Masters Degrees (Medical Microbiology)

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Browsing Masters Degrees (Medical Microbiology) by Author "Burt, Felicity"

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    Immunogenicity of Sindbis based replicons for Crimean-Congo hemorrhagic fever virus
    (University of the Free State, 2019-02) Tipih, Thomas; Burt, Felicity
    Introduction and Aim: Crimean-Congo hemorrhagic fever virus (CCHFV) infrequently causes hemorrhagic fever in humans with a case fatality rate of 30%. Currently, there is neither an internationally approved antiviral drug nor vaccine against the virus. In a move aimed at averting future epidemics, the World Health Organization has added the virus to the list of priority infectious organisms. The aim of the study was to investigate mechanisms of immunogenicity of Sindbis replicons encoding CCHFV glycoproteins and nucleoproteins for future development of an efficacious vaccine. Methodology: Genes encoding the complete open reading frames of the CCHFV nucleoprotein and glycoprotein precursor proteins of South African strains were amplified by the reverse transcription polymerase chain reaction technique and cloned into a Sindbis virus replicon vector. Sanger sequencing and next-generation sequencing were carried out to confirm gene sequences. Nucleoprotein and glycoprotein expression were demonstrated by transfecting baby hamster kidney cells and human embryonic kidney cells. Vaccine construct self-replication rates were assessed by transfecting BHK-21 cells and assaying for CCHFV RNA using gene-specific primers. Apoptosis induction in transfected BHK-21 cells was determined by measuring the enrichment of nucleosomes in the cytoplasm using an ELISA. Groups of three NIH mice were immunized with 100 μg of vaccine constructs three times intramuscularly three weeks apart with plasmid constructs pSinCCHF-31S, pSinCCHF-52S and pSinCCHF-52M. To augment cytokine responses the adjuvant poly (I:C) was co-inoculated with pSinCCHF-52S and pSinCCHF-52M separately. In addition, the constructs pSinCCHF-52M and pSinCCHF-52S were co-immunised with and without poly(I:C) to induce a response against both proteins simultaneously. Two weeks after receiving the third dose mice were sacrificed and blood was collected for determination of humoral immune responses while harvested splenocytes were stimulated with a CCHFV antigen for cytokine responses. Results: Two vaccine constructs (pSinCCHF-31S and pSinCCHF-52S) expressing CCHFV nucleoprotein and a construct (pSinCCHF-52M) expressing CCHFV glycoprotein were prepared. Recombinant protein expression was demonstrated by immunofluorescence assays targeting the histidine tag fused to the CCHFV proteins. Further confirmation of protein expression was performed by immunofluorescence assays using serum from CCHF survivors. All prepared vaccine constructs transcribed CCHFV RNA, as demonstrated by detection of protein using immunofluorescent antibody assays, and induced apoptosis in transfected cells. Immunized mice responded with the production of high titers of CCHFV IgG NP specific antibodies and higher levels of IgG2a in comparison to IgG1 responses were observed in responders suggesting a predominant Th1 antibody response. CCHFV IgG GP specific antibodies were not induced in vaccinated mice. Vaccine construct pSinCCHF-52S resulted in higher secretion of IL-2, (p = 0.0495) IFN-γ (p = 0.0369) and TNF-α (p = 0.0495) relative to immunisation with pSinGFP. An enhanced secretion of IFN-γ and IL-2 (p = 0.0463) was observed from splenocytes from mice co-immunised with pSinCCHF-52S and pSinCCHF-52M while vaccinating with pSinCCHF-52M increased IL-2 secretion (p = 0.0463). Co-administration of pSinCCHF-52M and pSinCCHF-52S constructs augmented IFN-γ (p = 0.0463) secretion. Co-inoculation of vaccine constructs with adjuvant poly (I:C) did not enhance cytokine secretion. Conclusion: The study demonstrated the expression of CCHFV nucleoproteins and glycoproteins by a Sindbis virus vector in mammalian cells. Vaccination of mice with construct pSinCCHF-52S induced type 1 immunity. Immunoglobulin G subtyping demonstrated IgG2a/IgG1 >1 as well as significantly higher IL-2, IFN- γ and TNF- α. Immunisation with pSinCCHF-31S and pSinCCHF-52M did not elicit specific antibody production and cytokines responses were weak. Further studies in CCHFV susceptible animals are necessary to determine whether the immune responses generated by vaccinating with pSinCCHF-52S are protective. However, this study shows the utility of Sindbis replicons in vaccine development against CCHFV.