Título de Ingeniero en Biotecnología Molecular / Diversos mecanismos de edición génica se han implementado a lo largo de los años para mutar el genoma del virus herpes simple tipo 2 (VHS-2). Éstos se han basado principalmente en el uso de procesos de recombinación homóloga. Un mecanismo comúnmente utilizado consiste en la realización de recombinación homóloga con un sustrato de intercambio alélico (AES) directamente sobre células transfectadas con genoma viral. Otra aproximación comúnmente utilizada es el desarrollo de recombinación homóloga en bacterias transformadas con un cromosoma artificial bacteriano (BAC) que contiene el genoma completo del virus herpes simple. La progenie viral mutante es luego obtenida mediante transfección de células con este material genético modificado. Si bien se han desarrollado diversas cepas mutantes de VHS-2 utilizando estas metodologías, ambas metodologías son laboriosas y presentan problemas técnicos tanto previos como posteriores a la generación de la mutación en el genoma. Dadas estas dificultades, se ha buscado desarrollar otros mecanismos más convenientes los cuales faciliten este proceso. Una metodología relativamente reciente que permite realizar modificaciones genéticas de manera más simple es aquella conocida como la tecnología de CRISPR-Cas9. Dada la mayor facilidad y especificidad de esta técnica, comparada a las más antiguas, nos propusimos mutar mediante el sistema CRISPR-Cas9, los genes virales US6, US4 y UL53 del VHS-2 para interrumpir la síntesis de las proteínas virales gD, gG y gK, respectivamente. Si bien se lograron desarrrollar los reactivos para realizar estas mutaciones, lamentablemente no se obtuvieron los aislados mutantes deseados. Se discuten los eventuales problemas que no permitieron lograr el objetivo

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Given the importance of innate defense mechanisms in the skin, we have examined the interactions of the arbovirus La Crosse virus (LACV) with serum factors that serve as a first line of antiviral defense, and the outcome of LACV infection of human keratinocytes, one of the main cell types present at viral entry. Incubation of LACV derived from insect cells (I-LACV) with normal human serum in vitro did not result in neutralization, but instead stabilized LACV virions and enhanced infectivity. Enhanced infectivity was also seen with heat inactivated serum devoid of complement activity and with serum from a range of animals including mouse, ferret and non-human primates. Depletion of antibodies from serum removed enhancement of I-LACV infectivity and sucrose gradient sedimentation assays showed IgG co-sedimenting with I-LACV particles. Serum-enhancement of LACV infectivity was not seen with virus derived from human cells, suggesting that insect cell-derived LACV is unique in its ability to subvert factors in serum to facilitate the initial infection of animal cells. In modeling initial replication following delivery of insect-derived virus to the skin, we show that I-LACV replication in HaCaT cells was restricted in culture by an antiviral response elicited by both IFN-β and IFN-λ. Media from I-LACV-infected cells induced killing of bystander non-infected HaCaT cells, and this cell death was relieved by blocking IFN-β signaling. Bystander cell killing was not seen with I-LACV infection of a human fibroblast cell line. Our data suggest that keratinocytes produce IFNs which limit virus spread through both antiviral signaling and by induction of cell death of potential new target cells for infection. These results are further evidence that virus and host immune interactions are complex and raises the question on how the combined outcome of these interactions determines the success of a virus infection and dissemination.

Hereditary breast and ovarian cancers (HBOC) represent 5-10% of breast and 10-15% of ovarian cancer cases. These cancers tend to be aggressive and curative treatment strategies are scarce. Poly(ADP-ribose) polymerase inhibitors (PARPi), a family of drugs that inhibit DNA repair, are a promising therapy for cancers harbouring mutations in their DNA repair machinery, such as HBOC. Unfortunately, nearly all patients ultimately become resistant to PARPi, leaving limited options for definitive treatment. Oncolytic or “cancer-killing” viruses are an innovative immunotherapeutic platform capable of selectively targeting cancer cells, leaving healthy tissues unharmed. Our group has demonstrated that oncolytic rhabdoviruses may be used to deliver therapeutic payloads by encoding targeting sequences to act on genes via RNA interference. In the present work, I have engineered the oncolytic virus, vesicular stomatitis virus (VSV), to express a variety of microRNA (miRNA) sequences that target genes essential for DNA repair, sensitizing resistant cancer cells to PARPi therapy. After initial experiments revealed hurdles concerning the functionality of artificial miRNAs which specifically target BRCA1 and BRCA2 I encoded the naturally occurring hsa-miR-182 into VSV to knockdown BRCA1 and additional genes essential for DNA repair. Using a 3D spheroid model, I have demonstrated sensitization of initially resistant MDA-MB-231 breast cancer cells to the PARPi, rucaparib. Complementary work exploring the shuttling of miRNAs into small extracellular vesicles, or EVs, has also shown that we can take advantage of the EV packaging facilities in infected cells, inducing the packaging of miRNAs over-expressed by VSV (EV-miRNAs) into EVs. Future work will address the functionality of these EV-miRNAs, testing their ability to knockdown targets in uninfected cancer cells.

Avian H5N1 viruses have perpetuated in poultry and caused sporadic human transmission since 1997. Vaccine candidates for the potential pandemic caused by H5N1 viruses have been continuously updated by World Health Organization. Multiple genetic lineages of H5N1 viruses which co-circulate and rapidly evolve in different regions, together with periodic population replacement of newly emerged genetic and antigenic variants in the field, pose great challenge for H5N1 vaccine candidate selection. The complexity of avian H5N1 viruses evolution raises an important issue for studying antigenic properties and also for projecting antigenic trend of this virus since the model established for the seasonal influenza viruses may not apply to H5N1 viruses which they are still in the animal phase. In contrast, the 2009 pandemic H1N1 viruses have established as another seasonal influenza viruses in humans. How will this swine originated viruses evolve genetically and antigenically in humans? For the first in human history, we are able to track the changes of pandemic viruses from the very beginning when they transmitted to human. This study focuses on antigenic and receptor binding properties of avian H5N1 viruses from 1997 to 2010 and 2009 pandemic H1N1 viruses from 2009 to 2011. It is found that avian H5N1 viruses continue to display highly diverse antigenic profile. The newly emerged H5N1 virus variants of clade 2.3.4 in 2008 and clade 2.3.2 in 2010 exhibit distinct antigenic properties as compared to the genetically similar viruses that were characterized previously. Receptor binding analysis showed H5N1 viruses still exhibit binding preference for avian type receptor. However, analysis of escape mutants selected from H5N1 viruses exposed to H5 monoclonal antibodies in cell based assay indicates that mutations in the conserved sites may cause switch of receptor binding specificity to human type or dual specificity for both human and avian. Based on antigenic and receptor binding analyses, it is found that the 2009 pandemic H1N1 viruses isolated from 2009 to 2011 are relatively stable. Most of the antigenic variants to monoclonal antibodies are transient and not able to become prevalent. It remains to be investigated if more significant antigenic variants may emerge in the coming seasons when population immunity prevails this virus. In conclusion, this study showed that clade 2.3 avian H5N1 viruses become increasingly antigenic distinct as compared to clade 2.1 and 2.2 viruses. Antigenic variation in antigenic sites may change receptor binding specificity in avian H5N1 viruses. The 2009 pandemic H1N1 viruses remain stable up to date but continue monitoring in coming seasons is necessary. / published_or_final_version / Microbiology / Master / Master of Philosophy

Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thèse (Ph. D.)--Université Laval, 2002. / Bibliogr.: f. 238-315. Publié aussi en version électronique.

Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.

Orientador : Elliot W. Kitajima / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-18T02:58:49Z (GMT). No. of bitstreams: 1 Camargo_IrineuJoseBarsantide_D.pdf: 9596167 bytes, checksum: 926cbf7f4d50de741cd0761e27ba0956 (MD5) Previous issue date: 1973 / Resumo: Não informado / Abstract: Not informed. / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular