Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects
Authors: Pedro R Lowenstein
Journal: Molecular Therapy (2004)

Administration of viral vectors induces innate inflammation and primes antivector adaptive immune responses, some of which are detrimental to viral vector-mediated gene transfer (reviewed in [1Lowenstein P.R. Immune responses to viral vectors.Gene Ther. 2003; 10: 933-998Crossref PubMed Scopus (21) Google Scholar]). Immune responses can also be generated against the protein encoded by the transgene, or against transcriptional regulatory elements if they are immunogenic. Innate inflammation occurs immediately upon vector injection, while adaptive cellular and humoral immune responses take up to two weeks to reach their peak. If vectors are adinistered to previously immunized subjects however, adaptive immune responses can be activated immediately, in conjunction with innate inflammation. Expression of transgenes encoded by adenovirus [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar, 3Maione D. et al.An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.Proc. Natl. Acad. Sci. 2001; 98: 5986-5991Crossref PubMed Scopus (79) Google Scholar], AAV [4Sanftner L.M. et al.Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV.Mol. Ther. 2004; 9: 403-409Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Peden C. Burger C. Muzyczka N. Mandel R.J. The effects of preimmunization to wtAAV on single and multiple administrations of rAAV in the brain.Mol. Ther. 2003; 7: S312Google Scholar, 6Peden C. Burger C. Muzyczka N. Mandel R.J. Circulating anti-wt- AAV2 antibodies inhibit rAAV2-, but not rAAV5-mediated gene transfer in the brain.J. Virol. 2004; (In Press.)Google Scholar], or lentivirus-derived vectors (unpublished data) is reduced in animals previously immunized against either a component of the particular vector or the transgene. Innate immune responses are triggered by recognition of pathogen-associated molecular patterns by cellular receptors (e.g. the Toll receptors) [7Medzhitov R. Janeway Jr., C.A. Innate immunity: the virtues of a non clonal system of recognition.Cell. 1997; 91: 295-298Abstract Full Text Full Text PDF PubMed Scopus (1979) Google Scholar] or the binding of complement proteins and antibodies to virions. Binding of virions to receptors on the plasma membrane of target cells stimulates intracellular signalling cascades that in turn influence both viral entry and how cells respond to viral infection [8Smith A.E. Helenius A. How viruses enter animal cells.Science. 2004; 304: 237-242Crossref PubMed Scopus (623) Google Scholar, 9Greber U.F. Signalling in viral entry.Cell Mol. Life Sci. 2002; 59: 608-626Crossref PubMed Scopus (155) Google Scholar]. Whereas antibodies recognize large three-dimensional antigenic structures, the T-cell receptor on CD8+ T cells recognizes short peptide epitopes derived from viral proteins presented in the context of MHC class I proteins (MHC-I) on the surface of antigen-presenting cells. These viral antigens are processed in the cytoplasm by the proteasome, transported into the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP), and loaded onto MHC-I that are then delivered to the plasma membrane. For the most part, these antigens are derived from newly synthesized viral proteins in infected cells. Therefore, viral vectors whose genomes do not encode any viral proteins would not be expected to present antigen to previously activated CD8+ T cells. However, what is the fate of the proteins that enter cells as physical components of the input virion—such as the protein shell, membrane proteins, and other proteins comprising the viral vector? Little attention has been paid to the possibility that viral input proteins are sufficient to either induce an immune response, or provide a source of antigenic epitopes to previously activated T cells [10Kafri T. et al.Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy.Proc. Natl. Acad. Sci. 1998; 95: 11377-11382Crossref PubMed Scopus (224) Google Scholar]. However, a series of recent papers, along with data presented at a recent Gordon Conference (Santa Barbara, February 2004), together strongly suggest that the turnover of virion input proteins may determine whether infected cells will be targeted by antivector T cells—and if so, for how long [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar, 5Peden C. Burger C. Muzyczka N. Mandel R.J. The effects of preimmunization to wtAAV on single and multiple administrations of rAAV in the brain.Mol. Ther. 2003; 7: S312Google Scholar, 6Peden C. Burger C. Muzyczka N. Mandel R.J. Circulating anti-wt- AAV2 antibodies inhibit rAAV2-, but not rAAV5-mediated gene transfer in the brain.J. Virol. 2004; (In Press.)Google Scholar, 11Thomas C.E. Storm T.A. Huang Z. Kay M.A. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno- associated virus vectors.J. Virol. 2004; 78: 3110-3122Crossref PubMed Scopus (301) Google Scholar, 12Sabatino D. et al.An animal model of transient transaminitis and diminished factor IX expression following AAV-mediated liver-directed gene transfer for hemophilia B.Blood. 2003; 102: 493aGoogle Scholar]. If a subject has been previously exposed to the parent virus or vector, then the immune system has already been ‘armed’ to recognize specific antigenic epitopes. In this case, injected vector virions will be targeted rapidly by the immune system as soon as they enter the body (by circulating antibodies), or they will be metabolized and presented on MHC molecules on transduced cells. Data from several laboratories using adenoviral or AAV vectors in different target tissues indicate that the turnover of input virion proteins correlates with, and may causally determine, the persistence of transgene expression in preimmunized subjects (Fig. 1). Injection of first generation adenoviral vectors into the brain of previously immunized rats is characterized by a reduction in transgene expression of over 90% within two weeks [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar]. However, injection of high-capacity adenoviral vectors (HC-Ad) into similar animals leads to persistent transgene expression for at least two months (Fig. 1a; [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar]; reviewed in [13Lowenstein P.R. Castro M.G. Inflammation and adaptive immune responses to adenoviral vectors injected into the brain: peculiarities, mechanisms, and consequences.Gene Ther. 2003; 10: 946-954Crossref PubMed Scopus (72) Google Scholar]). The main difference between these vectors is that first generation Ad continues to express viral proteins, whereas HC-Ad does not. However, infection with HC-Ad will lead to immune presentation of viral proteins, but these are exclusively derived from input virions [14Molinier-Frenkel V. et al.Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes.J. Virol. 2000; 74: 7678-7682Crossref PubMed Scopus (105) Google Scholar]. Once this limited source of antigenic epitopes has been metabolized and turned over, the HC-Ad genomes become invisible to the immune system. Therefore, immediately upon injection of HC-Ad into preimmunized animals, virion-derived proteins provide antigenic epitopes to activated T cells. However, this is a self-limiting process, since the capacity of activated T cells to encounter infected cells and reduce transgene expression is transient. Surprisingly, while injection of HC-Ad (derived from Ad5) results in long-term expression in subjects preimmunized against adenovirus [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar, 3Maione D. et al.An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.Proc. Natl. Acad. Sci. 2001; 98: 5986-5991Crossref PubMed Scopus (79) Google Scholar], gene expression from AAV2 vectors is greatly reduced in animals preimmunized against this vector [4Sanftner L.M. et al.Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV.Mol. Ther. 2004; 9: 403-409Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Peden C. Burger C. Muzyczka N. Mandel R.J. The effects of preimmunization to wtAAV on single and multiple administrations of rAAV in the brain.Mol. Ther. 2003; 7: S312Google Scholar, 6Peden C. Burger C. Muzyczka N. Mandel R.J. Circulating anti-wt- AAV2 antibodies inhibit rAAV2-, but not rAAV5-mediated gene transfer in the brain.J. Virol. 2004; (In Press.)Google Scholar, 15Halbert C.L. et al.Repeat transduction in the mouse lung by using adeno- associated virus vectors with different serotypes.J. Virol. 2000; 74: 1524-1532Crossref PubMed Scopus (225) Google Scholar]. In comparison, even first generation adenoviral vectors [16Bramson J.L. Hitt M. Gauldie J. Graham F.L. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination.Gene Ther. 1997; 4: 1069-1076Crossref PubMed Scopus (110) Google Scholar], and herpes simplex virus (HSV) vectors [17Brockman M.A. Knipe D.M. Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity.J. Virol. 2002; 76: 367887Crossref Scopus (59) Google Scholar], can achieve efficient gene transfer and expression in preimmunized subjects. The nearly complete inhibition of AAV2 expression in preimmunized animals is somewhat unexpected, because in the case of adenovirus, HSV-derived vectors, [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar, 3Maione D. et al.An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.Proc. Natl. Acad. Sci. 2001; 98: 5986-5991Crossref PubMed Scopus (79) Google Scholar, 16Bramson J.L. Hitt M. Gauldie J. Graham F.L. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination.Gene Ther. 1997; 4: 1069-1076Crossref PubMed Scopus (110) Google Scholar, 17Brockman M.A. Knipe D.M. Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity.J. Virol. 2002; 76: 367887Crossref Scopus (59) Google Scholar], and lentiviral vectors (unpublished observations), preimmunization is unable to eliminate vector entry and transgene expression. Neutralizing antibodies are thought to be the main factor inhibiting vector entry into a target cell. However, as indicated above, the efficiency of neutralization of infection by antibodies in vivo is not 100% [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar, 3Maione D. et al.An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.Proc. Natl. Acad. Sci. 2001; 98: 5986-5991Crossref PubMed Scopus (79) Google Scholar, 16Bramson J.L. Hitt M. Gauldie J. Graham F.L. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination.Gene Ther. 1997; 4: 1069-1076Crossref PubMed Scopus (110) Google Scholar, 17Brockman M.A. Knipe D.M. Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity.J. Virol. 2002; 76: 367887Crossref Scopus (59) Google Scholar]. Thus, other factors, such as cellular immune responses, could contribute to the inhibition of transduction and transgene expression mediated by AAV2 in preimmunized animals. In addition to the effect of neutralizing antibodies, the presence of activated T cells specific for virion-derived, antigenic epitopes presented on MHC molecules in transduced cells, could contribute to the elimination of transgene expression. Antigen presentation on transduced cells signals the cells’ presence to T cells, which can subsequently interfere with transgene expression. Virion-derived proteins would appear to be the only potential source of antigenic epitopes, since—as is the case with HC-Ad—the genomes of AAV vectors do not encode any viral proteins. Lack of transgene expression from AAV2 in AAV2 immunized animals suggests that virion input proteins are metabolized very slowly, remaining within infected cells for extended periods of time. This has now been addressed experimentally, at least for AAV-infected hepatocytes in vivo. Thomas et al. [11Thomas C.E. Storm T.A. Huang Z. Kay M.A. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno- associated virus vectors.J. Virol. 2004; 78: 3110-3122Crossref PubMed Scopus (301) Google Scholar], have recently shown that AAV2 uncoats very slowly, and that virions are still present eight weeks following infection. This finding predicts that antigenic epitopes will be expressed on the surface of infected cells for extended periods of time, thus exposing AAV2- infected cells to continued recognition and targeting by activated immune cells. Importantly, the same authors also showed that AAV6 and AAV8 uncoat more rapidly than AAV2. At forty-two days post-infection, the percentage of DNAse-resistant genomes present in the nucleus was reduced from 37% to 3% (Fig. 1d and 1e) if the AAV2 genome was packaged within an AAV8 virion. AAV6 was also uncoated more rapidly than AAV2, and it is likely that the kinetics of uncoating is serotype specific. The slow kinetics of uncoating by AAV2 also correlates with a protracted increase in transgene expression by this vector, as can be seen in transduced brain in Fig. 1c, where maximal expression is not reached until four weeks following vector injection. In turn, these results predict that, in the presence of anti- AAV immune responses, AAV vector genomes encoded within AAV8 or AAV6 capsids should be more stable, and should allow more sustained gene expression, compared to genomes encoded within slowly-uncoating capsids, such as AAV2. Thus, the kinetics of virion uncoating determines the strength of the immune response to viral vectors, and thus influences the clinical utility of different types and serotypes of such vectors for clinical gene therapy. The kinetics of uncoating could be used to predict how long antigenic virion epitopes will be expressed on the surface of infected cells. Faster kinetics would be associated with a shorter period of antigen presentation and therefore greater transduction stability in preimmunized subjects. Interestingly, the new data on the kinetics of AAV uncoating can be now used to offer alternative interpretations for the results published in 2000 by Halbert et al. [15Halbert C.L. et al.Repeat transduction in the mouse lung by using adeno- associated virus vectors with different serotypes.J. Virol. 2000; 74: 1524-1532Crossref PubMed Scopus (225) Google Scholar]. These authors demonstrated that injection of (slowly uncoating) AAV2 into animals preimmunized against AAV2 did not lead to detectable transgene expression thirty days later. However, significant transgene expression was observed when (rapidly uncoating) AAV6 vectors were injected into animals preimmunized against AAV6. Thus, results obtained from experiments using AAV2, AAV6 or HC-Ad vectors in animals preimmunized with the corresponding vector are compatible with a model whereby the kinetics of the turnover of virion capsid proteins is a key determinant of the persistence of transgene expression (Fig. 2). Although the turnover kinetics of endocytosed adenoviral virions is not known, extrapolations from published data indicate that it is at least 18.5 h for Ad2 [18Greber U.F. Webster P Weber J. Helenius A. The role of the adenovirus protease on virus entry into cells.Embo J. 1996; 15: 1766-1777Crossref PubMed Scopus (258) Google Scholar]. In addition, High and colleagues presented evidence at the recent Gordon Conference on Viral Vectors that demonstrated that adoptive transfer of T lymphocytes from animals immunized against AAV2 eliminates transgene expression from the liver of animals previously transduced with AAV2. Importantly, they showed that adoptive transfer eliminated transgene expression if performed at one week, but not four weeks, following delivery of AAV2 to the liver. These results are not only compatible with those of Thomas et al. [11Thomas C.E. Storm T.A. Huang Z. Kay M.A. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno- associated virus vectors.J. Virol. 2004; 78: 3110-3122Crossref PubMed Scopus (301) Google Scholar], Peden et al. [5Peden C. Burger C. Muzyczka N. Mandel R.J. The effects of preimmunization to wtAAV on single and multiple administrations of rAAV in the brain.Mol. Ther. 2003; 7: S312Google Scholar, 6Peden C. Burger C. Muzyczka N. Mandel R.J. Circulating anti-wt- AAV2 antibodies inhibit rAAV2-, but not rAAV5-mediated gene transfer in the brain.J. Virol. 2004; (In Press.)Google Scholar], Halbert et al. [15Halbert C.L. et al.Repeat transduction in the mouse lung by using adeno- associated virus vectors with different serotypes.J. Virol. 2000; 74: 1524-1532Crossref PubMed Scopus (225) Google Scholar], and Sanfter et al. [4Sanftner L.M. et al.Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV.Mol. Ther. 2004; 9: 403-409Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar], but furthermore provide evidence that T cells play a major role in mediating the inhibition of transgene expression, as they are thought to do in Ad-infected cells [2Thomas C.E. et al.Preexisting antiadenoviral immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-capacity adenovirus vectors.Hum. Gene Ther. 2001; 12: 839-846Crossref PubMed Scopus (92) Google Scholar, 13Lowenstein P.R. Castro M.G. Inflammation and adaptive immune responses to adenoviral vectors injected into the brain: peculiarities, mechanisms, and consequences.Gene Ther. 2003; 10: 946-954Crossref PubMed Scopus (72) Google Scholar, 19Thomas C.E. et al.Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases.Proc. Natl. Acad. Sci. 2000; 97: 7482-7487Crossref PubMed Scopus (233) Google Scholar, 20Yang Y. Ertl H.C. Wilson J.M. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses.Immunity. 1994; 1: 433-442Abstract Full Text PDF PubMed Scopus (580) Google Scholar, 21Wadsworth S.C. Zhou H. Smith A.E. Kaplan J.M. Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo.J. Virol. 1997; 71: 5189-5196PubMed Google Scholar]. Another prediction follows from our knowledge of the role of various proteins in antigen presentation. Ubiquitinating enzymes target proteins to be metabolized by proteosomes. In turn, proteosomes degrade larger proteins into smaller peptides, which are then transported into the ER by TAP. Once in the ER, antigenic epitopes are loaded onto MHC-I proteins (reviewed in [22Yewdell J.W. Hill A.B. Viral interference with antigen presentation.Nat. Immunol. 2002; 3: 1019-1025Crossref PubMed Scopus (202) Google Scholar]). Thus, if antigen presentation of capsid proteins regulates the kinetics of the elimination of transgene expression by T cells, then inhibition of protein ubiquination, proteosome activity, or TAP would be predicted to potentiate the longevity of transgene expression in preimmunized animals. Although these experiments have not yet been performed, it has been demonstrated that inhibitors of ubiquitination and proteasome activity increase AAV-mediated transduction of airway epithelia, both in vitro and in vivo [23Duan D. et al.Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus.J. Clin. Invest. 2000; 105: 1573-1587Crossref PubMed Scopus (312) Google Scholar, 24Yan Z. et al.Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors.J. Virol. 2002; 76: 2043-2053Crossref PubMed Scopus (187) Google Scholar, 25Yan Z. et al.Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.J. Virol. 2004; 78: 2863-2874Crossref PubMed Scopus (101) Google Scholar]. Administration of viral vectors leads to the production of both humoral and cellular immune responses, both of which are likely to influence vector transduction and transgene expression. Clearly, we need to examine the separate and individual roles of antibodies and T cells in regulating transduction and transgene expression. Importantly, the kinetics of viral vector uncoating and virion protein turnover, as well as its species, MOI and cell-type specificity, remain to be determined for most gene therapy vectors. Although hitherto overlooked, it may represent a vital factor influencing the ultimate clinical effectiveness of gene therapy vectors. Kinetics of virion uncoating and virion protein turnover may determine the effectiveness of viral vectors in the presence of immune responses against components of the viral capsid or other sources of potentially antigenic peptides encoded by viral vectors. The author thanks Maria Castro, John Engelhardt, John Forsayeth, Urs Greber, Kathy High, Mark Kay, Ron Mandel, and Dusty Miller for their generosity in sharing unpublished data, experimental results prior to publication, and many lively discussions. Nelson Jovel is acknowledged for his technical expertise in preparing figure 2 for publication. Also, Joe Glorioso and Mark Kay are acknowledged as organizers of the Gordon Conference on Viral Vectors (Santa Barbara, CA, February 8-13, 2004), which provided an open intellectual space for discussing new ideas about the larger gene therapy field. Work in the author's laboratory is funded by the National Institutes of Health and the Board of Governors of Cedars-Sinai Medical Center. PRL is Bram and Elaine Goldsmith Chair in Gene Therapeutics.