Systematic Engineering of Uniform, Highly Efficient, Targeted and Shielded Viral-Mimetic Nanoparticles

In the past decades, numerous types of nanomedicines have been developed for the efficient and safe delivery of nucleic acid-based drugs for cancer therapy. Given that the destination sites for nucleic acid-based drugs are inside cancer cells, delivery systems need to be both targeted and shielded in order to overcome the extracellular and intracellular barriers. One of the major obstacles that has hindered the translation of nanotechnology-based gene-delivery systems into the clinic has been the complexity of the design and assembly processes, resulting in non-uniform nanocarriers with unpredictable surface properties and efficiencies. Consequently, no product has reached the clinic yet. In order to address this shortcoming, a multifunctional targeted biopolymer is genetically engineered in one step, eliminating the need for multiple chemical conjugations. Then, by systematic modulation of the ratios of the targeted recombinant vector to PEGylated peptides of different sizes, a library of targeted-shielded viral-mimetic nanoparticles (VMNs) with diverse surface properties are assembled. Through the use of physicochemical and biological assays, targeted-shielded VMNs with remarkably high transfection efficiencies (>95%) are screened. In addition, the batch-to-batch variability of the assembled targeted-shielded VMNs in terms of uniformity and efficiency is examined and, in both cases, the coefficient of variation is calculated to be below 20%, indicating a highly reproducible and uniform system. These results provide design parameters for engineering uniform, targeted-shielded VMNs with very high cell transfection rates that exhibit the important characteristics for in vivo translation. These design parameters and principles could be used to tailor-make and assemble targeted-shielded VMNs that could deliver any nucleic acid payload to any mammalian cell type.

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3-and viral-induced expression of type i interferons

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression. © 2012 Nature America, Inc. All rights reserved.

The effect of inhaled IFN-β on worsening of asthma symptoms caused by viral infections. a randomized trial

Rationale: Ex vivo, bronchial epithelial cells from people with asthma are more susceptible to rhinovirus infection caused by deficient induction of the antiviral protein, IFN-b. Exogenous IFN-b restores antiviral activity.

Objectives: To compare the efficacy and safety of inhaled IFN-b with placebo administered to people with asthma after onset of cold symptoms to prevent or attenuate asthma symptoms caused by respiratory viruses.

Methods: A total of 147 people with asthma on inhaled corticosteroids (British Thoracic Society Steps 2–5), with a history of virus-associated exacerbations, were randomized to 14-day treatment with inhaled IFN-b (n = 72) or placebo (n = 75) within 24 hours of developing cold symptoms and were assessed clinically, with relevant samples collected to assess virus infection and antiviral responses.

Measurements and Main Results: A total of 91% of randomized patients developed a defined cold. In this modified intention-to-treat population, asthma symptoms did not get clinically significantly worse
(mean change in six-item Asthma Control Questionnaire ,0.5) and IFN-b treatment had no significant effect on this primary endpoint, although it enhanced morning peak expiratory flow recovery (P = 0.033), reduced the need for additional treatment, and boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory analysis of the subset ofmore difficult-to-treat, Step 4-5 peoplewith asthma (n = 27 IFN-b; n = 31 placebo), Asthma Control Questionnaire-6 increased significantly on placebo; this was prevented by IFN-b (P = 0.004).

Conclusions: Although the trial did not meet its primary endpoint, it suggests that inhaled IFN-b is a potential treatment for virus-induced deteriorations of asthma in difficult-to-treat people with asthma and supports the needforfurther, adequately powered, trialsin this population. Clinical trial registered with www.clinicaltrials.gov (NCT 01126177).

Identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine

Vaccination procedures within the cattle industry are important disease control tools to minimize economic and welfare burdens associated with respiratory pathogens. However, new vaccine, antigen and carrier technologies are required to combat emerging viral strains and enhance the efficacy of respiratory vaccines, particularly at the point of pathogen entry. New technologies, specifically metabolomic profiling, could be applied to identify metabolite immune-correlates representative of immune protection following vaccination aiding in the design and screening of vaccine candidates. This study for the first time demonstrates the ability of untargeted UPLC-MS metabolomic profiling to identify metabolite immune correlates characteristic of immune responses following mucosal vaccination in calves. Male Holstein Friesian calves were vaccinated with Pfizer Rispoval® PI3 + RSV intranasal vaccine and metabolomic profiling of post-vaccination plasma revealed 12 metabolites whose peak intensities differed significantly from controls. Plasma levels of glycocholic acid, N-[(3α,5β,12α)-3,12-Dihydroxy-7,24-dioxocholan-24-yl]glycine, uric acid and biliverdin were found to be significantly elevated in vaccinated animals following secondary vaccine administration, whereas hippuric acid significantly decreased. In contrast, significant upregulation of taurodeoxycholic acid and propionylcarnitine levels were confined to primary vaccine administration. Assessment of such metabolite markers may provide greater information on the immune pathways stimulated from vaccine formulations and benchmarking early metabolomic responses to highly immunogenic vaccine formulations could provide a means for rapidly assessing new vaccine formulations. Furthermore, the identification of metabolic systemic immune response markers which relate to specific cell signaling pathways of the immune system could allow for targeted vaccine design to stimulate key pathways which can be assessed at the metabolic level.

Respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials

As the most important viral cause of severe respiratory disease in infants and increasing recognition as important in the elderly and immunocompromised, respiratory syncytial virus (RSV) is responsible for a massive health burden worldwide. Prophylactic antibodies were successfully developed against RSV. However, their use is restricted to a small group of infants considered at high risk of severe RSV disease. There is still no specific therapeutics or vaccines to combat RSV. As such, it remains a major unmet medical need for most individuals. The World Health Organisations International Clinical Trials Registry Platform (WHO ICTRP) and PubMed were used to identify and review all RSV vaccine, prophylactic and therapeutic candidates currently in clinical trials. This review presents an expert commentary on all RSV-specific prophylactic and therapeutic candidates that have entered clinical trials since 2008.

Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype

The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses.

Neural dysfunction following respiratory viral infection as a cause of chronic cough hypersensitivity

Respiratory viral infections are a common cause of acute coughing, an irritating symptom for the patient and an important mechanism of transmission for the virus. Although poorly described, the inflammatory consequences of infection likely induce coughing by chemical (inflammatory mediator) or mechanical (mucous) activation of the cough-evoking sensory nerves that innervate the airway wall. For some individuals, acute cough can evolve into a chronic condition, in which cough and aberrant airway sensations long outlast the initial viral infection. This suggests that some viruses have the capacity to induce persistent plasticity in the neural pathways mediating cough. In this brief review we present the clinical evidence of acute and chronic neural dysfunction following viral respiratory tract infections and explore possible mechanisms by which the nervous system may undergo activation, sensitization and plasticity.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Metagenomic characterisation of the viral community of lough neagh, the largest freshwater lake in Ireland

Lough Neagh is the largest and the most economically important lake in Ireland. It is also one of the most nutrient rich amongst the world's major lakes. In this study, 16S rRNA analysis of total metagenomic DNA from the water column of Lough Neagh has revealed a high proportion of Cyanobacteria and low levels of Actinobacteria, Acidobacteria, Chloroflexi, and Firmicutes. The planktonic virome of Lough Neagh has been sequenced and 2,298,791 2×300 bp Illumina reads analysed. Comparison with previously characterised lakes demonstrates that the Lough Neagh viral community has the highest level of sequence diversity. Only about 15% of reads had homologs in the RefSeq database and tailed bacteriophages (Caudovirales) were identified as a major grouping. Within the Caudovirales, the Podoviridae and Siphoviridae were the two most dominant families (34.3% and 32.8% of the reads with sequence homology to the RefSeq database), while ssDNA bacteriophages constituted less than 1% of the virome. Putative cyanophages were found to be abundant. 66,450 viral contigs were assembled with the largest one being 58,805 bp; its existence, and that of another 34,467 bp contig, in the water column was confirmed. Analysis of the contigs confirmed the high abundance of cyanophages in the water column.

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

New approaches for the diagnosis of human papillomavirus infection:relevance for clinical practice and cancer prevention

Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2014