AAV is a members of the family Parvoviridae, a group of small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 46kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. A summary committee report on this family was prepared as part of the International Committee on Taxonomy of Viruses (ICTV) (Cotmore et al 2019) An important aspect of the use of AAV vectors is the host immune response and the binding of ligands to the particle. Toward defining this structure-function relationship we have determined the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, to 3.45- resolution (Govindasamy 2013). One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. We recently report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1- resolution (Jose et al 2019). Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity. Application of these vectors for disease with unmet clinical need are ongoing. We have previously shown that in vitro transduction with bovine adeno-associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin-deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. In collaboration with Dr. Mammano we have reported that by manipulating inner ear connexin expression in vivo using BAAV vectors, and identifying the optimal route of vector delivery we have tested the use of gene therapy to restore hearing and identified limitations to this approach (Crispino et al 2017). Primary Sjgrens syndrome (pSS) is an autoimmune disease, characterized by lymphoid cell infiltration into the salivary and lacrimal glands, and affects 0.5% of the population in the United States of which 90% are women. The consequence of chronic immune cell activation in these exocrine glands is diminished secretory function, which leads to symptoms of dry mouth and dry eyes (Vivio et al 2019). In order to understand the environment of the salivary gland that might contribute to the gender bias associated with pSS we as worked to develop the first epigenetic road maps of the salivary gland (Michael et al 2019). We integrated data from DNase1 digital genomic footprinting, RNA-seq, and gene expression microarrays to comprehensively characterize the cis- and trans-regulatory components controlling gene expression of the healthy submandibular salivary gland. Analysis of 32 human tissues and 87 mouse tissues was performed to identify the highly expressed and tissue-enriched transcription factors driving salivary gland gene expression. This data was combined with protein expression levels and subcellular localization of 39 salivary transcription factors were confirmed by immunohistochemistry. These expression analyses revealed that the salivary gland highly expresses transcription factors associated with endoplasmic reticulum stress, human T-cell lymphotrophic virus 1 expression, and Epstein-Barr virus reactivation. Analysis of the DNase1 gene regulatory network identified dense interconnectivity among PLAG1, MYB, and 13 other transcription factors associated with balanced chromosomal translocations and salivary gland tumors. Collectively, these analyses provide a comprehensive atlas of the cis- and trans-regulators of the salivary gland and highlight known aberrantly regulated pathways of diseases affecting the salivary glands. As part of this work we analyzed several common salivary gland cell lines and found that all tested HSG samples had STR profiles indicating >80% match with HeLa in both the ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) databases (Lin et al 2019). This confirmed that HSG and HSY shared a common ancestry (host) with HeLa, whereas NS-SV-AC, NS-SV-DC, and A253 had unique STR profiles and it has been difficult to authenticate an uncontaminated sample of HSG. The future direction for our epigenetics work is to integrate across disease states. This will require improved bioinformatics. We have utilized the NIH Biowulf supercomputing cluster to assess the impact of parameter selection on biological reproducibility and ChIP-seq recovery by analyzing 4560 pipeline configurations. We have worked to identify an optimized pipeline and made this available for other researchers (Pranzatelli et al 2018). Salivary gland dysfunction occurs in several autoimmune and immune-related conditions, including Sjgren syndrome (SS); immune checkpoint inhibitor-induced sicca (ICIS) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Autoantibodies are common in Sjogrens patients but the B-cell targets are not well explored. To better understand their role in salivary gland dysfunction autoantibodies were evaluated against Ro52, Ro60, and La, as well as against a panel of 22 proteins derived from the salivary proteome (Burbelo et al 2019). A high percentage of autoantibody seropositivity was detected againstRo52, Ro60, and La in Sjogrens, but only a few ICIS patients were seropositive for these autoantigens. A few APECED subjects also harbored autoantibodies to Ro52 and La, but only Ro60 autoantibodies were weakly associated with a small subset of APECED patients with sicca. Additional testing of the salivary panel failed to detect seropositive autoantibodies against any of the salivary-enriched proteins in the SS and ICIS subjects. However, APECED subjects selectively demonstrated seropositivity against BPI fold containing family A member 1 (BPIFA1), BPI fold containing family A member 2 (BPIFA2)/parotid salivary protein (PSP), and lactoperoxidase, 3 salivary-enriched proteins. Moreover, high levels of serum autoantibodies against BPIFA1 and BPIFA2/PSP occurred in 30% and 67% of the APECED patients with sicca symptoms, respectively, and were associated with an earlier age onset of oral dryness. These findings highlight the complexity of humoral responses in different sicca diseases and provide new insights and biomarkers for APECED-associated sicca In summary, the future directions for the AAV Biology Section will be to continue examination and development of gene transfer vectors for use in treating disease as well as refine our tools for studying interactions necessary for cellular transduction.
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