Investigation of adenoviral immune neutralization and tropism for improved targeted gene delivery

Human adenovirus serotype 5 (HAdV-5)-based gene delivery vectors are an attractive option for gene therapy applications because they can deliver large transgenes to a broad range of tissues, allow high level transgene expression, have negligible risk of insertional mutagenesis and are easily produced at high titers. Unfortunately, their high immunogenicity and liver and spleen-associated toxicity when intravascularly administered and high prevalence of neutralizing antibodies in patients remain challenges to be overcome for the generation of safe HAdV-5-based vectors for systemic gene therapy. The discovery that coagulation factor X (FX), a zymogen of a vitamin K-dependent serine protease that circulates in the bloodstream, simultaneously binds HAdV-5 hexon and heparan sulphate proteoglycans (HSPGs) to mediate hepatic transduction enabled the generation of HAdV-5 vectors with substantially reduced liver transduction via the manipulation of key amino acid residues in the adenoviral hexon protein. However, FX was also recently shown to protect HAdV-5 from neutralization by preventing binding of natural IgM antibodies to HAdV-5 capsids and it was reported that, in the absence of FX-binding and neutralization, HAdV-5 vectors can use alternative FX-independent transduction pathways for hepatocyte transduction. These findings highlight the complex interactions between adenoviruses and host blood factors and cells as well as the need for further research on these processes. Here, the interactions that mediate hepatic and splenic tropism of HAdV-5-based vectors and activation of the anti-viral immune response following intravascular delivery were investigated and targeted HAdV-5 delivery to the kidney was assessed. Liver and spleen transduction was assessed in immunocompetent C57BL/6 and immunocompromised Rag 2-/- or NSG mice lacking different components of the immune response, following intravascular administration of wild type or FX-binding deficient HAdV-5 vectors. HAdV-5 virions were neutralized in C57BL/6 mice in the absence of FX-binding, confirming a role for FX in protecting HAdV-5 from in vivo neutralization. However, NSG mice, which lack both innate and adaptive immunity, failed to neutralize FX-binding deficient HAdV-5 virions. Interestingly, administration of FX-binding deficient HAdV-5 vectors to IgM antibody-deficient Rag 2-/- mice revealed that IgM antibodies might not be required for in vivo neutralization of HAdV-5, indicating that innate immunity alone might be sufficient. In agreement with previous reports, exposure of FX-binding deficient HAdV-5 vectors to C57BL/6 serum or wild type HAdV-5 vectors to C57BL/6 serum pre-incubated with the FX inhibitor X-bp led to HAdV-5 neutralization in vitro. In contrast, Rag 2-/- and NSG serum failed to neutralize HAdV-5 in vitro in the absence of FX-binding, indicating that IgM antibodies are essential for in vitro HAdV-5 neutralization. This suggests in vitro and in vivo adenovirus neutralization is mediated by different mechanisms. Importantly, administration of FX-binding deficient HAdV-5 vectors to NSG mice, which were unable to neutralize HAdV-5, confirmed the existence of alternative FX-independent pathways for liver and spleen transduction in the absence of neutralization. CAR and αvβ3,5 integrins were assessed as possible host cell receptors for HAdV-5 transduction of these tissues in immunocompetent and immunocompromised mice. The use of CAR or αvβ3,5 integrin-binding ablated HAdV-5 vectors revealed that CAR and αvβ3,5 integrins might serve as receptors for HAdV-5 liver transduction in immunocompetent C57BL/6 mice in contrast to immunocompromised Rag 2-/- mice. Neither CAR nor αvβ3,5 integrins mediated HAdV-5 spleen transduction in either mouse strain. Furthermore, administration of HAdV-5 vectors simultaneously ablated for FX and αvβ3,5 integrin-binding to NSG mice showed that αvβ3,5 integrins play no role in liver or spleen transduction in the absence of neutralization. With the aim to define novel FX-independent pathways of HAdV-5 transduction in vitro that might be relevant in vivo, cell transduction was assessed for wild type or FX-binding deficient HAdV-5 vectors in the presence of immunocompromised Rag 2-/- serum or serum that had been pre-incubated with X-bp. These studies confirmed the existence of FX-independent mechanisms able to enhance HAdV-5 cell transduction in vitro in the presence of mouse serum and absence of neutralization. To identify the receptor(s) involved in in vitro HAdV-5 transduction in the presence of mouse serum, soluble recombinant HAdV-5 fiber knob was used to block access of virions to CAR and wild type or FX-binding deficient HAdV-5 cell transduction was assessed in high and low CAR-expressing cell lines in the presence of C57BL/6 or Rag 2-/- serum with or without X-bp. HAdV-5 predominantly used a FX-independent pathway for cell transduction of high CAR-expressing cell lines in the presence of Rag 2-/- serum and soluble fiber knob substantially reduced both C57BL/6 and Rag 2-/- serum-enhanced transduction in such cell lines, suggesting a role for CAR, Conversely, HAdV-5 used the FX-mediated pathway or other FX and CAR-independent pathways for low CAR-expressing cell line transduction. Importantly, the use of CAR-binding deficient HAdV-5 vectors demonstrated that CAR usage in this setting does not rely on direct interactions of HAdV-5 with CAR, thus implicating a role for a mouse serum protein(s) in this process. To investigate these different pathways further, virions were fluorescently labelled with Alexa Fluor 488 dye and HAdV-5 cell binding, uptake and endosomal membrane penetration were characterized in the presence of FX by microscopic assessment of individual virions at the single cell level. FX substantially enhanced virion cell binding but had minimal effect on virion uptake and it was suggested to decrease efficiency of endosomal membrane penetration, limiting escape of virions from endosomes into the cytosol. Finally, FX and CAR-binding deficient HAdV-5 vectors were engineered to incorporate renal-targeting peptides found by in vitro phage display into the HI loop of the fiber knob domain for kidney-specific gene therapy. The resultant mutant HAdV-5 vectors were tested for their specificity in mediating gene delivery to ligand-expressing cells in vitro but failed to achieve specific targeting. Together, these findings contribute to a deeper understanding of the interactions between HAdV-5 vectors and host blood components and cell receptors, and their implications for liver and spleen transduction and neutralization of virions. The studies presented in this thesis highlight the limitations of current re-targeting strategies and the need for further research to successfully develop efficient HAdV-5-based vectors for systemic gene therapy

Quantitative Assessment of Stress Through EEG During a Virtual Reality Stress-Relax Session

This work was supported by the project PGC2018-098813-B-C31 (the Spanish Ministry of Science, Innovation and Universities, by European Regional Development Funds).Recent studies have addressed stress level classification via electroencephalography (EEG) and machine learning. These works typically use EEG-based features, like power spectral density (PSD), to develop stress classifiers. Nonetheless, these classifiers are usually limited to the discrimination of two (stress and no stress) or three (low, medium, and high) stress levels. In this study we propose an alternative for quantitative stress assessment based on EEG and regression algorithms. To this aim, we conducted a group of 23 participants (mean age 22.65 5.48) over a stress-relax experience while monitoring their EEG. First, we stressed the participants via the Montreal imaging stress task (MIST), and then we led them through a 360-degree virtual reality (VR) relaxation experience. Throughout the session, the participants reported their self-perceived stress level (SPSL) via surveys. Subsequently, we extracted spectral features from the EEG of the participants and we developed individual models based on regression algorithms to predict their SPSL. We evaluated stress regression performance in terms of the mean squared percentage error (MSPE) and the correlation coefficient (R2). The results yielded from this evaluation (MSPE = 10.62 2.12, R2 = 0.92 0.02) suggest that our approach predicted the stress level of the participants with remarkable performance. These results may have a positive impact in diverse areas that could benefit from stress level quantitative prediction. These areas include research fields like neuromarketing, and training of professionals such as surgeons, industrial workers, or firefighters, that often face stressful situations.Spanish Ministry of Science, Innovation and Universities, by European Regional Development Funds PGC2018-098813-B-C3

Using Hindsight to Anchor Past Knowledge in Continual Learning

In continual learning, the learner faces a stream of data whose distribution changes over time. Modern neural networks are known to suffer under this setting, as they quickly forget previously acquired knowledge. To address such catastrophic forgetting, many continual learning methods implement different types of experience replay, re-learning on past data stored in a small buffer known as episodic memory. In this work, we complement experience replay with a new objective that we call anchoring, where the learner uses bilevel optimization to update its knowledge on the current task, while keeping intact the predictions on some anchor points of past tasks. These anchor points are learned using gradient-based optimization to maximize forgetting, which is approximated by fine-tuning the currently trained model on the episodic memory of past tasks. Experiments on several supervised learning benchmarks for continual learning demonstrate that our approach improves the standard experience replay in terms of both accuracy and forgetting metrics and for various sizes of episodic memories.Comment: Accepted at AAAI 202

Hydroxylation of N-acetylneuraminic Acid Influences the in vivo Tropism of N-linked Sialic Acid-Binding Adeno-Associated Viruses AAV1, AAV5, and AAV6

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy. However, a number of recent preclinical large animal studies failed to translate into the clinic. This illustrates the formidable challenge of choosing the animal models that promise the best chance of a successful translation into the clinic. Several of the most common AAV serotypes use sialic acid (SIA) as their primary receptor. However, in contrast to most mammals, humans lack the enzyme CMAH, which hydroxylates cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) into cytidine monophosphate-N-glycolylneuraminic acid (CMP-Neu5Gc). As a result, human glycans only contain Neu5Ac and not Neu5Gc. Here, we investigate the tropism of AAV1, 5, 6 and 9 in wild-type C57BL/6J (WT) and CMAH knock-out (CMAH−/−) mice. All N-linked SIA-binding serotypes (AAV1, 5 and 6) showed significantly lower transduction of the heart in CMAH−/− when compared to WT mice (5–5.8-fold) and, strikingly, skeletal muscle transduction by AAV5 was almost 30-fold higher in CMAH−/− compared to WT mice. Importantly, the AAV tropism or distribution of expression among different organs was also affected. For AAV1, AAV5 and AAV6, expression in the heart compared to the liver was 4.6–8-fold higher in WT than in CMAH−/− mice, and for AAV5 the expression in the heart compared to the skeletal muscle was 57.3-fold higher in WT than in CMAH−/− mice. These data thus strongly suggest that the relative abundance of Neu5Ac and Neu5Gc plays a role in AAV tropism, and that results obtained in commonly used animal models might not translate into the clinic.Fil: Lopez Gordo, Estrella. Icahn School of Medicine ; Estados UnidosFil: Orlowski, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina. Icahn School of Medicine ; Estados UnidosFil: Wang, Arthur. Icahn School of Medicine ; Estados UnidosFil: Weinberg, Alan. Icahn School of Medicine ; Estados UnidosFil: Sahoo, Susmita. Icahn School of Medicine ; Estados UnidosFil: Weber, Thomas. Icahn School of Medicine ; Estados Unido

BCI-Based Navigation in Virtual and Real Environments

A Brain-Computer Interface (BCI) is a system that enables people to control an external device with their brain activity, without the need of any muscular activity. Researchers in the BCI field aim to develop applications to improve the quality of life of severely disabled patients, for whom a BCI can be a useful channel for interaction with their environment. Some of these systems are intended to control a mobile device (e. g. a wheelchair). Virtual Reality is a powerful tool that can provide the subjects with an opportunity to train and to test different applications in a safe environment. This technical review will focus on systems aimed at navigation, both in virtual and real environments.This work was partially supported by the Innovation, Science and Enterprise Council of the Junta de Andalucía (Spain), project P07-TIC-03310, the Spanish Ministry of Science and Innovation, project TEC 2011-26395 and by the European fund ERDF

Circumventing antivector immunity: potential use of nonhuman adenoviral vectors

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles

Human adenovirus serotype 5 is sensitive to IgM-independent neutralization in vitro and in vivo

Human adenovirus 5 (HAdV-5) is used as a vector in gene therapy clinical trials, hence its interactions with the host immune system have been widely studied. Previous studies have demonstrated that HAdV-5 binds specifically to murine coagulation factor X (mFX), inhibiting IgM and complement-mediated neutralization. Here, we examined the physical binding of immune components to HAdV-5 by nanoparticle tracking analysis, neutralization assays, mass spectrometry analysis and in vivo experiments. We observed that purified mouse Immunoglobulin M (IgM) antibodies bound to HAdV-5 only in the presence of complement components. Active serum components were demonstrated to bind to HAdV-5 in the presence or absence of mFX, indicating that immune molecules and mFX might bind to different sites. Since binding of mFX to HAdV-5 blocks the neutralization cascade, these findings suggested that not all complement-binding sites may be involved in virion neutralization. Furthermore, the data obtained from serum neutralization experiments suggested that immune molecules other than IgM and IgG may trigger activation of the complement cascade in vitro. In vivo experiments were conducted in immunocompetent C57BL/6 or immuno-deficient Rag2-/- mice. HAdV-5T* (a mutant HAdV-5 unable to bind to human or mFX) was neutralized to some extent in both mouse models, suggesting that murine immunoglobulins were not required for neutralization of HAdV-5 in vivo. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis of HAdV-5 and HAdV-5T* after exposure to murine sera showed stable binding of C3 and C4b in the absence of mFX. In summary, these results suggest that HAdV-5 neutralization can be mediated by both the classical and alternative pathways and that, in the absence of immunoglobulins, the complement cascade can be activated by direct binding of C3 to the virion

Censo de Productores de Arándanos. Partido de San Pedro – 2004

Este estudio se realizó con el objetivo de conocer la situación de la producción de arándanos en el Partido de San Pedro. Se identifican y ubican territorialmente a los establecimientos, releva información estratégica relativa a indicadores tecnológicos, el panorama varietal (grado de adopción y nº de plantas por variedad) y los sistemas de protección contra contingencias climáticas (mallas antigranizo, defensas contra heladas)EEA San PedroFil: Ros, Patricio Guillermo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Nicolás; ArgentinaFil: Hansen, Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Pedro; ArgentinaFil: Marcozzi, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; ArgentinaFil: Gordó, Manuela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; ArgentinaFil: López Serrano, Fernando Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; ArgentinaFil: Heguiabeheri, Adolfo Ricardo. Instituto Nacional de Tecnología Agropecuaria. Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; ArgentinaFil: Biglia, Jorge Lorenzo. Instituto Nacional de Tecnología Agropecuaria. Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; ArgentinaFil: Bisi, Marcelo Alejandro. Instituto Nacional de Tecnología Agropecuaria. Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; ArgentinaFil: Basaldúa, Beatriz Blanca. Instituto Nacional de Tecnología Agropecuaria. Estación Experimental Agropecuaria San Pedro. Agencia de Extensión Rural San Pedro; Argentin