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In vitro immune responses to Sindbis virus
No full textSindbis virus (SINV) is an arthritogenic alphavirus belonging to the Togaviridae family. SINV was initially isolated in Sindbis, Egypt in 1952, since then the virus has been isolated in different parts of the world including Africa, Asia, Australia and Europe. Infection with SINV results in febrile self-limiting symptoms that are usually of short duration. However, some individuals develop prolonged incapacitating joint pain that may persist for months to years. The mechanism by which SINV and other arthritogenic alphaviruses cause chronic arthritis is poorly understood however previous studies suggest the involvement of monocytes and macrophages which result in the secretion of pro-inflammatory cytokines which are induced by virus replicating in or around joint tissue. The aim of the study was to investigate the innate immune response to in vitro infection of macrophages with SINV in order to determine if human macrophages from different individuals differ in their susceptibility to SINV infection and to determine the role of interferon (IFN) in SINV infected macrophages. Peripheral blood mononuclear cells (PBMCs) were isolated from ten SINV antibody naïve individuals using ficoll-paque density gradient method. The cells were stimulated with human-macrophage colony-stimulating factor (HM-CSF) to differentiate from monocytes to macrophages. Post-stimulation the macrophages were infected with SINV at a multiplicity of infection (MOI) of 0.1. Additionally, some macrophages cultures were pre-treated with ruxolitinib, an IFN inhibitor 2 hours or immediately prior to infection with SINV at an MOI of 0.1. Virus replication was determined at different time intervals for 24 hours using a two-step quantitative RT-PCR. To determine the secretion levels of pro-inflammatory cytokines post-SINV infection, cell-free supernatant was collected at different intervals post-infection and levels of proinflammatory cytokines including IFN-α, TNF-α, IL-1β, IL-6, IL-8 and IL-12 in the supernatant were tested by ELISA. A primer pair that targets the nsP2 region of SINV nsP2 protein together with the TaqMan hydrolysis probe were used to quantify viral loads of infected macrophages by qRT-PCR. An increase in viral loads was detected in macrophages of 5/10 participants, suggestive of viral replication. A decrease in viral load over time was observed in macrophages of the remaining five participants, suggestive of little to no viral replication. IFN inhibition resulted in SINV replication in macrophages from 7/10 and 8/10 participants when treated at time 0 of infection and 2 hours prior to infection, respectively. Infection elicited a strong innate immune response demonstrated by secretion of pro-inflammatory cytokines including IFN-α. IL-6 and IL-8. Infection with SINV results in a strong pro-inflammatory response which seems to control viral load. Results suggest that macrophages are among SINV targeted cells during human infection and macrophages from different individuals appear to differ in their susceptibility to SINV replication. The study also shows that type I IFN may play an important role in the protection of SINV as its inhibition rendered macrophages from more participants susceptible to viral replication.National Health Laboratory ServiceThe South African Chairs initiative National Research FoundationPoliomyelitis Research FoundationUniversity of the Free State School of MedicineUniversity of the Free State Postgraduate Schoo
Adaptive immune response in COVID-19 patients and innate immune modulation of SARS-CoV-2
No full textThesis (Ph.D.(Medical Virology))--University of the Free State, 2023In December 2019, a cluster of cases of atypical pneumonia were reported in Wuhan, China. All patients had a history of attending a large seafood market. Surveillance and detection methods established during the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV)-1 outbreak contributed towards the identification of the virus as a novel coronavirus (CoV). The virus was later named SARS-CoV-2 and identified as the causative agent of Coronavirus Disease 2019 (COVID-19). Despite massive attempts to contain the virus in China, the cases rapidly increased and spread globally, and the World Health Organization (WHO) declared COVID-19 a Public Health Emergency of International Concern on 30 January 2020 and characterized the outbreak as a pandemic on 11 March 2020. Assessing immunoglobulin (Ig)-G and neutralizing antibodies is essential to comprehensively evaluate the efficacy and duration of immunity conferred by natural infection and the COVID-19 vaccines. Effectively determining SARS-CoV-2 seroprevalence within a population is important as it helps to improve our understanding of virus circulation dynamics, identify individuals at risk of infection, and the extent of virus exposure in the community. Therefore, this study investigated the duration and kinetics of adaptive immunity, particularly the persistence of IgG and neutralizing antibodies, in patients who recovered from SARS-CoV-2 in the Free State, South Africa.
Commercial assays are expensive, and hence two anti-spike (S) inhouse assays, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), were developed and validated for detection of anti-S IgG. A total of 89 serum samples were collected from COVID-19 PCR-confirmed patients between 2-94 days post-symptom onset to validate the assay. A 100 prepandemic samples were used as a negative control panel to determine the cutoff of the assay. A cutoff value of 30% was considered accurate to differentiate between negative and positive samples using a two-graph receiver operating characteristic (TG-ROC). The assays exhibited a sensitivity of 100% for ELISA and 98.8% for IFA when testing samples collected more than one week after the onset of illness. The positive predictive values (ppv) were 92.1% for ELISA and 91.0% for IFA on PCR-confirmed positive samples. The assays were also compared to a commercially available SAPRHA-approved assay, where the ELISA showed a higher ppv of 95.8 %, while the Roche assay was 89.6 %, and the commercial lateral flow was 93.9 %. The two in-house assays also detected IgG antibodies in samples collected from waves in which new variants were circulating, showing that despite mutations of the SARS-CoV-2 S protein, the assay was still sensitive to detect IgG antibodies in circulating variants. This indicates that these assays could be used for surveillance of the South African population.
To investigate the duration of anti-S IgG and neutralizing antibodies against SARS-CoV-2, 100 individuals with previously confirmed COVID-19 infection were recruited for the study. The cohort included 64/100 vaccinated and 36/100 unvaccinated individuals. Initial samples were collected between March 2021 and January 2022, with confirmed infection between June 2020 and December 2021. Follow-up samples were collected from 82/100 in 2022 and 62/100 in 2023 of the initial cohort. Samples were tested for anti-S IgG antibodies and neutralizing antibodies against Ancestral strain, Delta, and Omicron variants. A total of 95/100 baseline samples tested positive for anti-S IgG and 79/82 and 53/62 for follow-up samples. A total of 99/100, 78/100, and 72/100 baseline samples tested positive for neutralizing antibodies against the Ancestral strain, Delta, and Omicron variants. A total of 80/82, 63/82, 77/82 in 2022 and 53/62, 50/62, 56/62 in 2023 tested positive for neutralizing antibodies against the Ancestral, Delta, and Omicron variants, respectively. Samples were grouped based on the time (days) the samples were collected post-onset of illness. Results showed that IgG antibodies were significantly higher directly after infection, between 1-180 days, then gradually declined significantly with time. Neutralizing antibody titers against the Ancestral strain and Delta variant were significantly higher early after infection, between 1-180 days, remained relatively stable for an extended period, and then waned gradually before declining significantly. In contrast, although not significant, neutralizing antibodies against the Omicron variant increased with time, but this may be because samples were collected when the Omicron variant was still prevalent. Results also showed that vaccinated individuals had significantly higher antibody titers than unvaccinated, highlighting the importance of continued vaccination. The study shows the longevity of antibodies against SARS-CoV-2, as most individuals still had detectable titers at least 24 months post-acute infection, possibly boosted by vaccination or reinfection. The study also shows that the dynamics of antibodies vary among individuals; most individuals display declining antibody titers with time, and a smaller proportion maintain stable antibodies or a fluctuation of antibodies over time.
Traditional medicinal plants have been proposed as promising, cost-effective treatments for SARS-CoV-2, with studies showing the potential to induce protection against different viral infections. The study also investigated the potential of Phela, a traditional medicine prepared from the extracts of four South African plants, and the individual components to modulate the release of cytokines in SARS-CoV-2 Omicron-infected mammalian cells and to investigate the influence of the plant extracts on viral replication. Cells were treated with the plant extracts before or after infection with SARS-CoV-2. Subsequently, cell culture media was collected at 12, 24, 48, and 72 hours post-infection and tested for virus replication and levels of Interleukin (IL)-1β, IL-2Rα, IL-6, IL-10, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ cytokines. There was no statistically significant difference in viral load between infected cells treated with plant extracts compared to infected and untreated cells, showing that the plant extracts may have little to no effect on virus replication. Treatment with plant extracts resulted in significantly lower release of IL-1β, IL-2Rα, and TNF-α, with better response post-treatment than pretreatment, showing that the plant extracts may have the potential to manage cytokine storms and be a potential source of treatment for SARS-CoV-2
Immunogenicity of Sindbis based replicons for Crimean-Congo hemorrhagic fever virus
No full textThesis (Ph.D. (Medical Virology))--University of the Free State, 2019Introduction 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.National Health Laboratory Service Research TrustPolio Research FoundationSouth African Chairs initiative National Research FoundationUniversity of the Free State School of Medicine, Dr. Edward Tiffy Scholarshi
Characterisation of the human papillomavirus genome and p53 mutations in head and neck squamous cell carcinomas
No full textHigh-risk human papillomaviruses (HR-HPV) are ubiquitous, sexually transmitted, aetiologic agents of head and neck cancer (HNC). To date, no large-scale South African studies report on HPV type distribution and prevalence associated with head and neck cancer. In a previous study from our research group, HR-HPV was detected in biopsy samples from histologically confirmed head and neck squamous cell carcinoma (HNSCC) patients in a South African cohort. This study went on to determine genetic changes that accumulate within HR-HPV genomes, other than the well-researched HPV 16, that confer differences in oncogenicity. Unlike cervical carcinomas, it is unknown whether HPV variant research in HNSCC translates into clinical application.The first complete genomes of HPV 18 and HPV 31 from HNC were amplified and subjected to deep sequencing analysis. VBD 17/15, the South African HPV 18 isolate, clustered in lineage A1. Mutations were identified in the E2 and long control region (LCR) that might lead to differences in oncogenicity. Evidence of how papillomaviruses evolved is shown in this study, in a phenomenon known as linkage disequilibrium. A novel mutation of the South African isolate is described and further investigations in a larger cohort will determine whether this is a single nucleotide polymorphism unique to variants that preferentially infect the head and neck region. Although geographic and ethnic associations have been described for HPV 16 and 18, this study supports the use of alphanumeric nomenclature. Having obtained the first complete genome of HPV 31, deep sequencing analysis showed that this laryngeal carcinoma was co-infected with a closely related viral variant. HPV quasispecies has recently been described in cervical carcinoma and this is the first evidence in the head and neck region. The quasispecies described belonged to HPV 31 lineage B2. A unique deletion within the E5 gene needs to be investigated further to determine what this deletion represents to viral fitness. Polymorphisms in the LCR were investigated with pBlue-Topo® vector, a reporter gene system. Increased β-galactosidase expression was observed in the mutant that possessed a single nucleotide change within the YY1 binding site. This study provides evidence of sequence variation within HPV 31 LCR having a functional effect on viral p97 promoter activity. HPV-HNSCC is complicated by the synergistic interaction with the host. The human tumour suppressor gene, p53 was investigated for mutations in a subset of HNSCC samples. As the p53 gene is frequently mutated in most cancers, it has been proposed as a biomarker to deintensify treatment of HPV-HNSCC patients. The involvement of high-risk HPV in HNSCC is an alternative mechanism to inactivate the p53 protein function. Evidence of p53 mutations was shown, with a predominance of substitution patterns that are induced by a carcinogen from tobacco smoke. Immunostaining of p16 as a biomarker of HPV infection did not correlate with HPV detection by PCR. It is unknown whether the genomes of participants of African descent are too diverse from the reference genome used in this study to accurately use frequency and functional data of known mutations. All of the mutations within this study were detected within intron 5. Whether there may be mutations lying outside of the area investigated or whether other cancer driver genes are involved in tumourigenesis of this cohort of HNSCC samples is still to be determined. Sequence data for South African isolates from patients with HNSCC adds to the global understanding of this virus-related epidemic and contributes to elucidating the underlying molecular mechanisms of HPV infection in HNC in sub-Saharan Africa, especially in light of high HPV burden in the cervix.National Research Foundation (NRF)Poliomyelitis Research Foundatio
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Development and application of molecular assays for mosquito-borne alphaviruses in South Africa
No full textDissertation (M.Sc. (Medical Microbiology and Virology))--University of the Free State, 2021Surveillance of mosquito-borne alphaviruses is critical for the prevention of diseases and the control of outbreaks caused by these viruses, especially with the absence of approved vaccines and antiviral treatments available. Hence, the continual development of rapid and reliable tools for the surveillance of alphaviruses is important. This will aid in the understanding of which viruses are currently circulating with the potential to cause outbreaks. Molecular nucleic acid amplification tests (NAATs), particularly conventional and real-time reverse transcription (RT)-polymerase chain reaction (PCR), are typically employed in epidemiological surveys. In this study, a conventional nested RT-PCR assay was developed to detect alphaviruses in South Africa. In addition, an isothermal amplification technique, specifically a RT-helicase dependent amplification (HDA) assay, which only requires a simple heating device, for instance a heating block, and lateral flow dipsticks/ cassettes for end point detection, was developed to detect alphaviruses currently circulating in South Africa, as an alternative to the RT-PCR assay for application in low resource settings or for field application. The conventional nested RT-PCR assay was able to detect ≥620 copies of RNA compared to the RT-HDA assay which had a minimum limit of detection of 4.8 x 105 copies of RNA. Both assays were tested for theoretical cross-reactivity with other alphaviruses, which include Sindbis virus (SINV) and chikungunya virus (CHIKV) isolates from other regions and genotypes, and isolates from alphaviruses such as Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and western equine encephalitis virus (WEEV) that are endemic to other parts of world. Alignment of the primers with the sequences of these isolates shows that both assays in theory would be able to detect SINV isolates from northern Europe, taking into account the transcontinental transmission of the virus between South Africa and northern Europe by migratory birds. The conventional nested RT-PCR assay may be able to detect most alphaviruses due to minimal mismatches (0 – 1) detected between the primers and the partial nsP4 sequences of the alphavirus isolates, while the RT-HDA assay may not be well suited to detect other alphaviruses due to the many mismatches (>4) detected between the primers and the partial nsP4 sequences of the alphavirus isolates. Nevertheless, this shows that the RT-HDA is theoretically more specific that the conventional nested RT-PCR assay. The RT-HDA however failed to detect any alphaviruses in the 42 mosquito pools tested, which was not unexpected as the assay could only detect up to 4.8 x 105 copies of RNA. In contrast, the conventional nested RT-PCR assay was able to detect alphaviral RNA in five out of the 42 mosquito pools tested, and the nucleotide sequences were determined to identify the alphavirus species. SINV RNA was detected in three mosquito pools and Middelburg virus (MIDV) was detected in two pools. Phylogenetic analysis was subsequently performed to determine the genetic relationship of these isolates from the Free State with previously published/ reported SINV and MIDV isolates in South Africa, Africa, and around the world. The conventional nested RT-PCR assay developed in this study has shown to be a useful surveillance tool for the detection of mosquito-borne alphavirus infections. Given the low sensitivity determined for the RT-HDA assay, improvements, or alternative rapid and fieldable NAATs should be considered in the future for alphavirus surveillance applications in low resource settings.National Research Foundation (NRF)Department of Science and Technology (DST)South African Research Chairs Initiative (SARChI)Poliomyelitis Research Foundatio
Preparation of recombinant antigen for serological detection of African hantaviruses
No full textUnlike other members of the Bunyaviridae family, hantaviruses are transmitted to humans through direct exposure or inhalation of virus contaminated urine or droppings from their reservoir hosts. Hantaviruses were first discovered in 1976 with the identification of Hantaan virus (HNTV) from the reservoir Apodemus agarius in Asia and later in North America. In 2006, Sangassou virus (SANGV) was the first to be isolated in Africa in the African house mouse, Hylomyscus sinus and subsequently followed by the identification of ten more African hantaviruses in both rodent and insectivore hosts through reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). Hantaviruses are a public health concern with annual cases of disease reported to be approximately 200,000 per year, with most cases reported in Asia. In Africa, disease associated with hantaviruses is not well defined.
Culturing the virus and preparing reagents using native virus requires the use of biosafety level (BSL) 3 or 4 laboratories limiting the number of facilities with capability to prepare serological assays. Hence, the use of recombinant antigens that are safe to use in a BSL 1 laboratory that have application as serological tools for surveillance are required.
The aim of the study was to develop serological assays to test for antibodies against hantaviruses in human serum samples collected in the Free State, South Africa using a recombinant nucleocapsid protein (NP) of SANGV as a representative of African hantaviruses. Transiently transfected cells were used to prepare antigen slides for IFA and expressed protein was used in an in-house enzyme linked immunosorbent assay (ELISA). In-house assays and commercially available ELISA kits were used to screen human serum samples.
There are limited seroprevalence studies performed in Africa to detect IgG antibodies against hantaviruses in humans and no commercial serological assays are available using an African antigen. Hence, it was considered that the preparation of a recombinant African hantavirus antigen based on SANGV could have application in serological surveillance studies.
The S gene segment of the SA14 strain of SANGV was modified and codon optimized for enhanced expression and detection. The construct was sequenced and aligned to the native S gene. It was used to transfect baby hamster kidney cells (BHK-21). Expression of a 50kDA
protein was confirmed by SDS-PAGE and Western blot assay. Antigen slides were prepared from transfected cells fixed on 12 well chamber slides. Positive controls from the commercially available ELISA kits were used in the IFA. Four of the 176 serum samples tested gave a positive test. For the in-house ELISA, protein was harvested from T75 culture flasks. The antigen was tested using positive and negative controls from the commercial ELISA kits. A suitable differentiation between positive and negative samples was not detectable despite attempts to optimize the in house ELISA. It is likely that the protein yield was insufficient for the ELISA and further attempts, beyond the scope of this project, to increase the protein yield will be investigated using a stable cell line. Commercially available ELISA kits comprising of HNTV, Dobrava (DOBV) and Puumala (PUUV) recombinant NP antigens for Europe and Asia group and Andes virus (ANDV) and SNV for the American group were used to screen acute human sera in the laboratory. Positive reactors were identified using both kits. The significance of the results is difficult to interpret as there was lack of concordance. However, it does suggest that hantaviruses are to be found in this area and that the use of a homologous antigen for serological surveillance is essential. Results confirmed some serological cross-reactivity between heterologous Asian, American and African hantaviruses and a potential application for an African hantavirus as a tool for surveillance.National Research Foundation (NRF)Polio Research Foundation (PRF)University of the Free State (UFS
Preparation and immunogenicity of a candidate replicon based yellow fever vaccine
No full textEnglish: Yellow fever virus (YFV), a mosquito-borne virus that belongs to the family Flaviviridae and genus Flavivirus, is a significant cause of morbidity and mortality in yellow fever endemic areas, especially in West Africa. In humans, YFV causes yellow fever, a disease characterised by renal failure, jaundice, and/or haemorrhage. The burden of disease is highest in Africa constituting approximately 90% of reported cases worldwide. Despite the availability of highly efficacious live attenuated vaccines against YFV, the estimated prevalence for yellow fever in Africa was 130 000 severe cases and 78 000 deaths for 2013. The available live attenuated vaccines have been contraindicated for use in immunocompromised patients and individuals with hypersensitivity to eggs and/or chicken. Vaccine-associated neurotropic adverse events that result in the development of meningoencephalitis in infants and vaccine-associated viscerotropic adverse events that result in disease resembling wild-type yellow fever have been reported. Vaccine-associated viscerotropic adverse events are associated with fatality rates exceeding 40%. Therefore, there is a need for a safer alternative to complement the use of the available live attenuated vaccines. The aim of this study was to construct a DNA-launched candidate vaccine against YFV and to determine the immunogenicity of the DNA-launched pSinED-lll replicon, which would provide information regarding the applicability of DNA-launched replicons as an approach to vaccine development.
The pSinGFP replicon encoding the green fluorescent protein (GFP) was kindly provided by Prof. Mark Heise. Expression of the GFP was confirmed in mammalian cell culture post-transfection with the pSinGFP replicon, thus confirming the functioning of the replicon elements and subsequent expression of the encoded protein. The gene encoding the GFP was excised and replaced with a synthesised codon-optimised gene encoding the YFV ED-lll protein using directional cloning. The nucleotide sequence was confirmed by bidirectional sequencing at the site of insertion and subsequently protein expression was confirmed in selected mammalian cell lines. Protein expression was confirmed by detection of the C-terminal histidine tag by immunofluorescence using anti-His6 mouse monoclonal antibody. Thereafter, the expressed protein was characterised by immunofluorescence using mouse immune sera previously shown to contain anti-YFV ED-lll antibody. Reactivity of the expressed protein with anti-YFV ED-lll antibody substantiated the use of the pSinED-lll replicon in a mouse immunisation study.
Mice were immunised intramuscularly according to pre-approved immunisation regimes consisting of DNA only, as well as mixed modal immunisation regimes. Serum samples were collected and spleens were harvested two weeks after the administration of the final immunisations. Serum samples were screened for anti-YFV antibodies by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to determine the induction of a humoral immune response. Five of the twenty mice in groups immunised with the DNA-launched pSinED-lll replicon developed low level antibody responses illustrating the potential of the pSinED-lll replicon to induce a humoral immune response. Splenocytes were stimulated in cell culture with concanavalin A, YFV ED-lll protein, or no stimulant to facilitate the detection of cytokine release using ELISA’s. A predominantly T-helper 1 cell-mediated immune response characterised by high level release of interferon-γ post-stimulation with the YFV ED-lll protein in vitro was elicited. The release of interleukin-10 post-stimulation may be associated with the prevention of immunologically mediated damage to the host by preventing an overzealous inflammatory immune response. Antibody-mediated anti-vector immunity should not negatively impact the immunogenicity of the DNA-launched pSinED-lll replicon as antibody directed against the encoded Sindbis virus non-structural proteins was not detected in mouse sera.
In conclusion, the pSinED-lll replicon was shown to have the potential to induce both a cell-mediated and a humoral immune response post-immunisation. However, optimisation of the humoral immune response will be required.Afrikaans: Geelkoorsvirus (GKV), ‘n muskiet-oordraagbare virus wat behoort tot die familie Flaviviridae en genus Flavivirus, is ‘n belangrike oorsaak van morbiditeit en mortaliteit in geelkoors endemiese gebiede, veral in Wes-Afrika. In die mens veroorsaak GKV, geelkoors, ‘n siekte wat gekenmerk word deur nierversaking, geelsug en/of bloeding. Die las van die siekte is die hoogste in Afrika waar ongeveerd 90% van aangemeldte gevalle wêreldwyd voorkom. Ten spyte van die beskikbaarheid van hoogs effektiewe lewend verswakte entstowwe teen GKV is die beraamde voorkoms van geelkoors in Afrika vir 2013 steeds 130 000 ernstige gevalle en 78 000 sterftes. Die beskikbare lewend verswakte entstowwe is teenaangedui vir gebruik in immuunonderdrukte pasiënte en individue met eier en/of hoenderallergieë. Entstof-verwante neurotropiese newe-effekte wat lei tot die ontwikkeling van meningoenkefalitis/breinvliesontsteking in babas en entstof-verwante viserotropiese newe-effekte wat lei tot siekte wat natuurlike infeksie met GKV naboots is aangemeld. Entstof-verwante viserotropiese newe-effekte word geassosieer met ‘n sterftekoers wat 40% oorskry. Daar is dus ‘n behoefte vir ‘n veiliger alternatief wat die gebruik van die beskikbare lewend verswakte entstowwe kan aanvul. Die doel van hierdie studie was die voorbereiding van ‘n DNS-geloodsde replikon teen GKV en om die immunogenisiteit van die DNS-geloodsde pSinED-lll replikon te bepaal wat insae sal verskaf tot die toepaslikheid van DNS-geloodsde replikons vir entstof ontwikkeling.
Die pSinGFP replikon enkodeer die groen fluoresserende proteïen (GFP) en is goedkunstiglik deur Prof. Mark Heise verskaf. Die uitdrukking van die GFP is bevestig in geselekteerde soogdierselle na transfeksie met die pSinGFP replikon en gevolglik was die werking van die replikon elemente en die uitdrukking van die kodeerde proteïen bevestig. Die geen wat die GFP kodeer was verwyder uit die pSinGFP replikon en vervang met ‘n vervaardigde kodon-geoptimiseerde geen wat die GKV omhulsel domain lll (OD-lll) proteïen kodeer met behulp van gerigte klonering. Die nukleotiedvolgorde was bevestig deur tweerigting volgordebepaling in die gebied wat die ingevoegde geen bevat. Daarna was die uitdrukking van die kodeerde proteïen bevestig in geselekteerde soogdierselle. Proteïen uitdrukking was bevestig deur die opsporing van die C-terminale histidien merker met behulp van teen-His6 muis monoklonale teenliggame. Die uitgedrukte proteïen is daarna gekarateriseer deur immunofluoressensie met muis immuunserum wat voorheen bepaal is om teen-GKV OD-lll teenliggame te bevat. Reaktiwiteit van die uitgedrukte proteïen met teen-GKV OD-lll teenliggame het die gebruik van die DNS-geloodsde pSinOD-lll replikon vir ‘n muis immunisering studie bekragtig. Muise was binnespiers ingeënt volgens ‘n vooraf goedgekeurde immuniseringskema wat bestaan het uit slegs DNS, sowel as gemengde modale immuniseringskemas. Serum monsters was ingesamel en die milt was asepties verwyder twee weke na die toediening van die finale inentings. Teenliggame teen GKV was opgespoor in serum monsters met behulp van ‘n ensiem-gekoppelde immunosorbent toets (ELISA) en immunofluoressensie om te bepaal of ‘n humorale immuunrespons ontlok was. In 5/20 muise in groepe ingeënt met pSinOD-lll replikon DNS was ‘n lae vlak teenliggaam respons opgespoor wat die potensiaal van die pSinOD-lll replikon om ‘n humorale immuunrespons te ontlok illustreer. Om die opsporing van sitokiene met behulp van ‘n ELISA te fasiliteer was miltselle met konkanavalien A, GKV OD-lll proteïen of met geen stimulant gestimuleer in selkultuur. ‘n Oorwegend T-helper 1 sel-bemiddelde immuunrespons gekenmerk deur ‘n hoë vlak van interferon-γ vrystelling was bepaal na stimulasie met die GKV OD-lll proteïen in vitro. Die vrystelling van interleukin-10 na stimulasie kan moontlik geassosieer word met die voorkoming van immunologies bemiddelde skade aan die gasheer deur die voorkoming van ‘n opruiende inflammatoriese immuunrespons. Teenliggaam-bemiddelde teen-vektor immuniteit behoort nie die immunogenisiteit van die DNS-geloodsde pSinOD-lll replikon te affekteer nie, aangesien geen teenliggame teen die kodeerde Sindbis virus nie-strukturele proteïene opgespoor was in die serum van die muise nie.
Ten slotte was die potensiaal van die DNS-geloodsde pSinOD-lll replikon om ‘n sel-bemiddelde en ‘n humorale immuunrespons na inenting te ontlok gedemonstreer. Die humorale immuunrespons wat ontlok was sal egter versterk moet word.National Health Laboratory Service Research TrustNational Research Foundation (NRF)Poliomyelitis Research FoundationUniversity of the Free State, School of Medicin
The development and validation of a reverse transcription recombinase polymerase amplification assay for detection of flaviviruses
No full textFlaviviruses have been of clinical importance since ancient times. Five flaviviruses are known to occur or have been identified historically in South Africa (SA) namely, West Nile virus (WNV), Usutu virus (USUV), Wesselsbron virus (WSLV), Spondweni virus (SPOV) and Banzi virus (BANV). Medically significant flaviviruses, WNV and WSLV, are known to occur annually in SA. Development of isothermal assays, such as recombinase polymerase amplification (RPA), plays an important role in performing surveillance studies and increasing diagnostic capacity for emerging viral pathogens in limited resource settings. In their native form, WNV and WSLV can only be handled in a biosafety laboratory level 3 and this restricts laboratories that lack such resources, hence transcribed RNA controls were successfully prepared for WNV, USUV and WSLV to develop and validate a RT-RPA for the detection of flaviviruses. A lateral-flow RT-RPA was developed by identifying theoretical cross reactivity between the probe and primer candidates by sequence alignments of the conserved NS5 protein of WNV, USUV and WSLV. It was determined that a few mismatches were present between WNV and USUV in the probe binding region and in the reverse primer, as well as between WNV and WSLV, hence different probe and reverse primer regions were identified for WNV/USUV and WSLV. A limitation of the study was the selection of a reference strain of WNV belonging to lineage 1 as a lineage 2 isolate would have been a more suitable representative of SA lineages. Nonetheless, RNA from SA isolate 93/01 was amplified using the RT-RPA. The sensitivity of the assay was determined by diluting RNA control ten-fold, and was found that the WNV RT-RPA could detect WNV and USUV transcribed RNA diluted 109 fold, whereas the WSLV RT-RPA detected WSLV transcribed RNA diluted 1010 fold. Testing RNA from other arboviruses suggested that despite the binding tolerability of the assay there was good specificity as no other arboviruses were amplified. However because of similarity in sequence data, USUV transcribed RNA was detected with the WNV RT-RPA and WSLV RT-RPA. Theoretical cross reactivities with other flaviviruses were determined by sequence alignments of the NS5 region and it was proposed that Japanse encephalitis virus (JEV), Zika virus (ZIKV) and dengue virus (DENV) RNA will not be detected by either the WNV RT-RPA or WSLV RT-RPA. Seventeen pools of Culex spp mosquitoes were screened for flavivirus RNA, although no flaviviruses were expected within such a small cohort. Lack of amplification inhibitors in the mosquito samples was confirmed by spiking known negative mosquito samples with transcribed flavivirus RNA and performing a RT-RPA using the spiked samples. Detecting WNV by RT-RPA will not only be useful for surveillance studies in SA, but can also be used as a diagnostic tool for veterinary diagnostics, especially equine. In conclusion, the development and validation of a RT-RPA for the detection of WNV, WSLV and USUV flaviviruses was successful. The RT-RPA proved to be a robust, rapid and sensitive assay that might have potential as a diagnostic tool in the field or resource limited settings.National Research Foundation (NRF)Poliomyelitis Research Foundation (PRF
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