Oncolytic viruses (OVs) are effective anti-cancer agents, however their abilities to induce anti-tumor immunity are not yet optimal. Mutanome epitopes are a novel source of tumor antigen formed as a result of mutations within the tumor genome. Within this project we attempted to combine B16F10 mutanome vaccination with OV therapy. We confirmed previous findings that significant immune responses to these epitopes can be generated. Furthermore, we designed and cloned a multi-epitope mutanome construct into MG1 Maraba virus and E1-/E3- deleted type 5 Adenovirus to use for heterologous prime-boost vaccination. While we demonstrated that these viruses induced T-cell responses to one mutanome epitope, we failed to detect responses to the other epitopes. Furthermore there was no effect seen on overall survival. This approach warrants further investigation because coupling mutanome vaccination with OV therapy has the potential to exploit the therapeutic effects of the OV while inducing anti-tumor immunity to tumor-unique antigens.

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Mes travaux de thèse ont pour but d’évaluer l’approche « épitope display » pour sa capacité d'induire les réponses cellulaires. Ad à capside modifiée par insertion de différents épitopes T issus de la ovalbumine ont étés produit. Après administration à des souris C57BL/6, j'ai mis en évidence l'induction de la réponse cellulaire dirigée contre les épitope insérés (grâce aux techniques ELISPOT, tétramères, ou bien en quantifiant la production d’IFNg par les splénocytes restimulés in vitro). J’ai démontré que ces réponses sont limités chez des souris préalablement immunisées avec Ad. Des manipulations en course ont pour but de confirmer ces résultats et d'évaluer la cinétique de ces réponses. Mon 2ème objectif est de comprendre les paramètres qui contrôlent l’immunogénicité des Ad présentant des épitopes. Ainsi, j’ai montré que l’ablation des interactions des Ad porteurs d’un épitope issu de l’ovalbumine avec les récepteurs/facteurs (intégrines, facteur X de la coagulation) impliqués dans l’entrée de l’Ad dans les cellules ne modifiait pas leur capacité à induire une réponse humorale contre l’ovalbumine. Ces résultats suggèrent que le processus d’infection virale n’est pas requis pour l’induction d’une réponse humorale par les Ad porteurs d’épitopes. / Recombinant adenoviruses (Ad) have recently been employed for a wide range of vaccination strategies. Unfortunately, highly prevalent pre-existing neutralizing antibodies (Abs), reduce their ability to trigger transgene expression. To avoid the step of gene transfer a new vaccination strategy has been proposed based on the use of Ad displaying epitopes inserted into their capsid proteins. Using an ovalbumin-derived B cell epitope, our group demonstrated that vaccination efficiency depends on both the site of peptide insertion and the host immune status towards Ad (Lanzi et al; 2011). The present work aims at (1) evaluating the potency of Ad displaying T-cell epitopes from ovalbumin to elicit cellular responses and (2) understanding the molecular bases controlling the efficacy of this vaccination strategy. 1) Ad displaying T-cell epitopes from ovalbumin were constructed, produced and characterized in vitro. First in vivo experiments in naive mice showed induction of cellular responses, assessed with techniques like ELISPOT, tetramer staining and in vitro splenocyte restimulation. Subsequent experiments showed that pre-exisitng anti-vector immunity is hampering the potent induction of anti-epitope cellular responses. Current work is aiming at confirming the obtained results as well as at evaluating the kinetics of cellular responses induced upon "epitope display" vaccination. 2)First, the influence of interactions of Ad (displaying OVA peptide) with their natural receptors was investigated. Different detargeted Ads were produced and characterized in vitro. Upon mice immunization these vectors led to unmodified anti-epitope humoral responses, suggesting that their efficacy does not depend on the ability to transduce cells. In parallel we sought to evaluate the impact of innate immunity on the outcome of anti-epitope adaptive immune responses. Upon immunization of WT and MyD88-/- mice with Ad displaying OVA epitope we observed that cellular responses induced in MyD88-/- mice are significantly diminished while humoral responses were not altered. These results remain to be confirmed but question the role of other innate immunity sensors in the immunogenicity of Ad-based vaccines. Altogether, our work is expected to provide the foundations for the development of Ad-based vaccines with minimized side effects and unaltered adjuvant properties.

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Acute gastroenteritis (AGE) is a global public health problem causing considerable morbidity and mortality among infants and children, especially in low-income settings. Viruses including group A rotaviruses (RVA), noroviruses (NoV), adenoviruses (AdV), sapoviruses (SaV) and astroviruses (AstV) are widely acknowledged to be the most common cause of AGE in children. The importance of newly recognised viruses such as human bocavirus (HBoV) as an aetiological agent of AGE is becoming increasingly evident. The aim of this study was to investigate the molecular epidemiology of HAdV and HBoV in children aged ≤5 years hospitalised for AGE in South Africa (SA) from April 2009 to April 2015. Clinical and demographic data, along with stool specimens were collected from hospitalised children who presented with AGE. Real-time polymerase chain reaction (PCR) was used to screen for the presence of enteric DNA viruses. Genotyping was achieved by nucleotide sequence analysis or multiplex PCR. Whole genome sequencing was performed on selected strains to characterise their genetic variation and evolution. Between April 2009 and December 2014, the prevalence of HAdV in hospitalised children with AGE in SA was 18.1% (656/3623); 62.3% of the HAdV_positive children were 7–24 months of age. Human AdV was detected year round. Co-infections were found in 76.3% (222/291) cases of the HAdV_positive specimens with full enteric screening and AstV was detected most frequently as a co-infecting pathogen. Prolonged hospital stay was observed in human immunodeficiency virus (HIV)-infected children with HAdV. Human AdV-F was the most common species identified (254/603, 42.1%), with almost equally distribution of -40 and -41. Recombination breakpoints of the five HAdV41 strains varied in the number and location, indicating different evolution origins. Between April 2009 and April 2015, the prevalence of HBoV in hospitalised children with AGE in SA was 5.6% (212/3765); the majority of which were from children ≤2-year of age (92%, 195/212). Viral co-infections were found in 67% (142/212) of HBoV cases, while in fully screened specimens (virus, bacteria and parasites), 83.1% (74/89) had evidence of co-infections. In all co-infections, only HAdV was significantly associated with HBoV (adjusted Odds Ratio (aOR))=1.68; (95% CI 1.10-2.52; p=0.015) in multivariate analysis. Human BoV infections were reported throughout the year. All four HBoV genotypes were detected with HBoV1 being the most prevalent (79.6% (152/191). The variation in total number of specimens screened for HAdV and HBoV is because HAdV screening was done until December 2014; while HBoV screening was done until April 2015. The current study highlights the genetic diversity of HAdV-40 and -41 strains circulating in SA and suggests possible evolution from inter-strain recombination. Furthermore, the present study highlights the wide spectrum of HBoV genotypes in children with AGE in SA. This study presents the most comprehensive recent data on HAdV diversity in SA, and new baseline data on a HBoV-associated gastroenteritis in a country where no previous report is available. / Thesis (PhD (Medical Virology))--University of Pretoria, 2019. / Medical Virology / PhD (Medical Virology) / Unrestricted

Background: Acute respiratory infections (ARIs) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under 5 years old from low-income countries. Unfortunately, information regarding their epidemiology is still limited in Peru. Methods: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. Results: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation, the most prevelant microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). Conclusions: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment. / Revisión por pares

<p> Human adenovirus, type 2, was utilized to investigate its effects on the host cell population. The progression of KB cells through the various phases of the cell cycle after infection was studied, with special emphasis of DNA metabolism. At different times after infection, the rate and the amount of viral DNA synthesized was determined and their efficiency of incorporation into virious was investigated.</p> / Thesis / Master of Science (MSc)

The Fragile X-related mental retardation 1 (FXR1) is an N6-Methyladenosine reader involved in mRNAs metabolism like mRNA splicing, stability, transport, and miRNA regulation. It is also important in transcription, cell proliferation, differentiation, translation, polysome assembly and stress granule assembly. The protein is present in all eukaryotic cells, but so far it has been specifically essential for correct neural function. Until now, FXR1 has not been investigated in the concept of Human Adenovirus infection but we have observed an upregulation of FXR1 during the late phase of the Human Adenovirus 5 (HAdV-5) infection and an upregulation of some late HAdV-5 proteins in HeLa cells overexpressing FXR1. Our results furthermore showed that a FXR1 knockdown resulted in a reduced level of some HAdV-5 proteins at the same time as HAdV-5 mRNA were stabilized, indicating that FXR1 might be involved in translation of HAdV-5 late genes. Further investigation of the mechanism behind FXR1 mediated translation, a Death-associated protein 5 (DAP5) was founded to have an overall effect on the translation of HAdV-5 late proteins. / Part of a post-doctoral research

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