized that anti-HSV antibodies via their Fab domains recognize gB, gC, and/or gD but that simulta- These highly evolved and intricate mechanisms of

neous Fc binding by the gE–gI complex may result in a immune system evasion appear to be an indication that masking effect, termed ‘antibody bipolar bridging.’14 host immune responses would not impose a major This would lead to an inability of the bound IgG to burden on the in vivo employment of HSV-based gene activate the classical complement cascade and would transfer vectors. However, depending upon the iteration, result in failure to induce phagocyte function.15,16 HSV-based vectors have not only lost many of these Glycoprotein C of HSV-1 acts via a different mechanism virally encoded immunomodulatory activities, but can in that it possesses the capacity to bind the C3 factor produce cellular toxicity at the site of administration that resulting in ‘short-circuiting’ of the complement path- lead to significant inflammatory responses. In addition, way.17 delivery of viral vectors to the CNS is typically In addition, wild-type HSV evades the host immune performed by stereotactic surgery resulting in a tempor- system by interfering with MHC class I cytotoxic T- ary breach of the blood–brain barrier (BBB) and a lymphocyte (CTL) recognition of the infected cell. One potential influx of immune effectors from the periphery. manner by which HSV achieves this is by extinguishing Two disparate HSV-1-based delivery platforms capable global protein synthesis. The tegument of the HSV virion of gene transfer have been developed: recombinant and harbors several viral proteins that act to initiate viral amplicon vectors.26–32 Each vector demonstrates efficient gene expression and/or to establish a favorable environ- gene delivery to a variety of tissues and cell types, ment for viral propagation within infected cells. One of including cells resident in the CNS. However, because of these proteins, designated virion host shutoff (vhs), acts the inherent qualities specific to each of the platforms, as an mRNase to disrupt polysomes and degrade host recombinant and amplicon vectors are engaged differ- cell mRNA. During the process of global protein ently by the host immune system. The following will synthesis shutdown, MHC class I gene expression is detail the two HSV vector types and the information that diminished early during infection, inherently leading to is available regarding the immune responses elicited by a decreased ability of circulating CTLs to recognize and them in the setting of the CNS. destroy HSV-infected cells. The vhs protein is a highly regulated viral gene product since if levels are exces- sively high, vhs can be a source of cellular toxicity Recombinant HSV vectors because of its global mRNase activity.18,19 Another clever mechanism employed by HSV to evade Recombinant HSV vectors comprise a wild-type HSV MHC Class I CTL surveillance relates to the ability of this genome rendered replication defective via disruption/ virus to inhibit antigenic peptide transport to the deletion of an indispensable viral gene(s). Typically, the endoplasmic reticulum (ER). This mechanism was immediate-early gene loci, which encode for potent initially proposed because the occurrence of CTLs transactivation proteins that initiate the viral , specific to wild-type HSV is relatively low, and that later are targeted for insertion of therapeutic transcription during the infection process, MHC class I is retained in units via .33,34 Following the ER in a misfolded conformation.18,19,20 An HSV- construction, recombinant vectors are packaged into derived immediate-early gene product, designated infectious virions using an engineered eucaryotic cell ICP47, mediates this process by interrupting the function line that supplies the absent viral gene product(s) in

Gene Therapy Immune responses to HSV vectors within CNS WJ Bowers et al 943 trans.26,27 The genome of recombinant vectors, at present, propagation (ie, BHK, Vero) with packaging-incompetent can accommodate approximately 30 kb of genetic mate- HSV genomic DNA, amplicon DNA, and any accessory rial. Recombinant vectors are also attractive gene transfer HSV shown to enhance amplicon titers.51 Crude vehicles for CNS disorders because they can be propa- vector lysates are then purified by a series of ultracen- gated to relatively high titers (108–109 PFU/ml). In trifugation steps and titered by expression or transduc- addition, the threat of insertional mutagenesis is greatly tion-based methodologies.52 The titers obtained from diminished as the vector genome persists episomally -free amplicon packaging typically range within postmitotic cell nuclei. from 107 to 108 expressing virus particles/ml. The lack of Immune responses arising from infusion of recombi- contaminating helper virus in these stocks, and thus loss nant HSV vectors can arise from any of the following of immunosuppressive proteins like ICP47, has made the sources: viral particle components, copurified packaging HSV amplicon a powerful delivery platform for infec- cell debris, low-level de novo viral gene product expres- tious and vaccines.53–56 sion, and transgene expression itself. What appears to be Similar to recombinant HSV vectors, immune re- a major source of immune response elicitation is related sponses arising from infusion of HSV amplicon stocks to de novo expression of the intact viral transcription can arise from several sources and such responses are units. Recombinant HSV vectors, although replication- dependent upon the packaging system employed. defective, harbor virtually intact HSV . These Immune response-eliciting sources include viral particle contain ORFs that are expressed at low levels even in the components, copurified packaging cell debris, low-level absence of immediate-early gene products, augmenting de novo viral gene product expression (helper virus-based the potential for antigen processing and subsequent packaging only), and the expressed transgene. Early MHC Class I presentation.35,36 Detailed assessment of generation helper virus-based packaging methods lead immune responses elicited against HSV recombinant to vector stocks that contain substantive levels of vectors within the brain have been lacking. However, contaminating helper virus. Owing to the identical immunological insights may be gleaned from studies physical properties of amplicon and helper virus employing the amplicon vector platform, which have particles, preferential purification of amplicon particles received more investigational attention. is not possible. The replication-defective helper virus, comparable to the recombinant HSV vectors described above, expresses viral proteins at low levels within HSV amplicon vectors transduced cells. These viral proteins exhibit cytotoxic activity and can potentially undergo antigenic processing The HSV-1 amplicon is a uniquely designed eucaryotic and subsequent immune presentation. expression that harbors two nonprotein encod- Wood et al57 were the first investigators to examine the ing virus-derived elements: an HSV origin of DNA immune responses elicited against early iterations of replication (OriS) and the cleavage/packaging sequence packaged HSV amplicon stocks. For their studies they (‘a’ sequence).37–45 Both cis sequences are specifically utilized an amplicon expressing the reporter gene recognized by HSV proteins to promote the replication product b-galactosidase and packaged using a recombi- and incorporation of the vector genome into viable viral nant HSV helper virus (tsK), which possessed a particles, respectively. This highly versatile plasmid can temperature-sensitive mutation in the ICP4 gene locus.57 be readily manipulated to contain desired promoters, Although tsK is replication-defective at the nonpermis- enhancers, and transgenes of substantial size sive temperature of 391C, viral gene product-associated (B130 kb).46 Heterologous transcription units either cytotoxicity remains observable. Administration of these singly or in combination can be cloned into the amplicon amplicon stocks induced a vigorous inflammatory plasmid using conventional molecular cloning techni- response. Elevated MHC Class I expression and micro- ques, and the resultant construct is packaged into glial activation was evident by 2 days postinfection, enveloped viral particles for subsequent transduction of which was followed by MHC Class II cell recruitment, T- cells or tissues. cell activation, and macrophage influx at 4 days follow- Amplicon are dependent upon helper virus ing delivery of helper virus-contaminated amplicon function to provide the replication machinery and stocks. structural proteins necessary for packaging amplicon In a more recent study, our laboratories described the vector DNA into viral particles. An engineered replica- innate responses elicited upon stereotactic delivery of tion-defective HSV derivative that lacks an essential viral HSV amplicon vectors packaged via two different regulatory gene has conventionally provided helper methods.58 C57Bl/6 mice were injected with sterile packaging function. These helper are similar to saline, b-galactosidase-expressing amplicon (HSVlac) the recombinant HSV vectors discussed above in that packaged by a conventional helper virus-based techni- they retain a majority of the HSV genome. The final que, or a helper virus-free HSVlac preparation. product of helper virus-based packaging contains a were killed at 1 or 5 days post-transduction and analyzed mixture of varying ratios of helper and amplicon virions. by immunocytochemistry and quantitative RT-PCR for The titers obtained from helper virus-based amplicon various chemokine, cytokine, and adhesion molecule packaging range from 108 to 109 expressing virus gene transcripts. All injections induced inflammation particles/ml. Recently, helper virus-free amplicon packa- with BBB opening on day 1 that was similarly enhanced ging methods have been developed by providing a following all treatments. By day 5, mRNA levels for the packaging-deficient helper virus genome via a set of five proinflammatory cytokines (IL-1b, TNF-a, IFN-g), che- overlapping or a bacterial artificial chromo- mokines (MCP-1, IP-10) and an adhesion molecule some.47–50 This packaging strategy requires the cotrans- (ICAM-1) had fully resolved in saline-injected mice and fection of eucaryotic cells that are receptive to HSV to near baseline levels in mice receiving helper virus-free

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