Hepatitis Delta Virus Detected in Salivary Glands of Sjögren's Syndrome Patients and Recapitulates a Sjögren's Syndrome-Like Phenotype in Vivo.

BACKGROUND: Low-level, chronic viral infections have been suspect in the development of select autoimmune diseases, including primary Sjögren's syndrome (pSS). Multiple studies have shown stimulation of antiviral response pathways in pSS tissues suggestive of a viral infection. Yet, with this data in hand, a causal link between a viral infection and development of pSS had not been identified. Therefore, a study was designed to further define the viral landscape within pSS-affected salivary gland tissue to identify potential viral-mediated triggers in the pathogenesis of this autoimmune disease. METHODS: A viral microarray was utilized to measure viral transcripts present in salivary gland tissue from patients diagnosed with pSS compared to healthy controls. Murine models of salivary gland localized HDV antigen expression were developed to evaluate the capacity of a chronic HDV signature to trigger the development of a pSS-like phenotype. RESULTS: Through this analysis, two distinct viral profiles were identified, including the increased presence of hepatitis delta virus (HDV) in 50% of pSS patients evaluated. Presence of HDV antigen and sequence were confirmed in minor salivary gland tissue. Patients with elevated HDV levels in salivary gland tissue were negative for detectible hepatitis B virus (HBV) surface antigen and antibodies to HBV or HDV. Expression of HDV antigens in vivo resulted in reduced stimulated saliva flow, increase in focal lymphocytic infiltrates, and development of autoantibodies. CONCLUSION: Identification of HDV in pSS patients and induction of a complete pSS-like phenotype in vivo provides further support of a viral-mediated etiopathology in the development of pSS.

Transduction, tropism, and biodistribution of AAV vectors in the lacrimal gland.

PURPOSE: The lacrimal gland (LG) delivers defensive and metabolic factors to the ocular surface. These functions may be disrupted in several diseases, and for most of them there is no cure. The aim of this study is to investigate conditions and limitations for using adeno-associated virus (AAV) vectors as gene transfer agents to LG. METHODS: Eight-week-old Balb/c mice were used to investigate route, gene expression, and time course of AAV gene vector transfer to LG. AAV vectors encoding firefly luciferase were administered to the LG and luciferase expression was evaluated in vivo by immunohistochemistry. Ocular surface and neutralizing antibodies were also evaluated. RESULTS: The present work revealed that AAV vectors are able to delivery DNA to the LGs of mice. Direct injection had the highest level of transduction, and topical ocular drops the lowest. Overall, the AAV strain with highest transduction activity as measured by both luminescence and immunohistochemistry was AAV9, followed by AAV 5w8 and AAV5. Transduction was not different between sexes, could be detected as soon as 24 hours after injection, and lasted for at least 30 days (study termination). No tissue damage was observed when compared with controls. All vectors with detectable LG transduction induced neutralizing antibodies. CONCLUSIONS: LG gene delivery by AAV vectors appears to be both safe and well tolerated. The choice of vector influences both the overall transduction activity, as well as the spread of vector to other organs. This work supports the use of AAV-mediated gene therapy for dry eye.

Scalable purification of adeno-associated virus type 2, 4, or 5 using ion-exchange chromatography.

The availability of high-titer, high-purity, adeno-associated virus type 2 (AAV2) stocks has dramatically increased our understanding of this virus and its utility as a gene transfer vector. Current methods of purification take advantage of the stable interaction of AAV2 with heparin sulfate. This affinity chromatography, however, is not useful for purifying AAV4 and AAV5, because these serotypes lack heparin-binding activity. We have developed simple ion exchange high-performance liquid chromatography (HPLC) method for purifying different AAV serotypes that does not rely on the affinity of the viruses for heparin. The protocol is fast, efficient, and yields highly infectious material. Analysis of the highly purified virus indicated that more than 90% of the particles contained genomes and were more active than virus purified by cesium chloride (CsCl) gradient purification. This procedure is scalable and can easily be streamlined for large-scale production of recombinant adeno-associated virus (rAAV), regardless of the serotype. Ultimately, the new purification method will further the characterization of rAAV of different serotypes as vectors for gene therapy applications.