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HSV-replication defective based vector as vaccines against Rotavirus infections

Facciolo, Sabrina (2011) HSV-replication defective based vector as vaccines against Rotavirus infections. PhD Thesis , Università degli Studi di Ferrara.

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    Background: Rotaviruses (RVs) are the most important cause of acute gastroenteritis in humans and animals. These viruses cause diarrheal disease primarily in the young, but infection and disease in older children and adults can occur, resulting in more than 600 000 deaths per annum, mainly in developing countries. RV particle is nonenveloped, with the viral genome of 11 segments of double-stranded RNA surrounded by three concentric protein layers: the outer layer is composed of VP (Viral Protein) 7 and VP4, the intermediate layer is formed by VP6, and the innermost layer is composed by VP2. Studies of natural infection in children indicated that primary infections can protect against severe disease upon secondary infection. For these reasons the most frequent approach to Rotavirus vaccine design was based on the use of attenuated animal or human Rotavirus strains, orally delivered to mimic natural infection. However, several major drawbacks have affected the development and application of these live Rotavirus-based oral vaccines, including the withdrawal from market of one of these vaccines because of adverse effects (gut intussusception). Moreover Rotaviruses are constantly and rapidly evolving, due both to their segmented genomes and to the fact that their genes can reassort between strains coinfecting a same host, and this fact is an important threat to the rational of using live attenuated Rotavirus strains as vaccines, both because these strains can revert to more virulent phenotypes and because these vaccine strains are being released to the nature in the faeces of the inoculated person, raising a considerable ecological concern. It is therefore critical to develop alternatives to this classical approach, both to generate a deeper understanding and to explore the potential of novel vaccination strategies. Aim: the goal of this project is the production of a collection of plasmids and HSV-1-based vectors expressing Rotavirus antigens as tool for research on Rotaviruses and the development of innovative genetic vaccines to fight against Rotavirus, based on the use of replication-defective HSV-1 vectors. Methods: the Rotavirus genes (vp7, vp4, vp6 and vp2) were subcloned first in basic plasmids (pcDNA Hygro 3.1+ or 3.1-, or in pBSSK) then in plasmids that have HSV specific loci sequences (pB41, pB5, pgJHE), where the Rotavirus cassettes were inserted between these Herpes sequences in order to recombine them into the viral genomes. Homologous recombinations have been carried out using standard calcium phosphate transfection. The recombinant backbones used (T0ZGFP, THZ and S0ZgJGFP) are replication-defective HSV-1 viral vectors that has low toxicity due to the deletion of one or more immediate early genes essential for viral replication; in addition they contain marker genes (gfp gene or lacZ gene) useful for the identification of the recombinant backbone which has integrated the transgene in its genome. Transfection and isolation of the recombinant viruses were performed in modified Vero cells (7b or E5), capable of providing the essential immediate early gene products in trans. The recombinant viruses containing the Rotavirus transgenes were identified by isolation of a clear plaque phenotype for GFP under the fluorescent microscope or after X-gal staining. All the recombinant viruses have been purified by three rounds of limiting dilution and the presence of the transgenes was verified by Southern blot analysis. The protein expression was evaluated by immunofluorescence and Western blot techniques with specific mono/polyclonal antibodies. Main results: a lot of plasmids containing Rotavirus genes of human, mice or simian origin and a large set of HSV-1-based vectors expressing Rotavirus proteins were constructed. The expression of VP6, VP2 and VP7 Rotavirus proteins (of murine or simian origin, under ICP0 promoter or HCMV promoter control, and in different loci of HSV genome) was visible in Western blot analysis. Moreover, nanotubes formed by VP6 protein were detected after immunofluorescence assays. Conclusions: plasmids and recombinant HSV-vectors carrying Rotavirus genes represent a tool to improve the research on Rotavirus and to elucidate the features of individual Rotavirus antigens. HSV-1-based vectors expressing Rotavirus antigens could represent an attractive alternative strategy to current vaccines. In addition, the possibility to cross recombinant HSV-vectors carrying Rotavirus genes in different loci of HSV could enhance the immune response by the construction of virus-like particles (VLPs); VLPs are a highly effective type of subunit vaccines that mimic the overall structure of virus particles without any requirement that they contain infectious genetic material.

    Item Type:Thesis (PhD Thesis)
    Date:22 February 2011
    Tutor:Manservigi, Roberto
    Coordinator:Bernardi, Francesco
    Institution:Università degli Studi di Ferrara
    Divisions:Dipartimento > Biochimica e biologia molecolare
    Subjects:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
    Uncontrolled Keywords:Rotavirus, HSV vectors, antigens, vaccines
    Deposited on:13 Apr 2012 14:56


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