Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.<br /

Global-Local Finite Element Analysis

114 σ.Η αναλυτική επίλυση πολύπλοκων προβλημάτων της μηχανικής στις μέρες μας καθίσταται δυσχερής εως αδύνατη χωρίς την εφαρμογή αριθμητικών μεθόδων και τη χρήση ηλεκτρονικού υπολογιστή. Η μέθοδος των πεπερασμένων στοιχείων αποτελεί σήμερα ένα ισχυρό εργαλείο για την επίλυση τέτοιων προβλημάτων και εξελίσσεται με μεγάλη ταχύτητα τόσο σε ακαδημαϊκό όσο και σε επαγγελματικό επίπεδο. Ενδεικτικά, αν και επινοήθηκε και χρησιμοποιήθηκε για τη στατική ανάλυση φορέων, έχει καθολικότερη εφαρμογή σε μια ευρύτερη κατηγορία προβλημάτων του μηχανικού, όπως στη ρευστομηχανική, στη μεταφορά θερμότητας, στην ακουστική, στον ηλεκτρομαγνητισμό και στην εμβιομηχανική. Επιπλέον, η εξέλιξη στων Η/Υ με τις ολοένα μεγαλύτερες δυνατότητες διαχείρισης όγκου δεδομένων αλλά και με την αύξηση της ταχύτητας εκτέλεσης των αριθμητικών πράξεων κατέστησε εφικτή την επίλυση σύνθετων προβλημάτων τα οποία θεωρούνταν απροσπέλαστα πριν μερικά χρόνια. Στην κατηγορία αυτή, των προβλημάτων αυξημένου υπολογιστικού κόστους, ανήκει και η καταστατική περιγραφή πολυφασικών υλικών. Είναι γεγονός ότι το μεγαλύτερο μέρος των παραγώμενων δομικών υλικών σήμερα, παρουσιάζει κάποιο είδος ανομοιογένειας, διακριτή ή μη στην κλίμακα δομικών έργων. Χαρακτηριστικά παραδείγματα αποτελούν τα κράματα μετάλλων, τα πορώδη, τα πολυκρυσταλλικά και τα σύνθετα υλικά στα οποία το μέγεθος, το σχήμα και οι ιδιότητες των συστατικών τους μερών καθορίζουν άμεσα τη συνολική τους μηχανική συμπεριφορά. Διάφορες τεχνικές έχουν αναπτυχθεί για την προσομοίωση και την περιγραφή της απόκρισης ανομοιογενών υλικών. Η παρούσα εργασία επικεντρώνεται στη μέθοδο ομογενοποίησης πολλαπλών κλιμάκων η οποία συνίσταται στην επίλυση δύο εμφωλευμένων προβλημάτων συνοριακών τιμών, για τη μακροκλίμακα και τη μικροκλίμακα αντίστοιχα. Τα βασικά χαρακτηριστικά μιας τέτοιας μεθόδου είναι ότι - Δεν απαιτείται η περιγραφή των καταστατικών νόμων του μακροφορέα. - Παρέχει τη δυνατότητα ενσωμάτωσης μεγάλων παραμορφώσεων και στροφών τόσο στην προσομοίωση της μικροκλίμακας όσο και του μακροφορέα. - Παρέχει τη δυνατότητα λεπτομερούς προσομοίωσης των συστατικών μερών της μικροκλίμακας. - Επιτρέπει οποιαδήποτε τεχνική επίλυσης στην κλίμακα του μικροφορέα. Αναλυτικά, σύμφωνα με τη μέθοδο αυτή. υπολογίζεται το διάνυσμα ανηγμένων παραμορφώσεων σε κάθε υλικό σημείο του μακροφορέα το οποίο στη συνέχεια χρησιμοποιείται για τη μόρφωση των συνοριακών συνθηκών του αντιπροσωπευτικού μικροφορέα στο αντίστοιχο σημείο. Μετά την επίλυση του προβλήματος συνοριακών τιμών της μικροκλίμακας, το διάνυσμα των τάσεων του μακροφορέα υπολογίζεται μέσα από τη διαδικασία ομογενοποίησης του πεδίου των τάσεων και κατά τον τρόπο αυτό υπολογίζεται η σχέση τάσεων ανηγμένων παραμορφώσεων για κάθε υλικό σημείο Ωστόσο, υπάρχουν κάποιοι περιορισμοί στην εφαρμογή της εν λόγω υπολογιστικής τεχνικής. Συγκεκριμένα, παρά το ότι κατά την προσομοίωση λαμβάνονται υπ' όψην οι διάφορες παράμετροι της μικροκλίμακας όπως το ποσοστό όγκου, η κατανομή και η μορφολογία των συστατικών μερών του υλικού, τα αποτελέσματα της μεθόδου είναι ανεξάρτητα από το απόλυτο μέγεθος του αντιπροσωπευτικού όγκου της μικροκλίμακας. Παρ' όλα αυτά, η τεχνική ομογενοποίησης στα πλαίσια ανάλυσης πολλαπλών κλιμάκων αποτελεί ένα σημαντικό εργαλείο για τον υπολογισμό των καταστατικών σχέσεων πολυφασικών υλικών στα οποία είναι αδύνατη η εφαρμογή οποιασδήποτε άλλης μεθόδου.Nowadays, analysis of complicated problems in the domain of mechanics consti- tutes a hard and even impossible task without the implementation of numerical methods and the employment of computational machines. Finite element method is a powerful tool for the solution of such problems and is rapidly developed in an academic and professional sense. Even if it was developed and implemented for structural analysis, it is widely employed in several domains such as in fluid mechanics, heat transfer, acoustics and electromagnetism. Furthermore, the development of computer hardware in terms of data processing, has significantly contributed to the solution of problems that were considered inaccessible a few years ago. Most of the materials produced in industry are heterogeneous on one or another spatial scale. Typical examples include metal alloy systems, porous media and polycrystalline materials and composites. The overall response of these micro heterogeneous materials depends strongly on the size, shape properties and spatial distribution of the microstructural components. Several techniques have been developed for the prediction of the macroscopic behavior of such materials. The present work is concentrated on the first order homogenization technique in the framework of a multi-scale approach which consists of the solution of two nested boundary value problems, for the macro-scale and the micro-scale respectively. Methods of this type - Do not require any constitutive assumption with respect to the overall ma- terial behavior. - Enable the incorporation of large deformations and rotations on both micro and macrolevel. - Provide the possibility to introduce detailed microstructural information. - Allow the use of any modelling technique at the microlevel. Concretely, according to this approach, the macroscopic deformation tensor is calculated for every integration point of the macrostructure and then is used to formulate the kinematic boundary conditions for the associated microstructural representative volume element (RVE). After the solution of the microstructural boundary value problem, the macroscopic stress tensor is computed by averaging the resulting microstructural stress field over the volume of the RVE and as a result, we obtain the stress-strain relation at every macroscopic point. However, there is a major disadvantage of the existing first-order computational homogenization. More specifically, this technique can account for the volume fraction, distribution and morphology of the micro-components however, it cannot take into account the absolute size of the microstructure making it thus impossible to treat microstructural size effects. Nevertheless, computational homogenization provides a significant strategy to obtain micro-macro structure-property relations for materials for which the overall macroscopic response cannot be computed by any other method.Κωνσταντινος Ε. Τατση

Enhanced immunogenicity following miR-155 incorporation into the influenza A virus genome

Influenza A vaccine efficacy in the elderly is generally poor and so identification of novel molecular adjuvants to improve immunogenicity is important to reduce the overall burden of disease. Short non-coding RNAs, known as microRNAs (miRNAs) are known to regulate gene expression and have the potential to influence immune responses. One such miRNA, miR-155, has been shown to modulate T and B cell development and function. We incorporated miR-155 into the influenza A virus (IAV) genome creating a self-adjuvanting \u27live vaccine\u27 with the ability to modify immunogenicity. Infection of mice with a recombinant influenza virus encoding miR-155 in the NS gene segment altered epitope-specific expansion of influenza-specific CD8(+) T cells and induced significantly higher levels of neutralising antibody

Evaluation of recombinant influenza virus-simian immunodeficiency virus vaccines in macaques

There is an urgent need for human immunodeficiency virus (HIV) vaccines that induce robust mucosal immunity. Influenza A viruses (both H1N1 and H3N2) were engineered to express simian immunodeficiency virus (SIV) CD8 T-cell epitopes and evaluated following administration to the respiratory tracts of 11 pigtail macaques. Influenza virus was readily detected from respiratory tract secretions, although the infections were asymptomatic. Animals seroconverted to influenza virus and generated CD8 and CD4 T-cell responses to influenza virus proteins. SIV-specific CD8 T-cell responses bearing the mucosal homing marker 7 integrin were induced by vaccination of naïve animals. Further, SIV-specific CD8 T-cell responses could be boosted by recombinant influenza virus-SIV vaccination of animals with already-established SIV infection. Sequential vaccination with influenza virus-SIV recombinants of different subtypes (H1N1 followed by H3N2 or vice versa) produced only a limited boost in immunity, probably reflecting T-cell immunity to conserved internal proteins of influenza A virus. SIV challenge of macaques vaccinated with an influenza virus expressing a single SIV CD8 T cell resulted in a large anamnestic recall CD8 T-cell response, but immune escape rapidly ensued and there was no impact on chronic SIV viremia. Although our results suggest that influenza virus-HIV vaccines hold promise for the induction of mucosal immunity to HIV, broader antigen cover will be needed to limit cytotoxic T-lymphocyte escape

Clade, Country and Region-specific HIV-1 Vaccines: Are they necessary?

Today, scientists are often encouraged to custom-design vaccines based on a particular country or clade. Here, we review the scientific literature and then suggest that the overwhelming endeavor to produce a unique vaccine for every world region or virus subtype may not be necessary

Mucosal Application of gp140 Encoding DNA Polyplexes to Different Tissues Results in Altered Immunological Outcomes in Mice

Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG(+) and IgA(+) antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge

ADAMTS5 is a critical regulator of virus-specific T cell immunity

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality

Evolution of high pathogenicity of H5 avian influenza virus: haemagglutinin cleavage site selection of reverse-genetics mutants during passage in chickens

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms

Inhibition of Nox2 Oxidase Activity Ameliorates Influenza A Virus-Induced Lung Inflammation

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2−/y mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8+ T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2−/y mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2−/y mice. The numbers of influenza-specific CD8+DbNP366+ and DbPA224+ T cells in the BALF and spleen were comparable in WT and Nox2−/y mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner