Simian adeno-associated virus (AAV) serotype rh.10 is a promising gene therapy tool, achieving safe, sustained transgene expression in the nervous system, lung, liver and heart in animal models. To date, preexisting immunity in humans has not been confirmed, though exposure is unexpected. We compared the humoral immune response with serotypes AAVrh.10 and AAV9 in mice, and AAVrh.10, AAV9 and AAV2 in 100 healthy humans. Mice, injected-intravenously, raised significantly more anti-AAV9 than anti-AAVrh.10 IgG (immunoglobulins), and sera demonstrated greater neutralizing capacity, correspondingly. Antibody cross-binding studies in mice showed negligible cross-recognition between AAVrh.10, AAV9 and AAV2. In humans, IgG prevalence against the most common human serotype, AAV2, was 72%; AAV9, 47% and AAVrh.10, a surprising, 59%. Yet, neutralizing-antibody seroprevalences were 71% for AAV2, 18% for AAV9 and 21% for AAVrh.10. Thus, most anti-AAV9 and anti-AAVrh.10 IgG were nonneutralizing. Indeed, sera generally neutralized AAV2 more strongly than AAVrh.10. Further, all samples neutralizing AAVrh.10 or AAV9 also neutralized AAV2, suggesting antibody cross-recognition. This contrasts with the results in mice, and highlights the complexity of tailoring gene therapy to minimize the immune response in humans, when multiple-mixed infections during a lifetime evoke a broad repertoire of preexisting antibodies capable of cross reacting with non-human serotypes.

Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of different sets of genes in response to stimuli, playing an active role in the homeostasis of the fish immune system. Nowadays, vaccination is one of the main ways to control and prevent viral diseases in aquaculture and the development of novel vaccination approaches is a focal point in fish vaccinology. One of the strategies that has recently emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines have already been shown to immunostimulate and protect fish against bacterial infections. To explore the role of RBCs in the immune response to two nanostructured recombinant proteins, TNF α and a G-VHSV protein fragment, we performed different in vitro and in vivo studies. We show for the first time that rainbow trout RBCs are able to endocytose nanostructured TNF α and G-VHSV protein fragment in vitro , despite not being phagocytic cells, and in response to nanostructured TNF α and G-VHSV fragment, the expression of different immune genes could be modulated.

Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous system (CNS) disorders because of their preferential transduction of neurons and efficient retrograde axonal transport. Here we tested the potential of a helper-dependent CAV-2 vector expressing β-glucuronidase (HD-RIGIE) in a mouse model of mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disease caused by deficiency in β-glucuronidase activity. MPS VII leads to glycosaminoglycan accumulation into enlarged vesicles in peripheral tissues and the CNS, resulting in peripheral and neuronal dysfunction. After intracranial administration of HD-RIGIE, we show long-term expression of β-glucuronidase that led to correction of neuropathology around the injection site and in distal areas. This phenotypic correction correlated with a decrease in secondary-elevated lysosomal enzyme activity and glycosaminoglycan levels, consistent with global biochemical correction. Moreover, HD-RIGIE-treated mice show significant cognitive improvement. Thus, injections of HD-CAV-2 vectors in the brain allow a global and sustained expression and may have implications for brain therapy in patients with lysosomal storage disease.

In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as “nanopellets” (NPs). These are inclusion bodies of fish viral antigenic proteins produced in Escherichia coli. Soluble recombinant proteins are too labile to endure the in vivo environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up in vitro by zebrafish ZFL cells and in vivo by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated.

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