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Immune responses to adenovirus vectors in the nervous system
REVIEW
M.J.A. Wood et al. – Immune responses to adenovirus vectors
Immune responses to adenovirus vectors in the nervous system Matthew J.A. Wood, Harry M. Charlton, Kathryn J. Wood, Koji Kajiwara and Andrew P. Byrnes Non-replicating adenovirus vectors are being developed as vehicles for gene transfer into cells of the nervous system. An important requirement for successful gene transfer is the absence of deleterious cytotoxic or inflammatory side effects of the delivery system. Despite offering relatively stable reporter gene expression, currently available adenovirus vectors also elicit immune responses in the brain, both at the site of vector delivery and at synaptically linked distant sites. However, although an anti-viral T-lymphocyte response eliminates the vector and damages local tissue in many peripheral organs, the immune response to adenovirus in the brain is less effective and enables the vector to persist. Nevertheless, in this persistent state the adenovirus vector remains a potential target for a destructive immune response that can also cause local demyelination. The development of strategies to minimize this damaging immune response, through either vector modification or immunomodulation, will be crucial for the future success of genetic therapies in the brain. Trends Neurosci. (1996) 19, 497–501
G
ENE THERAPY has attracted considerable interest as a molecular tool for neurobiological research and also as a potential treatment for human neurological diseases that range from simple monogenic disorders (such as spinal muscular atrophy and Lesch–Nyhan syndrome) to complex diseases, including the common neurodegenerative disorders and malignancy1,2. The only reliable approach that has so far been developed to transfect significant numbers of adult brain cells is the use of a non-replicating virus as a vector. Several viral vector systems based on DNA viruses like herpes simplex virus (HSV)3, adeno-associated virus (AAV)4 and adenovirus5–7, and very recently a retroviral system based on the human immunodeficiency virus (HIV)8, have each demonstrated potential for direct gene transfer to the nervous system in vivo. Similar requirements for success pertain to each system; namely, that they should ideally provide long-term regulated gene expression at therapeutic levels, targeted in a cell-specific fashion, in the absence of harmful cytotoxic or immunological side effects. At present, none of these criteria has been satisfied completely. The immunological constraints in particular represent a significant obstacle to success, not least because the regulation of immunity in the brain is currently poorly understood.
Immune responses to adenovirus vectors The immune system plays a crucial role in peripheral organs in limiting the duration of transgene expression from adenoviral vectors9,10. For example, in normal mouse liver, transgene expression declines dramatically over the initial few weeks and is accompanied by inflammation; both of these effects are due to anti-vector immune responses10,11. In sharp contrast, transgene expression occurs for considerably longer in young animals with underdeveloped Copyright © 1996, Elsevier Science Ltd. All rights reserved. 0166 - 2236/96/$15.00
immune systems or in those that are deficient in T lymphocytes or are immunosuppressed9,10,12. Unlike the rapid decline in transgene expression observed in peripheral organs, adenovirus-mediated transduction of adult brain parenchyma leads to reasonably stable gene expression5–7,13. (The same does not necessarily hold true for the transduction of cells lining the ventricular cavities14.) However, despite this optimistic early observation, initial studies noted histological evidence of focal pathology in the brain5,15. It is still commonly believed that the brain is an organ protected from the effects of destructive immune responses. However, it is quite clear that the immune system can and does respond to antigenic stimuli in the brain16–18, albeit less robustly than is usually observed in other organs, and that such antigenic stimuli may include non-replicating viral vectors. Immune responses to HSV-1 vectors have been described19,20; on the other hand, AAV vectors are thought to be relatively non-immunogenic4, but a systematic immune analysis of these vectors in the brain has yet to be undertaken. First-generation vectors based on human adenovirus type-5 contain a deletion of the E1 region (Fig. 1) to render the virus non-replicating. When these vectors are inoculated into the striatum, they stimulate a strong inflammatory reaction13. This response displays biphasic kinetics, comprising an early nonspecific phase and a later T-cell-mediated phase21. During the early phase, T cells and macrophages are recruited, local microglia and astrocytes are activated strongly, and high levels of major histocompatibility complex (MHC) class-I antigens are expressed (Fig. 2A). Negligible inflammation occurs in control animals, indicating that the trauma associated with vector delivery plays little part in the genesis of the observed early inflammation. (MHC class-I proteins PII: S0166-2236(96)10060-6
TINS Vol. 19, No. 11, 1996
Matthew J.A. Wood, Harry M. Charlton, Koji Kajiwara and Andrew P. Byrnes are at the Dept of Human Anatomy, Oxford University, Oxford, UK OX1 3QX. Kathryn J. Wood and Koji Kajiwara are at the Nuffield Dept of Surgery, John Radcliffe Hospital, Oxford, UK OX3 9DU.
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M.J.A. Wood et al. – Immune responses to adenovirus vectors
Fig. 1. Simplified transcription map of the genome of human adenovirus type 5. The genome of adenovirus type 5 is a 36 kb linear duplex structure. The early transcription units (E1–E4) and the major late transcription region are shown, and these are expressed in a temporally ordered sequence. E1 and E3 are the most convenient loci at which to insert new genes. In this figure a 2.9 kb deletion in the E1 region enabled the insertion of a transcription unit encoding the lacZ reporter gene. New vectors containing mutations in the E2 region or deletions in the E4 region may provide improved transgene expression compared with vectors deleted only in E1.
are cell-surface heterodimers, normally expressed at very low levels in the brain, whose function is to present intracellular antigens to CD8+ T cells. In contrast, extracellular antigens are presented via MHC class-II proteins to CD4+ T cells. Expression of MHC class-II is restricted to specific cell populations, including dendritic cells and cells of the macrophage–monocyte lineage17 .) The later T-cell-mediated immune response to adenovirus appears to be directed mainly against viral proteins, since vectors that do not express a transgene elicit similar responses13, and others that have been irradiated with UV (to abolish de novo viral gene expression) provoke the early, nonspecific, inflammatory reaction but not the later T-cell-mediated phase21. This immune response has declined greatly 1–2 months after the vector was inoculated into the brain, despite the fact that expression of transgene and viral proteins can still be detected13. The strength of the immune response to adenovirus is to some extent modifiable by genetic factors because it has been shown to be dependent to some degree on the strain of rodent inoculated. Precisely what such genetic factors might be is currently unknown. From the above, it can be concluded that adenovirus vectors can offer long-term transgene expression in the brain despite provoking an immune response.
Immune responses in distant synaptically linked brain sites An important and interesting property of viral vectors is their capacity for rapid retrograde axonal transport in the brain, which suggests that they might be useful for gene delivery to distant synaptically linked brain regions. This behaviour has been particularly well characterized for HSV vectors22 and, more recently, for adenovirus5,23,24, a virus that is not naturally neurotropic. While most cell types in the brain appear to be susceptible to adenovirus infection, it is not yet clear that the same is true of all retrograde pathways. The cellular mechanisms underlying 498
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adenoviral infection have not been fully identified but it is thought to proceed in two phases25. The first step involves the attachment of the viral fibre protein to an unknown cellular receptor. Infection then progresses by the binding of the viral penton protein to cellular integrins, followed by endocytosis26. The expression of these viral molecules is not well characterized in the brain, and so there can be no certainty that adenoviral infection occurs by specific receptor-mediated uptake in all cases. Nevertheless, this type of retrograde gene delivery might prove to be a valuable strategy for cellspecific gene transfer in the brain. Gene delivery to distant synaptically linked sites using HSV vectors has been shown to be associated with inflammation in these linked areas19; similar, although milder, inflammation occurs when adenovirus vectors are used13. Inoculation of an adenoviral vector into the striatum results in transgene expression in neurones of the synaptically linked ipsilateral substantia nigra pars compacta (SNc), but it also induces inflammation at this site (Fig. 2B). This immune response is less strong and is typically delayed compared with that occurring at the primary inoculation site, but it still features locally upregulated MHC class-I expression and microglial activation in the early phase. Only relatively small numbers of T cells are detected in the SNc; precisely what the mechanism might be for the inflammation at this site and for the low level of lymphocyte recruitment is not known. The low concentration of T cells is of particular interest, given that the vector is only present in neurones and that even minor trauma is not associated with vector delivery to these distant sites – both of these facts suggest the possibility of a vector-specific immune response. (This idea is discussed below.) In summary, the retrograde targeting of genes in the brain has great potential as a strategy for cell-specific gene delivery, but at present it is an obligatory and not optional feature of adenovirus vectors and carries with it the risk of initiating unwanted inflammation in distant afferent brain regions.
Quantification of immune responses to non-replicating vectors The development of methods to quantify immunological parameters following the injection of nonreplicating viral vectors into the brain is important for several reasons. Such techniques will enable standard functional immune assays to be applied to questions of viral vector neuroimmunology, and will therefore give further insight into important aspects of immune regulation in the brain. Perhaps more crucially, the advent of such methods will be vital for the accurate evaluation of the immunological risk of new or improved viral vectors. Immune responses to nonreplicating viral vectors are typically much weaker than those encountered in the various types of viral encephalitis, such as HSV encephalitis27 or Borna disease28, making accurate quantification all the more difficult. Sensitive methods for isolating infiltrating leukocytes from the brain after viral vector injection have now been established, based on enzymatic digestion of brain tissue followed by density gradient centrifugation (K. Kajiwara, M.J.A. Wood and K.J. Wood, unpublished observations). Such leukocytes can now be studied by fluorescence-activated cell-sorting (FACS) analysis and in vitro immune assays (such as
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M.J.A. Wood et al. – Immune responses to adenovirus vectors
precursor, cytotoxic and proliferation assays). Using these methods in the study of the effects of E1-deleted adenovirus vectors, the numbers of infiltrating leukocytes are found to peak at around 9 days after inoculation and to decline slowly thereafter. Activated T lymphocytes [expressing receptors for interleukin-2 (IL2)] only begin to constitute a significant component of the infiltrate after 6 days, and therefore form part of the second, so-called adaptive, phase of the immune response. Interestingly, CD8+ T cells are dominant only in the early phase; thereafter, CD4+ cells are more common (Fig. 3).
Role of T-cell subsets in the immune response From the quantitative studies referred to above, the predominant T cells present during the adaptive phase of the immune response to adenovirus vectors in the brain are CD4+ T lymphocytes. To examine the functional role of CD4+ and CD8+ cells more closely, monoclonal antibodies were used to deplete each subset in turn21. Significantly, the early inflammatory response was undiminished even when both subsets of T cells were depleted. Given that irradiation of the adenovirus vector with UV also failed to diminish this inflammation, it appears that this early phase requires neither viral gene transcription nor T lymphocytes for its generation. Similar findings have been obtained in the lung29,30. Potential inflammatory stimuli during the early phase include the release of cytokines by virally infected cells, a well-documented phenomenon in the case of peripheral organs30. Similarly, cytokine release in the brain might activate cerebral vascular endothelium and initiate leukocyte chemoattraction. Studies of this early inflammatory phase in the brain indicate that neither vector modification by further limiting viral gene expression nor T-cell immunotherapy will be able to eliminate this inflammation. A better understanding of the mechanisms underlying this inflammatory phase and the future development of anti-inflammatory strategies seem to be necessary to reduce or eliminate this risk. In contrast, the later T-cell-mediated phase of the immune response to adenovirus vectors in the brain (from 6 days onwards) is markedly reduced following CD8+ cell depletion and eliminated completely by CD4+ T-cell depletion21. This implicates the CD4+ subset as functionally dominant in this inflammatory phase. In peripheral organs, both T-cell subsets are important during this phase, as shown by subset depletion and adoptive T-cell transfer studies (that is, transplanting T cells from one animal to another)31–33. However, unique to the brain, and of significance in this discussion, is the observation that vector transgene expression is unaltered in level and duration whether T cells are depleted during this phase or not. This suggests that any decline in gene expression during this phase of the immune response is probably not due to T lymphocytes. It is possible that the early phase of inflammation, which occurs independently of T cells, could contribute in some way to the later decline in transgene expression; however, other possibilities, such as the promoter inactivation observed in some retroviral vectors34 or the gradual loss of episomal adenoviral elements during cell division, should also be considered. It would appear that the mechanism underlying the persistent transgene expression in the brain, as distinct from the
Fig. 2. Immune responses to adenovirus vectors in the brain. (A) Induction of major histocompatibility complex (MHC) class-I expression is observed after adenovirus injection into the caudate nucleus of the striatum of rats, and is maximal 48 h after inoculation. (B) Upregulated MHC class-I expression can also be observed 9–15 days after inoculation in brain regions that are synaptically linked to the caudate, such as the ipsilateral substantia nigra pars compacta (between dashed lines). (C) High levels of persistent MHC class-I expression are seen in and extending beyond the caudate nucleus after peripheral sensitization with adenovirus vector in animals that had previously received vector injected into the caudate. (D) In an adjacent section to that shown in C, histochemical staining for myelin reveals demyelination (arrow) in sensitized animals – in this case, in the corpus callosum. Scale bar, 800 mm in A, 300 mm in B, and 600 mm in C and D. Abbreviation: lv, lateral ventricle.
periphery, is an ineffective T-cell response that fails to clear the vector or transgene product from the brain.
Immune responses to adenovirus vectors in sensitized animals The conclusion drawn at the end of the previous section prompts the obvious question of why the T-cell immune response to a non-replicating adenovirus vector in the brain is ineffective and incapable of clearing the virus from the brain. Where precisely is the defect in this response? Alternatively, under what conditions might the immune system act effectively to clear the vector and eliminate transgene expression from the brain, and what effector mechanisms might be used in so doing? It is known that foreign antigens can survive in the brain for extended periods without inducing a cellmediated immune response35, even though the same TINS Vol. 19, No. 11, 1996
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M.J.A. Wood et al. – Immune responses to adenovirus vectors
Fig. 3. Relative abundance of CD4+ and CD8+ T lymphocytes in the brain after adenovirus injection. Leukocytes were isolated from the rat brain at times ranging from 0 to 60 days after adenovirus vector injection into the caudate nucleus. Cells were labelled with antibodies to CD4 and CD8, and the numbers of these cells were determined by fluorescence-activated cell-sorting (FACS) analysis. The relative abundance of the CD4+ and CD8+ T-cell subsets during this period of time is shown, expressed as a proportion of total leukocytes isolated from the brain (CD45+ cells). CD8+ T cells increase rapidly during the early inflammatory phase. After approximately day 9, and correlating with the onset of the T-cell-mediated phase of the immune response, CD4+ T lymphocytes are found in greatest abundance.
antigens stimulate an effective response when injected into the periphery. The defect underlying such an ineffective T-cell response might be located in either the afferent or efferent limbs of the immune system, both of which are less robust in the brain compared with the periphery. Inadequate immune activation (afferent limb defect) can occur when foreign transplantation antigens placed in the brain survive by failing to stimulate the immune system36. However, a subsequent transplant (syngeneic to the original) placed in the periphery can potentiate a response to the graft placed earlier in the brain37,38, in some cases
Fig. 4. Inflammation in regions afferent to the caudate nucleus (C). Parasagittal representation of rat-brain regions synaptically linked to the caudate nucleus of the striatum in which severe inflammation is observed following peripheral sensitization to adenovirus in animals that have previously received vector injected into the caudate (arrow). These areas include frontal cortex (FC), globus pallidus (GP), thalamus (T), ventral tegmental area (VTA), substantia nigra (SN), retrorubral field of the mesencephalic reticular nucleus (MRN) and the locus coeruleus (LC). Abbreviation: lv, lateral ventricle.
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leading to graft rejection in the brain36. Thus, it is not that the T cells are incapable of destroying the foreign tissue but that the foreign transplantation antigens placed in the brain do not on their own elicit a sufficiently strong T-cell response. A T-cell response strong enough to eliminate adenoviral vectors in the brain can be stimulated by subsequent peripheral subcutaneous exposure to the vector13,39. In this case, vector transgene expression in the brain is found to be curtailed rapidly and is accompanied by severe inflammation, suggesting strongly that an effective T-cell response has been stimulated. The notable features of this process are severe mononuclear and T-cell inflammation, associated with dendritic cell recruitment and strong microglial activation at the inflammatory focus. More importantly, this intense inflammation is also accompanied by evidence of local demyelination (Fig. 2D). Such demyelination is not seen in control animals, indicating that the minor trauma involved in vector delivery plays no part in its generation; neither is demyelination found in synaptically linked distant sites. The observed demyelination resembles the neuropathology seen after the immune system has been peripherally sensitized to mycobacterial antigens that were previously inoculated into the brain35. This severe inflammation occurs not only at the original site of vector delivery but also in areas containing synaptically linked neuronal populations39. For example, following primary inoculation into the striatum, secondary foci of severe inflammation include frontal cortex, ipsilateral SNc and ventral tegmental area, globus pallidus, thalamus, locus coeruleus and the retrorubral field of the mesencephalic reticular nucleus (Fig. 4). The intense inflammation in these areas, similar in nature to that found in the striatum and including numerous T cells, implies a vector-specific immune response. Although it is known that activated T cells enter the brain, they fail to accumulate significantly in the absence of antigen recognition40. Any mechanism proposed to account for the interesting hypothesis of a vectorspecific immune response would be speculative at present since it is debatable whether neurones are able to present antigens via MHC class-I to T cells in vivo. Although there is some evidence defining the circumstances under which neurones might present antigens in vitro, it is possible that they lack some crucial proteins necessary for effective antigen presentation in the living animal41,42. Mechanisms for this inflammation other than direct antigen presentation by neurones should also be considered, and this adenovirus model system offers the opportunity to distinguish between these possibilities in future experiments. What mechanisms might underlie the rapid elimination of transgene expression and the demyelinating pathology seen in the sensitized animals? The elimination of reporter gene expression in the periphery is accompanied by vector elimination10. MHC class Irestricted CD8+ cytotoxic T cells are required for this elimination since persistent expression is seen in mice deficient in b2-microglobulin or perforin33,43 (deficiencies of which disable antigen presentation via the class-I route). Such direct cytotoxicity is a possible explanation for the decline in transgene expression in the brain but whether it occurs at all, and if it does so whether it occurs with or without the elimination of
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M.J.A. Wood et al. – Immune responses to adenovirus vectors
the transduced cells, has yet to be established. Other indirect non-cytotoxic mechanisms (for example, cytokine-mediated mechanisms like those found in lymphocytic choriomeningitis virus infection44) also require investigation. Similarly, the mechanism for the demyelination could be either direct cytotoxic immune damage to virus-infected oligodendrocytes or indirect damage through other routes.
Concluding remarks Since the T-cell response to adenovirus in the brain is generally ineffective, long-term transgene expression is possible with currently available vectors. Furthermore, adenovirus vectors can deliver genes to synaptically linked brain areas, which is a valuable potential strategy for gene targeting in the complex environment of the adult brain. However, against these putative advantages must be set the definite disadvantage that long-lasting transgene expression in the brain is seriously at risk if the immune response is reactivated. Therefore, adenovirus vectors are currently unsafe for humans, in whom prior or subsequent infection with adenovirus would lead to inflammation, transgene elimination from the brain and local damage. Such severe inflammation would be located not only at the site of vector delivery but also in synaptically linked brain regions. Future generations of adenovirus vectors from which more viral genes have been deleted are likely to be of lower immunogenicity. This optimism is supported by the observation that UV-irradiated adenovirus inoculated into the brain fails to stimulate severe inflammation after peripheral sensitization with an E1-deleted vector39. Vectors incorporating mutations in the E2 region45 or deletions in the E4 region46 could offer improvements in transgene expression, although this has yet to be shown unequivocally. In addition, the development of strategies for immunotherapy might lead to the effective control of the early inflammatory and later T-cell-mediated phases of the immune response that are directed against the vector and also the transgene. As has been alluded to above, immunosuppression can enhance transgene expression in the periphery9. The construction of adenovirus vectors incorporating genes encoding cytokines47, particularly those with immunosuppressive potential (such as IL4, IL10 or transforming growth factor-b), is an interesting and potentially elegant strategy for immunotherapy in many organs, including the brain. Not only would the testing of such a strategy advance our understanding of immune regulation in the brain, but it might also speed up the development of safer vectors that are necessary for gene therapy in the nervous system to proceed. In addition, such immunotherapy might have a direct application in
the treatment of neurological disorders associated with inflammatory or immune-mediated pathology. Selected references 1 Karpati, G. et al. (1996) Trends Neurosci. 19, 49–54 2 Friedmann, T. (1994) Trends Genet. 10, 210–214 3 Glorioso, J.C., Goins, W.F. and Fink, D.J. (1992) Sem. Virol. 3, 265–276 4 Kaplitt, M.G. et al. (1994) Nat. Genet. 8, 148–154 5 Akli, S. et al. (1993) Nat. Genet. 3, 224–228 6 Davidson, B.L. et al. (1993) Nat. Genet. 3, 219–223 7 Le Gal La Salle, G. et al. (1993) Science 259, 988–990 8 Naldini, L., Blomer, U. and Gallay, P. (1996) Science 272, 263–267 9 Kass-Eisler, A. et al. (1994) Gene Ther. 1, 395– 402 10 Yang, Y. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4407– 4411 11 Yang, Y. et al. (1996) Gene Ther. 3, 137–144 12 Kay, M.A. et al. (1995) Nat. Genet. 11, 191–197 13 Byrnes, A.P. et al. (1995) Neuroscience 66, 1015–1024 14 Bajocchi, G. et al. (1993) Nat. Genet. 3, 229–234 15 Davidson, B.L. et al. (1994) Exp. Neurol. 125, 258–267 16 Perry, V.H., Andersson, P-B. and Gordon, S. (1993) Trends Neurosci. 16, 268–273 17 Sloan, D.J., Wood, M.J. and Charlton, H.M. (1991) Trends Neurosci. 14, 341–346 18 Fabry, Z., Raine, C.S. and Hart, M.S. (1994) Immunol. Today 15, 218–224 19 Wood, M.J.A. et al. (1994) Gene Ther. 1, 283–291 20 Wood, M.J.A. et al. (1996) in Protocols for Gene Transfer in Neuroscience: Towards Gene Therapy of Neurological Disorders (Lowenstein, P.R. and Enquist, L.W., eds), pp. 365–376, John Wiley and Sons 21 Byrnes, A.P., Wood, M.J.A. and Charlton, H.M. Gene Ther. (in press) 22 Wood, M.J.A. et al. (1994) Exp. Neurol. 130, 127–140 23 Ridoux, V. et al. (1994) Brain Res. 648, 171–175 24 Kuo, H. et al. (1995) Brain Res. 705, 31–38 25 Nemerow, G.R., Cheresh, D.A. and Wickham, T.J. (1994) Trends Cell Biol. 4, 52–55 26 Wickham, T.J. et al. (1993) Cell 73, 309–319 27 McFarland, D.J., Sikora, E. and Hotchin, J. (1986) J. Neurol. Sci. 72, 307–318 28 de la Torre, J.C. (1994) J. Virol. 68, 7669–7675 29 McCoy, R.D. et al. (1995) Hum. Gene Ther. 6, 1553–1560 30 Ginsberg, H.S. et al. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1651–1655 31 Yang, Y. et al. (1995) J. Virol. 69, 2004–2015 32 Yang, Y. and Wilson, J.M. (1995) J. Immunol. 155, 2564–2570 33 Yang, Y. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7257–7261 34 Rettinger, S.D. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1460–1464 35 Matyszak, M.K. and Perry, V.H. (1995) Neuroscience 64, 967–977 36 Mason, D.W. et al. (1986) Neuroscience 19, 685–694 37 Duan, W-M. et al. (1995) Neuroscience 64, 629–641 38 Nicholas, M.K. et al. (1987) J. Immunol. 139, 2275–2283 39 Byrnes, A.P., MacLaren, R.E. and Charlton, H.M. (1996) J. Neurosci. 16, 3045–3055 40 Hickey, W.F., Hsu, B.L. and Kimura, H. (1991) J. Neurosci. Res. 28, 254–260 41 Neumann, H. et al. (1995) Science 269, 549–552 42 Joly, E. and Oldstone, M.B.A. (1992) Neuron 8, 1185–1190 43 Yang, Y., Ertl, H.C.J. and Wilson, J.M. (1994) Immunity 1, 433– 442 44 Tishon, A. et al. (1995) Virology 212, 244–250 45 Yang, Y. et al. (1994) Nat. Genet. 7, 362–369 46 Krougliak, V. and Graham, F.L. (1995) Hum. Gene Ther. 6, 1575–1586 47 Kolls, J. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 215–219
Acknowledgements We thank P. Belk for his help in the preparation of the manuscript and M. McMenamin for her assistance with some of the figures. This work is supported by grants from the MRC (UK), BBSRC (UK), the Wellcome Trust and by the award of a Wellcome Prize Studentship to APB.
TINS in Washington TINS will be at the Society for Neuroscience meeting in Washington in November. Be sure to visit the Elsevier Science stand (Booth Nos 215–220) to pick up FREE sample copies of TINS and other Elsevier journals. New personal subscribers to TINS receive a 20% discount when they subscribe at the meeting and there will also be some very special give-aways! Don’t miss out this year!
TINS Vol. 19, No. 11, 1996
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M.J.A. Wood et al. – Immune responses to adenovirus vectors
Immune responses to adenovirus vectors in the nervous system Matthew J.A. Wood, Harry M. Charlton, Kathryn J. Wood, Koji Kajiwara and Andrew P. Byrnes Non-replicating adenovirus vectors are being developed as vehicles for gene transfer into cells of the nervous system. An important requirement for successful gene transfer is the absence of deleterious cytotoxic or inflammatory side effects of the delivery system. Despite offering relatively stable reporter gene expression, currently available adenovirus vectors also elicit immune responses in the brain, both at the site of vector delivery and at synaptically linked distant sites. However, although an anti-viral T-lymphocyte response eliminates the vector and damages local tissue in many peripheral organs, the immune response to adenovirus in the brain is less effective and enables the vector to persist. Nevertheless, in this persistent state the adenovirus vector remains a potential target for a destructive immune response that can also cause local demyelination. The development of strategies to minimize this damaging immune response, through either vector modification or immunomodulation, will be crucial for the future success of genetic therapies in the brain. Trends Neurosci. (1996) 19, 497–501
G
ENE THERAPY has attracted considerable interest as a molecular tool for neurobiological research and also as a potential treatment for human neurological diseases that range from simple monogenic disorders (such as spinal muscular atrophy and Lesch–Nyhan syndrome) to complex diseases, including the common neurodegenerative disorders and malignancy1,2. The only reliable approach that has so far been developed to transfect significant numbers of adult brain cells is the use of a non-replicating virus as a vector. Several viral vector systems based on DNA viruses like herpes simplex virus (HSV)3, adeno-associated virus (AAV)4 and adenovirus5–7, and very recently a retroviral system based on the human immunodeficiency virus (HIV)8, have each demonstrated potential for direct gene transfer to the nervous system in vivo. Similar requirements for success pertain to each system; namely, that they should ideally provide long-term regulated gene expression at therapeutic levels, targeted in a cell-specific fashion, in the absence of harmful cytotoxic or immunological side effects. At present, none of these criteria has been satisfied completely. The immunological constraints in particular represent a significant obstacle to success, not least because the regulation of immunity in the brain is currently poorly understood.
Immune responses to adenovirus vectors The immune system plays a crucial role in peripheral organs in limiting the duration of transgene expression from adenoviral vectors9,10. For example, in normal mouse liver, transgene expression declines dramatically over the initial few weeks and is accompanied by inflammation; both of these effects are due to anti-vector immune responses10,11. In sharp contrast, transgene expression occurs for considerably longer in young animals with underdeveloped Copyright © 1996, Elsevier Science Ltd. All rights reserved. 0166 - 2236/96/$15.00
immune systems or in those that are deficient in T lymphocytes or are immunosuppressed9,10,12. Unlike the rapid decline in transgene expression observed in peripheral organs, adenovirus-mediated transduction of adult brain parenchyma leads to reasonably stable gene expression5–7,13. (The same does not necessarily hold true for the transduction of cells lining the ventricular cavities14.) However, despite this optimistic early observation, initial studies noted histological evidence of focal pathology in the brain5,15. It is still commonly believed that the brain is an organ protected from the effects of destructive immune responses. However, it is quite clear that the immune system can and does respond to antigenic stimuli in the brain16–18, albeit less robustly than is usually observed in other organs, and that such antigenic stimuli may include non-replicating viral vectors. Immune responses to HSV-1 vectors have been described19,20; on the other hand, AAV vectors are thought to be relatively non-immunogenic4, but a systematic immune analysis of these vectors in the brain has yet to be undertaken. First-generation vectors based on human adenovirus type-5 contain a deletion of the E1 region (Fig. 1) to render the virus non-replicating. When these vectors are inoculated into the striatum, they stimulate a strong inflammatory reaction13. This response displays biphasic kinetics, comprising an early nonspecific phase and a later T-cell-mediated phase21. During the early phase, T cells and macrophages are recruited, local microglia and astrocytes are activated strongly, and high levels of major histocompatibility complex (MHC) class-I antigens are expressed (Fig. 2A). Negligible inflammation occurs in control animals, indicating that the trauma associated with vector delivery plays little part in the genesis of the observed early inflammation. (MHC class-I proteins PII: S0166-2236(96)10060-6
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Matthew J.A. Wood, Harry M. Charlton, Koji Kajiwara and Andrew P. Byrnes are at the Dept of Human Anatomy, Oxford University, Oxford, UK OX1 3QX. Kathryn J. Wood and Koji Kajiwara are at the Nuffield Dept of Surgery, John Radcliffe Hospital, Oxford, UK OX3 9DU.
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Fig. 1. Simplified transcription map of the genome of human adenovirus type 5. The genome of adenovirus type 5 is a 36 kb linear duplex structure. The early transcription units (E1–E4) and the major late transcription region are shown, and these are expressed in a temporally ordered sequence. E1 and E3 are the most convenient loci at which to insert new genes. In this figure a 2.9 kb deletion in the E1 region enabled the insertion of a transcription unit encoding the lacZ reporter gene. New vectors containing mutations in the E2 region or deletions in the E4 region may provide improved transgene expression compared with vectors deleted only in E1.
are cell-surface heterodimers, normally expressed at very low levels in the brain, whose function is to present intracellular antigens to CD8+ T cells. In contrast, extracellular antigens are presented via MHC class-II proteins to CD4+ T cells. Expression of MHC class-II is restricted to specific cell populations, including dendritic cells and cells of the macrophage–monocyte lineage17 .) The later T-cell-mediated immune response to adenovirus appears to be directed mainly against viral proteins, since vectors that do not express a transgene elicit similar responses13, and others that have been irradiated with UV (to abolish de novo viral gene expression) provoke the early, nonspecific, inflammatory reaction but not the later T-cell-mediated phase21. This immune response has declined greatly 1–2 months after the vector was inoculated into the brain, despite the fact that expression of transgene and viral proteins can still be detected13. The strength of the immune response to adenovirus is to some extent modifiable by genetic factors because it has been shown to be dependent to some degree on the strain of rodent inoculated. Precisely what such genetic factors might be is currently unknown. From the above, it can be concluded that adenovirus vectors can offer long-term transgene expression in the brain despite provoking an immune response.
Immune responses in distant synaptically linked brain sites An important and interesting property of viral vectors is their capacity for rapid retrograde axonal transport in the brain, which suggests that they might be useful for gene delivery to distant synaptically linked brain regions. This behaviour has been particularly well characterized for HSV vectors22 and, more recently, for adenovirus5,23,24, a virus that is not naturally neurotropic. While most cell types in the brain appear to be susceptible to adenovirus infection, it is not yet clear that the same is true of all retrograde pathways. The cellular mechanisms underlying 498
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adenoviral infection have not been fully identified but it is thought to proceed in two phases25. The first step involves the attachment of the viral fibre protein to an unknown cellular receptor. Infection then progresses by the binding of the viral penton protein to cellular integrins, followed by endocytosis26. The expression of these viral molecules is not well characterized in the brain, and so there can be no certainty that adenoviral infection occurs by specific receptor-mediated uptake in all cases. Nevertheless, this type of retrograde gene delivery might prove to be a valuable strategy for cellspecific gene transfer in the brain. Gene delivery to distant synaptically linked sites using HSV vectors has been shown to be associated with inflammation in these linked areas19; similar, although milder, inflammation occurs when adenovirus vectors are used13. Inoculation of an adenoviral vector into the striatum results in transgene expression in neurones of the synaptically linked ipsilateral substantia nigra pars compacta (SNc), but it also induces inflammation at this site (Fig. 2B). This immune response is less strong and is typically delayed compared with that occurring at the primary inoculation site, but it still features locally upregulated MHC class-I expression and microglial activation in the early phase. Only relatively small numbers of T cells are detected in the SNc; precisely what the mechanism might be for the inflammation at this site and for the low level of lymphocyte recruitment is not known. The low concentration of T cells is of particular interest, given that the vector is only present in neurones and that even minor trauma is not associated with vector delivery to these distant sites – both of these facts suggest the possibility of a vector-specific immune response. (This idea is discussed below.) In summary, the retrograde targeting of genes in the brain has great potential as a strategy for cell-specific gene delivery, but at present it is an obligatory and not optional feature of adenovirus vectors and carries with it the risk of initiating unwanted inflammation in distant afferent brain regions.
Quantification of immune responses to non-replicating vectors The development of methods to quantify immunological parameters following the injection of nonreplicating viral vectors into the brain is important for several reasons. Such techniques will enable standard functional immune assays to be applied to questions of viral vector neuroimmunology, and will therefore give further insight into important aspects of immune regulation in the brain. Perhaps more crucially, the advent of such methods will be vital for the accurate evaluation of the immunological risk of new or improved viral vectors. Immune responses to nonreplicating viral vectors are typically much weaker than those encountered in the various types of viral encephalitis, such as HSV encephalitis27 or Borna disease28, making accurate quantification all the more difficult. Sensitive methods for isolating infiltrating leukocytes from the brain after viral vector injection have now been established, based on enzymatic digestion of brain tissue followed by density gradient centrifugation (K. Kajiwara, M.J.A. Wood and K.J. Wood, unpublished observations). Such leukocytes can now be studied by fluorescence-activated cell-sorting (FACS) analysis and in vitro immune assays (such as
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precursor, cytotoxic and proliferation assays). Using these methods in the study of the effects of E1-deleted adenovirus vectors, the numbers of infiltrating leukocytes are found to peak at around 9 days after inoculation and to decline slowly thereafter. Activated T lymphocytes [expressing receptors for interleukin-2 (IL2)] only begin to constitute a significant component of the infiltrate after 6 days, and therefore form part of the second, so-called adaptive, phase of the immune response. Interestingly, CD8+ T cells are dominant only in the early phase; thereafter, CD4+ cells are more common (Fig. 3).
Role of T-cell subsets in the immune response From the quantitative studies referred to above, the predominant T cells present during the adaptive phase of the immune response to adenovirus vectors in the brain are CD4+ T lymphocytes. To examine the functional role of CD4+ and CD8+ cells more closely, monoclonal antibodies were used to deplete each subset in turn21. Significantly, the early inflammatory response was undiminished even when both subsets of T cells were depleted. Given that irradiation of the adenovirus vector with UV also failed to diminish this inflammation, it appears that this early phase requires neither viral gene transcription nor T lymphocytes for its generation. Similar findings have been obtained in the lung29,30. Potential inflammatory stimuli during the early phase include the release of cytokines by virally infected cells, a well-documented phenomenon in the case of peripheral organs30. Similarly, cytokine release in the brain might activate cerebral vascular endothelium and initiate leukocyte chemoattraction. Studies of this early inflammatory phase in the brain indicate that neither vector modification by further limiting viral gene expression nor T-cell immunotherapy will be able to eliminate this inflammation. A better understanding of the mechanisms underlying this inflammatory phase and the future development of anti-inflammatory strategies seem to be necessary to reduce or eliminate this risk. In contrast, the later T-cell-mediated phase of the immune response to adenovirus vectors in the brain (from 6 days onwards) is markedly reduced following CD8+ cell depletion and eliminated completely by CD4+ T-cell depletion21. This implicates the CD4+ subset as functionally dominant in this inflammatory phase. In peripheral organs, both T-cell subsets are important during this phase, as shown by subset depletion and adoptive T-cell transfer studies (that is, transplanting T cells from one animal to another)31–33. However, unique to the brain, and of significance in this discussion, is the observation that vector transgene expression is unaltered in level and duration whether T cells are depleted during this phase or not. This suggests that any decline in gene expression during this phase of the immune response is probably not due to T lymphocytes. It is possible that the early phase of inflammation, which occurs independently of T cells, could contribute in some way to the later decline in transgene expression; however, other possibilities, such as the promoter inactivation observed in some retroviral vectors34 or the gradual loss of episomal adenoviral elements during cell division, should also be considered. It would appear that the mechanism underlying the persistent transgene expression in the brain, as distinct from the
Fig. 2. Immune responses to adenovirus vectors in the brain. (A) Induction of major histocompatibility complex (MHC) class-I expression is observed after adenovirus injection into the caudate nucleus of the striatum of rats, and is maximal 48 h after inoculation. (B) Upregulated MHC class-I expression can also be observed 9–15 days after inoculation in brain regions that are synaptically linked to the caudate, such as the ipsilateral substantia nigra pars compacta (between dashed lines). (C) High levels of persistent MHC class-I expression are seen in and extending beyond the caudate nucleus after peripheral sensitization with adenovirus vector in animals that had previously received vector injected into the caudate. (D) In an adjacent section to that shown in C, histochemical staining for myelin reveals demyelination (arrow) in sensitized animals – in this case, in the corpus callosum. Scale bar, 800 mm in A, 300 mm in B, and 600 mm in C and D. Abbreviation: lv, lateral ventricle.
periphery, is an ineffective T-cell response that fails to clear the vector or transgene product from the brain.
Immune responses to adenovirus vectors in sensitized animals The conclusion drawn at the end of the previous section prompts the obvious question of why the T-cell immune response to a non-replicating adenovirus vector in the brain is ineffective and incapable of clearing the virus from the brain. Where precisely is the defect in this response? Alternatively, under what conditions might the immune system act effectively to clear the vector and eliminate transgene expression from the brain, and what effector mechanisms might be used in so doing? It is known that foreign antigens can survive in the brain for extended periods without inducing a cellmediated immune response35, even though the same TINS Vol. 19, No. 11, 1996
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Fig. 3. Relative abundance of CD4+ and CD8+ T lymphocytes in the brain after adenovirus injection. Leukocytes were isolated from the rat brain at times ranging from 0 to 60 days after adenovirus vector injection into the caudate nucleus. Cells were labelled with antibodies to CD4 and CD8, and the numbers of these cells were determined by fluorescence-activated cell-sorting (FACS) analysis. The relative abundance of the CD4+ and CD8+ T-cell subsets during this period of time is shown, expressed as a proportion of total leukocytes isolated from the brain (CD45+ cells). CD8+ T cells increase rapidly during the early inflammatory phase. After approximately day 9, and correlating with the onset of the T-cell-mediated phase of the immune response, CD4+ T lymphocytes are found in greatest abundance.
antigens stimulate an effective response when injected into the periphery. The defect underlying such an ineffective T-cell response might be located in either the afferent or efferent limbs of the immune system, both of which are less robust in the brain compared with the periphery. Inadequate immune activation (afferent limb defect) can occur when foreign transplantation antigens placed in the brain survive by failing to stimulate the immune system36. However, a subsequent transplant (syngeneic to the original) placed in the periphery can potentiate a response to the graft placed earlier in the brain37,38, in some cases
Fig. 4. Inflammation in regions afferent to the caudate nucleus (C). Parasagittal representation of rat-brain regions synaptically linked to the caudate nucleus of the striatum in which severe inflammation is observed following peripheral sensitization to adenovirus in animals that have previously received vector injected into the caudate (arrow). These areas include frontal cortex (FC), globus pallidus (GP), thalamus (T), ventral tegmental area (VTA), substantia nigra (SN), retrorubral field of the mesencephalic reticular nucleus (MRN) and the locus coeruleus (LC). Abbreviation: lv, lateral ventricle.
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leading to graft rejection in the brain36. Thus, it is not that the T cells are incapable of destroying the foreign tissue but that the foreign transplantation antigens placed in the brain do not on their own elicit a sufficiently strong T-cell response. A T-cell response strong enough to eliminate adenoviral vectors in the brain can be stimulated by subsequent peripheral subcutaneous exposure to the vector13,39. In this case, vector transgene expression in the brain is found to be curtailed rapidly and is accompanied by severe inflammation, suggesting strongly that an effective T-cell response has been stimulated. The notable features of this process are severe mononuclear and T-cell inflammation, associated with dendritic cell recruitment and strong microglial activation at the inflammatory focus. More importantly, this intense inflammation is also accompanied by evidence of local demyelination (Fig. 2D). Such demyelination is not seen in control animals, indicating that the minor trauma involved in vector delivery plays no part in its generation; neither is demyelination found in synaptically linked distant sites. The observed demyelination resembles the neuropathology seen after the immune system has been peripherally sensitized to mycobacterial antigens that were previously inoculated into the brain35. This severe inflammation occurs not only at the original site of vector delivery but also in areas containing synaptically linked neuronal populations39. For example, following primary inoculation into the striatum, secondary foci of severe inflammation include frontal cortex, ipsilateral SNc and ventral tegmental area, globus pallidus, thalamus, locus coeruleus and the retrorubral field of the mesencephalic reticular nucleus (Fig. 4). The intense inflammation in these areas, similar in nature to that found in the striatum and including numerous T cells, implies a vector-specific immune response. Although it is known that activated T cells enter the brain, they fail to accumulate significantly in the absence of antigen recognition40. Any mechanism proposed to account for the interesting hypothesis of a vectorspecific immune response would be speculative at present since it is debatable whether neurones are able to present antigens via MHC class-I to T cells in vivo. Although there is some evidence defining the circumstances under which neurones might present antigens in vitro, it is possible that they lack some crucial proteins necessary for effective antigen presentation in the living animal41,42. Mechanisms for this inflammation other than direct antigen presentation by neurones should also be considered, and this adenovirus model system offers the opportunity to distinguish between these possibilities in future experiments. What mechanisms might underlie the rapid elimination of transgene expression and the demyelinating pathology seen in the sensitized animals? The elimination of reporter gene expression in the periphery is accompanied by vector elimination10. MHC class Irestricted CD8+ cytotoxic T cells are required for this elimination since persistent expression is seen in mice deficient in b2-microglobulin or perforin33,43 (deficiencies of which disable antigen presentation via the class-I route). Such direct cytotoxicity is a possible explanation for the decline in transgene expression in the brain but whether it occurs at all, and if it does so whether it occurs with or without the elimination of
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the transduced cells, has yet to be established. Other indirect non-cytotoxic mechanisms (for example, cytokine-mediated mechanisms like those found in lymphocytic choriomeningitis virus infection44) also require investigation. Similarly, the mechanism for the demyelination could be either direct cytotoxic immune damage to virus-infected oligodendrocytes or indirect damage through other routes.
Concluding remarks Since the T-cell response to adenovirus in the brain is generally ineffective, long-term transgene expression is possible with currently available vectors. Furthermore, adenovirus vectors can deliver genes to synaptically linked brain areas, which is a valuable potential strategy for gene targeting in the complex environment of the adult brain. However, against these putative advantages must be set the definite disadvantage that long-lasting transgene expression in the brain is seriously at risk if the immune response is reactivated. Therefore, adenovirus vectors are currently unsafe for humans, in whom prior or subsequent infection with adenovirus would lead to inflammation, transgene elimination from the brain and local damage. Such severe inflammation would be located not only at the site of vector delivery but also in synaptically linked brain regions. Future generations of adenovirus vectors from which more viral genes have been deleted are likely to be of lower immunogenicity. This optimism is supported by the observation that UV-irradiated adenovirus inoculated into the brain fails to stimulate severe inflammation after peripheral sensitization with an E1-deleted vector39. Vectors incorporating mutations in the E2 region45 or deletions in the E4 region46 could offer improvements in transgene expression, although this has yet to be shown unequivocally. In addition, the development of strategies for immunotherapy might lead to the effective control of the early inflammatory and later T-cell-mediated phases of the immune response that are directed against the vector and also the transgene. As has been alluded to above, immunosuppression can enhance transgene expression in the periphery9. The construction of adenovirus vectors incorporating genes encoding cytokines47, particularly those with immunosuppressive potential (such as IL4, IL10 or transforming growth factor-b), is an interesting and potentially elegant strategy for immunotherapy in many organs, including the brain. Not only would the testing of such a strategy advance our understanding of immune regulation in the brain, but it might also speed up the development of safer vectors that are necessary for gene therapy in the nervous system to proceed. In addition, such immunotherapy might have a direct application in
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Acknowledgements We thank P. Belk for his help in the preparation of the manuscript and M. McMenamin for her assistance with some of the figures. This work is supported by grants from the MRC (UK), BBSRC (UK), the Wellcome Trust and by the award of a Wellcome Prize Studentship to APB.
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