Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

In order to study the immune response elicited by asymptomatic carriage of Neisseria meningitidis, samples of serum, peripheral blood mononuclear cells (PBMCs), and saliva were collected from a cohort of more than 200 undergraduate students in Nottingham, United Kingdom, who were subject to high rates of acquisition and carriage of meningococci. Serum immunoglobulin G levels were elevated following increases in the rate of carriage, and these responses were specific for the colonizing strains. ؉ CD4 ؉ population, leading to the conclusion that an unbiased T-helper subset response was elicited by meningococcal carriage.

2050 Adeno-associated viral (AAV) vector-mediated gene transfer has shown great potential as a therapeutic platform for inherited and metabolic diseases. Systemic delivery of AAV vectors through the bloodstream is a safe, non-invasive, and potentially effective strategy to target a variety of organs, including liver, muscle, and brain. However, neutralizing antibodies (NAb) to AAV, highly prevalent in humans, constitute a major obstacle to successful gene transfer, particularly when a vector is delivered through the vasculature. Thus far, the liver was targeted to express the coagulation factor IX (F.IX) transgene in two clinical studies. In one study, a single-stranded AAV2 vector expressing the F.IX transgene was delivered through the hepatic artery to severe hemophilia B subjects at doses of 8×1010, 4×1011, and 2×1012 vector genomes (vg)/kg. Efficacy was observed in one subject from the high-dose cohort, who achieved peak F.IX transgene plasma levels of ∼10% of normal. The subjects infused at lower doses did not show any evidence of transgene expression, despite the fact that they did not have detectable NAb to AAV. In a second study, a self-complementary AAV8 vector expressing the F.IX transgene was delivered through peripheral vein infusion to severe hemophilia B subjects at doses similar to those administered in the AAV2 study, 2×1011, 6×1011, and 2×1012 vg/kg. All subjects enrolled in the AAV8 trial had evidence of transgene expression above baseline levels, despite the fact that some of the subjects had low-but-detectable anti-AAV8 NAb. Peak F.IX plasma levels at the high vector dose were 8–12% of normal, similar to the high dose of the AAV2 trial, suggesting that the vectors used in the two studies had comparable potency. Importantly, the vectors used in the two studies differed in empty capsid content, as the AAV2 vector preparation was essentially empty capsid-free and the AAV8 vector contained a 5–10 fold excess of empty capsids. The current study was undertaken to explore the role of empty capsids as a factor in the difference in outcome in the low- and mid- dose cohorts of the two trials. Our underlying hypothesis was that the presence of an excess of empty capsids effectively absorbs low-level neutralizing and non-neutralizing antibodies, and permits transduction even in their presence. Using a newly developed AAV antibody dot-blot assay, we demonstrate that adult human subjects with a low to undetectable NAb titer (1:1) as assessed by a commonly used assay do, in fact, carry significant amounts of anti-AAV antibodies. Conversely, children aged one year appear to be truly naïve for anti-AAV humoral immunity. Using C57BL/6 mice passively immunized with purified human IgG injected intraperitoneally 24 hours before vector administration, we further demonstrate that the same low levels of anti-AAV antibodies found in humans (NAb titer of 1:1–1:3) can block >90% of liver transduction after peripheral vein delivery of AAV8 vectors expressing F.IX at doses of 1×1012 vg/kg, comparable to those tested in the clinic. We next demonstrated that the inhibitory effect of low titer (1:1–1:3) anti-AAV antibodies can be overcome by adding a 5 to 10-fold excess of empty capsids to the final formulation of AAV8 vector, and that empty capsid content can be carefully titrated as a function of the animal's anti-AAV NAb in order to achieve efficient target organ transduction, even at titers >1:100. However, the beneficial effect of empty capsids on liver transduction is lost when a 1000-fold excess of AAV8 empty capsids are added to the formulation of AAV8 vectors, due to receptor binding competition. This inhibitory effect could be avoided by using AAV2 empty capsids, which efficiently protect AAV8 vectors from NAb without inhibiting transduction. These results were confirmed in non-human primates, a natural host for AAV8, in which a 5 to 6-fold increase in liver transduction was achieved by formulating vector in 5–10 fold excess AAV8 empty capsids, reaching levels of F.IX expression of 10 to 20% of normal. Application of these findings to the development of personalized formulations of vector product for intravascular delivery will facilitate safe, effective AAV-mediated gene transfer in settings in which vectors are delivered through the systemic circulation. Disclosures: Mingozzi: Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Anguela:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Wright:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. High:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties.

The 90th general meeting of the Association of Clinical Pathologists was held at Churchill College, Cambridge, from 4 to 6 April 1973. The guest lecture was given by Professor R. R. A. Coombs on 'The significance of recently developed rosetting techniques in immunology'. There were two symposia, the first on 'Renal transplantations' and the second on 'Data processing in haematology'.

Adult male guinea pigs were immunized with hepatitis B virus polypeptides prepared by Triton X-100 solubilization of purified 22-nm hepatitis B surface antigen (HBsAg) particles. A virus-specific subunit containing both the 28,000 molecular weight glycoprotein and the 23,000 molecular weight protein stimulated both cell-mediated and humoral immunity. A whole-blood-cell transformation assay additionally showed that the 64,000 molecular weight component of HBsAg, previously shown to contain host-specific antigens, also stimulated a cellular response to purified intact HBsAg particles, suggesting the additional presence of virus-specific material in this fraction. Immunization of animals with the 28,000–23,000 molecular weight polypeptides subsequently prepared in micelle form enhanced the demonstration of a cell-mediated reaction after only a single dose of immunogen. These results provide further evidence as to the suitability of HBsAg polypeptides prepared by Triton X-100 solubilization for hepatitis B prophylaxis.