CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.
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ABSTRACT:
SUBMITTER: Wu TL
PROVIDER: S-EPMC3978806 | biostudies-other | 2014 Jan
REPOSITORIES: biostudies-other
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Project description:Using adoptive transfer models we determined that an adeno-associated viral vector of serotype 2 (AAV2) induces in mice proliferation of CD8(+) T cells that recognize an epitope within the viral capsid. Proliferation to an endogenous epitope within viral protein (VP)3 could be observed for at least 3 weeks while a foreign epitope placed at multiple copies within VP2 elicited CD8(+) T cell expansion for at least 10 weeks. These data show that capsid antigens of AAV2 degrade slowly over a period of weeks and during this period provide targets to CD8(+) T cells.
| S-EPMC3048183 | biostudies-other
Project description:Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.
| S-EPMC6972890 | biostudies-literature
Project description:Adeno associated vectors (AAV) have shown considerable promise to treat various genetic disorders in both preclinical and clinical settings mainly because of its safety profile. However, efficient use of AAV to deliver genes in immune-competent sites like muscles and liver requires very high doses which are associated with concomitant cellular immune response against the viral capsids leading to destruction of the transduced cells. Coupled with that, there are enough evidences that at high doses, AAV particles are subjected to increased cellular phosphorylation/uniquitination leading to proteasome mediated degradation and loss of the viral particles. The presence of preexisting immunity against AAV further adds on to the problem which is acting as a major roadblock to efficiently use it as a gene therapy vector in the clinics. To overcome this, rational bioengineering of AAV capsid becomes a prime tool by which specific amino acid residue(s) can be suitably modified/replaced by compatible residue(s) to create vectors having lower host immune response and higher intracellular trafficking rate. This article reviews the various aspects of rationally designing AAV capsids like by site-directed mutagenesis, directed evolution and combinatorial libraries which can create vectors having not only immune evasive property but also enhanced gene expression and transduction capability. One or more combinations of these strategies have strong potential to create novel vectors which will have suitable clinical efficiency even at a low dose.
| S-EPMC4251935 | biostudies-literature
Project description:De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8+ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.
| S-EPMC10485635 | biostudies-literature
Project description:Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better "communication" between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.
| S-EPMC6950324 | biostudies-literature
Project description:Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.
| S-EPMC7120696 | biostudies-literature
Project description:BackgroundAdeno-associated virus (AAV) is an ideal gene therapy vector and is non-immunogenic in many small animal models. The stable gene expression commonly seen in murine models does not necessarily translate to nonhuman primates and higher-order species, highlighting the need for a better understanding of immune activation to these vectors. One capsid variant, AAVrh32.33, demonstrates a unique phenotype in murine muscle, reminiscent of what is often seen in higher-order species. AAVrh32.33 generates a strong CD8+ T-cell response to both capsid and encoded transgene antigens in a manner independent of transgene product or major histocompatability complex haplotype, making it an ideal candidate for studying immune activation to AAV in the mouse.MethodsTo map the H-2b and H-2d dominant epitopes of the AAVrh32.33 capsid, C57BL/6 or Balb/C mice received an intramuscular injection of 1 x 10(11) genome copies of AAV2/rh32.33.CB.nLacZ. Three weeks later, splenocytes were harvested and stimulated in vitro with pooled or individual peptides from the AAVrh32.33 capsid peptide library and analysed by an interferon (IFN)-gamma enzyme-linked immunosorbent spot assay or intracellular cytokine staining.ResultsThe immunodominant epitopes within the AAVrh32.33 capsid responsible for driving CD8+ T-cell responses to the capsid protein in C57BL/6 (SSYELPYVM) and Balb/C (KIPASGGNAL) mice were defined.ConclusionsIdentification of dominant capsid epitopes will make it possible to monitor cellular responses to the AAV capsid in vivo, facilitating mechanistic studies critical to defining how cellular immunity to the AAV capsid arises and, ultimately, how the generation of capsid-specific T cells can be avoided to ensure safety in a gene therapy setting.
| S-EPMC10694856 | biostudies-literature
Project description:Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid-derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.
| S-EPMC2689109 | biostudies-other
Project description:CD8(+) T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of antipathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing rhesus cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8(+) T cells that recognize unusual, diverse, and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8(+) T cell responses is suppressed by the RhCMV-encoded Rh189 gene (corresponding to human CMV US11), and the promiscuous MHC class I- and class II-restricted CD8(+) T cell responses occur only in the absence of the Rh157.5, Rh157.4, and Rh157.6 (human CMV UL128, UL130, and UL131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8(+) T cell epitope recognition.
| S-EPMC3816976 | biostudies-literature
Project description:Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.
| S-EPMC4588448 | biostudies-literature