Neutralizing antibodies against adeno-associated viruses in Sjögren's patients: implications for gene therapy.

One potential setback to the use of gene therapy for the treatment of Sjögren's syndrome is the presence of neutralizing antibodies (nAb) against adeno-associated virus (AAV) serotypes. In order to evaluate the efficacy of this treatment option, nAb titers were measured in both healthy individuals and Sjögren's patients. Several serotypes with known transduction activity in mouse salivary glands were tested and only AAV5 showed a statistically significant change in the prevalence of nAbs between Sjögren's and healthy participants. Both groups showed a higher rate of nAbs for AAV2 compared with most of the other serotypes tested, except for bovine AAV (BAAV). Although a similar rate of seropositivity was seen against BAAV and AAV2, the percentage of samples with high titer was significantly lower with BAAV. Furthermore, the majority of positive samples exhibited low nAb titers in the primary Sjögren's syndrome (pSS) group for all serotypes except for AAV2. AAV5 was the only serotype that showed a statistically significant shift in the percentage of medium or high neutralizing titer. Based on these results, many serotypes are viable vectors in a gene therapy approach and pSS patients do not have a statistically significant higher rate of seropositivity or titer compared with healthy donors.

Repetitive adenovirus administration to the parotid gland: role of immunological barriers and induction of oral tolerance.

This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-gamma (IFN-gamma) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.

Isolation and characterization of a rat HSP 27 gene.

A gene encoding the rat heat shock protein 27 protein was isolated and characterized. The gene is composed of three exons and two introns. The proximal 5' untranslated region of this gene contains consensus heat shock elements, multiple SP1 sites, and basal regulatory elements including a CCAAT box and a TATA box. Comparison of this heat shock gene with the murine heat shock protein 25 and human heat shock protein 27 genes revealed the conserved presence of a putative heat shock element in the first intron of these mammalian small M(r) heat shock protein genes. While preliminary expression studies indicate the promoter directs heat shock induced expression of heat shock protein 27, the functional significance of the intronic heat shock element to the expression of this gene remains to be determined.

Binding of murine osteoblastic cells to titanium disks and collagen I gels: implications for alternative interpretations of osseointegration.

Using 3H-thymidine-labeled MC3T3-El osteoblastic cells, the number of osteoblasts bound to titanium surfaces after various surface treatments and incubation periods was directly measured. MC3T3-El cell binding to titanium surfaces was saturable at a low level (approximately 10,000 cells/cm2). Although treatment of these surfaces with fibronectin, keratin sulfate, and the fibronectin-derived peptide GRGDS (glycine-arginine-glycine-glutamate-serine) increases cellular binding by 29% to 31%, the relative binding to titanium was 5 to 10 times lower than binding to collagen I gels. A collagen I matrix competed with the commercially pure titanium surfaces for cell binding from solution, suggesting that direct binding of osteoblasts to titanium surfaces present within an organic matrix may not be favored. The significance of immediate and direct bone cell attachment to titanium surfaces for osseointegration should be reevaluated.

Further studies of salivary inhibition of HIV-1 infectivity.

An HIV-1/ATH8-cell cytopathic system was used to characterize the previously reported anti-HIV-1 activity of human saliva. Inhibitory activity was demonstrated by monitoring viable cell counts, HIV-1 p24 core antigen, and reverse transcriptase levels. Nonfiltered whole saliva, sterilized by irradiation, protected the ATH8 cells from HIV-1 infection. When HIV-1/saliva mixtures were filtered following incubation, the quantity of virus was significantly less (approximately 50%) than in HIV-1/media-filtered controls, suggesting that salivary aggregation and/or agglutination may be involved in the inhibitory activity. However, a sufficient number of apparently morphologically intact viral particles were still present in the HIV-1/saliva filtrates to lead to infection. When saliva was filtered prior to incubation with HIV-1, these filtrates showed substantial inhibitory activity, although reduced compared with that of non-prefiltered saliva. We conclude that saliva likely has several means by which to inhibit HIV-1 infectivity.