Covert Warfare Against the Immune System: Decoy Capsids, Stealth Genomes, and Suppressors
Authors: Brad E Hoffman, Roland W Herzog
Journal: Molecular Therapy (2013)

In part owing to their low immune profile, adeno-associated virus (AAV) vectors have long been viewed as ideal for in vivo gene transfer. They are based on a small, nonpathogenic virus that is composed of a single-stranded DNA genome and a protein capsid. As compared with many other viral vectors, recombinant AAVs are better at avoiding activation signals, are less efficient in transducing professional antigen-presenting cells, and elicit weaker and more transient innate inflammatory responses.1Mays LE Wilson JM The complex and evolving story of T cell activation to AAV vector-encoded transgene products.Mol Ther. 2011; 19: 16-27Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Ultimately, these naturally stealthy vectors have been able to express various transgenes for months if not years in numerous immune-competent animal models. Many serotypes with differing tropism that are capable of transducing various cell types in vivo have been identified. Induction of immune tolerance to transgene products has been demonstrated for hepatic gene transfer, which induces regulatory T cells (Tregs).2Mingozzi F High KA Immune responses to AAV vectors: overcoming barriers to successful gene therapy.Blood. 2013; 122: 23-36Crossref PubMed Scopus (534) Google Scholar,3Nietupski JB Hurlbut GD Ziegler RJ Chu Q Hodges BL Ashe KM et al.Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression.Mol Ther. 2011; 19: 1999-2011Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Despite all this, immune responses are currently viewed as the main obstacle for many AAV-based gene therapies in humans.2Mingozzi F High KA Immune responses to AAV vectors: overcoming barriers to successful gene therapy.Blood. 2013; 122: 23-36Crossref PubMed Scopus (534) Google Scholar One immune complication revealed in clinical trials is the activation of CD8+ T-cell responses against AAV capsid. These capsid-specific cytotoxic T lymphocytes (CTLs) are probably derived from prior natural infection of patients and are phenotypically different from those that may be found in animal models.2Mingozzi F High KA Immune responses to AAV vectors: overcoming barriers to successful gene therapy.Blood. 2013; 122: 23-36Crossref PubMed Scopus (534) Google Scholar,4Li H Lasaro MO Jia B Lin SW Haut LH High KA et al.Capsid-specific T-cell responses to natural infections with adeno-associated viruses in humans differ from those of nonhuman primates.Mol Ther. 2011; 19: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,5Herzog RW Davidoff AM Markusic DM Nathwani AC AAV vector biology in primates: finding the missing link?.Mol Ther. 2011; 19: 1923-1924Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Following cellular entry, a portion of the input capsid antigen is routed for proteasomal degradation. The resulting major histocompatibility complex class I (MHC I) presentation may create a target for capsid-specific CTLs, which therefore can limit or even entirely eliminate therapeutic gene expression upon AAV gene transfer. More recently, an effective countermeasure against this response has emerged in the form of transient immune suppression with steroid drugs.6Nathwani AC Tuddenham EG Rangarajan S Rosales C McIntosh J Linch DC et al.Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.N Engl J Med. 2011; 365: 2357-2365Crossref PubMed Scopus (1352) Google Scholar However, rather than borrowing from the playbook of immunologists working in the transplant or autoimmune arena, gene therapists are poised to develop vector-specific approaches to tackle this problem. For example, the capsid sequence can be altered to limit proteasomal processing, thereby reducing MHC I presentation and limiting the ability of CD8+ T cells to kill their targets.7Martino AT Basner-Tschakarjan E Markusic DM Finn JD Hinderer C Zhou S et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (117) Google Scholar Additional clues that could lead to a solution may come from innate immune recognition of AAV particles, which provide the activation signals for subsequent antigen-specific responses. Only recently has it become obvious that the very limited innate response to AAV does indeed have consequences for adaptive immune responses to vector and transgene. A key observation has been the discovery by Yang and colleagues that Toll-like receptors (TLRs) recognize molecular patterns of the vector.8Zhu J Huang X Yang Y The TLR9–MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice.J Clin Invest. 2009; 119: 2388-2398Crossref PubMed Scopus (207) Google Scholar Specifically, the endosomal DNA receptor TLR9 senses vector genomes as AAV particles traffic through endosomes following cellular entry. The resulting signaling through the cytosolic adaptor MyD88 triggers inflammatory and type I interferon responses, which ultimately modulate adaptive responses.8Zhu J Huang X Yang Y The TLR9–MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice.J Clin Invest. 2009; 119: 2388-2398Crossref PubMed Scopus (207) Google Scholar,9Martino AT Suzuki M Markusic DM Zolotukhin I Ryals RC Moghimi B et al.The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver.Blood. 2011; 117: 6459-6468Crossref PubMed Scopus (145) Google Scholar Faust et al. interrogated the mechanisms by which one of the newer AAV serotypes (rh33.32, isolated from nonhuman primates) activates immune responses.10Faust SM Bell P Cutler BJ Ashley SN Zuh Y Rabinowitz JE et al.CpG-depleted adeno-associated virus vectors evade immune detection.J Clin Invest. 2013; 123: 2994-3001Crossref PubMed Scopus (145) Google Scholar In contrast to most other characterized AAVs, rh33.32 effectively induces CD8+ T-cell responses to transgenes following intramuscular administration and may thus be a good vaccine carrier. However, TLR9-deficient mice failed to mount this T-cell response. While CD8+ T cells destroyed transduced muscle fibers of wild-type mice, mice lacking TLR9 showed sustained transgene expression without infiltrates. TLR9, being a pathogen sensor, most strongly responds to unmethylated DNA such as that of bacterial or viral genomes. Therefore, the authors hypothesized that a vector genome devoid of CpG motifs would be mostly invisible to TLR9. Sure enough, a vector containing such a stealthy genome packaged into the identical capsid failed to elicit CD8+ T-cell responses to a reporter gene, thus directing long-term expression even in wild-type mice with intact TLR9. The same concept applies to less immunogenic serotypes, where already weak responses can be further reduced. Future studies are likely to adapt this concept to therapeutic gene constructs. Interestingly, use of the "stealth genome" also substantially reduced CD8+ T-cell responses against capsid antigens, suggesting a more general requirement for sensing of the viral genome in CTL activation.10Faust SM Bell P Cutler BJ Ashley SN Zuh Y Rabinowitz JE et al.CpG-depleted adeno-associated virus vectors evade immune detection.J Clin Invest. 2013; 123: 2994-3001Crossref PubMed Scopus (145) Google Scholar This is in agreement with other work, showing blockage of capsid-specific T-cell responses by co-administration of a vector with a conventional genome and an oligonucleotide that inhibits TLR9 (ref. 9Martino AT Suzuki M Markusic DM Zolotukhin I Ryals RC Moghimi B et al.The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver.Blood. 2011; 117: 6459-6468Crossref PubMed Scopus (145) Google Scholar). Blockers of TLR9 or other innate immune sensors may represent an alternative strategy, if complete elimination of CpG motifs from the expression cassette is not possible. As reported in this issue, Hui et al. took a very different approach to the problem of capsid-specific T-cell responses.11Hui DJ Basner-Tschakarjan E Chen Y Davidson RJ Buchlis G Yazicioglu M et al.Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.Mol Ther. 2013; 21: 1727-1737Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar They sought to suppress T cells by tapping into naturally occurring immune regulatory pathways rather than by using pharmacological intervention with immune-suppressive drugs that may have various side effects and increase the risk of opportunistic infections. As proof of concept in a human cell system, immunoglobulin-derived peptide epitopes were employed to stimulate naturally occurring CD4+CD25+FoxP3+ Tregs via MHC class II presentation. As a result, these suppressor cells proliferated in culture and effectively prevented CTL responses against human leukocyte antigen–matched target cells displaying AAV capsid antigen via MHC I. In this system, peripheral blood mononuclear cells had been simultaneously stimulated with capsid-specific CD8+ T-cell epitopes and the suppressive "tregitopes." Expanded Tregs induced anergy in effector CD8+ T cells in a cell contact–dependent manner but were unable to suppress CTLs against a different viral antigen. Simultaneous exposure of the peripheral blood mononuclear cells to the specific viral and Treg epitopes was required for suppression, assuring a level of antigen specificity. This work may lead to novel reagents for suppression of T-cell responses against viral vectors, such as capsid–Tregitope fusion proteins. Another problem posed by the immune system is the formation of neutralizing antibodies (NABs) against capsid that prevents readministration of vector, a process that is less dependent on TLR9 activation.10Faust SM Bell P Cutler BJ Ashley SN Zuh Y Rabinowitz JE et al.CpG-depleted adeno-associated virus vectors evade immune detection.J Clin Invest. 2013; 123: 2994-3001Crossref PubMed Scopus (145) Google Scholar,12Sudres M Ciré S Vasseur V Brault L Da Rocha S Boisgérault F et al.MyD88 signaling in B cells regulates the production of Th1-dependent antibodies to AAV.Mol Ther. 2012; 20: 1571-1581Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Unfortunately, many humans develop NABs to various AAV serotypes progressively with age through natural infection. Such preexisting NABs can block hepatic gene transfer even at fairly low titers.2Mingozzi F High KA Immune responses to AAV vectors: overcoming barriers to successful gene therapy.Blood. 2013; 122: 23-36Crossref PubMed Scopus (534) Google Scholar Options around this problem include physical removal of the antibodies by plasmapheresis or adjustment of the injection technique.13Mimuro J Mizukami H Hishikawa S Ikemoto T Ishiwata A Sakata A et al.Minimizing the inhibitory effect of neutralizing antibody for efficient gene expression in the liver with adeno-associated virus 8 vectors.Mol Ther. 2013; 21: 318-323Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,14Monteilhet V Saheb S Boutin S Leborgne C Veron P Montus MF et al.A 10-patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8.Mol Ther. 2011; 19: 2084-2091Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar Neither approach is without drawbacks and limitations. Mingozzi et al. have now developed a simpler and more widely applicable method, taking advantage of a byproduct of AAV vector production: "empty" capsids.15Mingozzi F Anguela XM Pavani G Chen Y Davidson RJ Hui DJ et al.Overcoming pre-existing humoral immunity to AAV using capsid decoy.Sci Transl Med. 2013; 5: 194ra92Crossref PubMed Scopus (222) Google Scholar These nontherapeutic vector particles, lacking the genome, can be removed from the DNA-containing particles by centrifugation as they differ in density. Interestingly, when mixed with DNA-containing particles, empty capsids increase transduction efficiency in the presence of NABs. Apparently they can function as decoys, binding up the antibodies, thereby allowing "full" particles to transfer the therapeutic gene to hepatocytes. Moreover, optimal transduction efficiency can easily be obtained for a specific NAB titer by adjusting the ratio of empty to full particles. In other words, depending on the level of preexisting immunity in a particular patient, an optimal mixture can be created and injected intravenously. Remarkably, up to 50% of control transgene expression was obtained even in the presence of NAB titers of >1:3,000. A potential concern with the approach is the possibility of increasing anti-capsid T-cell responses because of the additional antigen load that is being introduced. To counter this concern, empty mutant capsids were created that still absorb the antibodies but lack receptor binding and therefore do not enter hepatocytes. Hence, these capsid antigens will not be presented by hepatocytes. In addition, lack of TLR9 stimulation by empty capsids should minimize CD8+ T-cell activation as discussed above.10Faust SM Bell P Cutler BJ Ashley SN Zuh Y Rabinowitz JE et al.CpG-depleted adeno-associated virus vectors evade immune detection.J Clin Invest. 2013; 123: 2994-3001Crossref PubMed Scopus (145) Google Scholar In conclusion, careful mechanistic studies combined with innovative designs and unconventional and creative approaches are providing a new spectrum of countermeasures against immune responses directed at AAV gene transfer (Figure 1). These methods are specifically designed for gene therapy protocols rather than merely borrowed from other applications. An ideal vector preparation of the future may contain decoy capsids to make it through the antibody blockade while being cloaked and less visible to the immune system, using stealth genomes packaged into a modified capsid to minimize MHC I presentation and CD8+ T-cell activation, and combined with blockers of innate immune sensors or stimulators of Tregs to blind and suppress the immune system. Much of that, however, will depend on how similar innate immune regulation is between humans and the animal models.