Medical Microbiology

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Browsing Medical Microbiology by Author "Burt, Felicity"

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    ItemOpen Access
    Adaptive immune response in COVID-19 patients and innate immune modulation of SARS-CoV-2
    (University of the Free State, 2023) Litabe, Matefo Millicent; Burt, Felicity
    In 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.
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    Immunogenicity and serological applications of flavivirus ED III proteins and multiplex RT-PCR for detecting novel Southern African viruses
    (University of the Free State, 2015-01) Mathengtheng, Lehlohonolo; Burt, Felicity; University of the Free State, Grow Our Own Timber Fellowship
    English: West Nile virus (WNV) is endemic to southern Africa but the true burden of disease associated with WNV infection remains unknown in this region. The presence of the mosquito-borne Wesselsbron virus (WESSV) has also been established in southern Africa. Although not considered a serious human pathogen, WESSV has been associated with encephalitis in humans. No routine testing is performed for WESSV diagnosis in South African patients and hence, similar to WNV infections, the virus remains unreported and overlooked. The presence of tick-borne flaviviruses in southern Africa on the other hand, has not been established despite the presence of suitable vectors. A challenge associated with serological identification of flaviruses is the high level of cross-reactivity between members of flaviviruses and the impracticality of using neutralization assays. Serological assays using reagents that can be handled in a biosafety level 2, or lower facility, were developed and evaluated for the detection and differentiation of tick- and mosquito-borne flaviviruses in the Free State province of South Africa. A total of 2393 serum samples from a variety of species including humans, cattle and sheep were tested using Kunjin virus (KUNV) cell lysate antigen for the detection of anti-flavivirus antibodies in an indirect IgG enzyme-linked immonosorbent assay (ELISA). To further differentiate positive reactors on KUNV assay for antibodies against tick- or mosquito-borne flaviviruses, recombinant envelope domain III (r-EDIII) proteins of Langat virus (LGTV), WNV and WESSV were expressed in a bacterial expression system and used in ELISA. A total of 722 samples were positive on the KUNV assay of which 71, 457 and 431 were positive on the r-LGTVEDIII, r-WNVEDIII and r-WESSVEDIII assays, respectively. A total of 70 samples were reactive on the KUNV assay but not on any of the other assays, suggesting that there are other flaviviruses circulating in the Free State province for which specific r-EDIII assays were not available. Collectively, the results suggest a strong presence of flaviviruses co-circulating in the Free State province with an abundance of mosquito-borne flaviviruses. There is evidence suggesting the presence of tick-borne flaviviruses but it has yet to be confirmed. The EDIII protein is a useful tool that can be utilized in the detection and differentiation of flaviviruses in resource-limited laboratories. Vertebrate hosts play a role in the maintenance and circulation of flaviviruses and, although not involved in the direct transmission of tick- and mosquito-borne flaviviruses, form a link for virus transmission between vectors. In addition to rodent involvement in maintenance of flaviviruses, rodents have also been implicated in the transmission of other medically significant viruses such as arenaviruses, lyssaviruses and hantaviruses. Arboviruses and viral heamorrhagic fevers are among the most pathogenic and devastating disease agents in many parts of the world. It is therefore important for surveillance of such pathogens to be conducted as they may result in considerable public health implications. Molecular assays were developed for the detection of a selected number of arboviruses and viral heamorrhagic fevers, specifically Crimean-Congo haemorrhgaic fever virus (CCHFV), mosquito-borne and tick-borne flaviviruses, as well as hantaviruses. To date, the presence of hantaviruses have not been confirmed in southern Africa despite their emergence in the western and eastern parts of Africa in recent years. In our study, serum samples of patients presenting with a tick-bite and febrile illness without diagnosis were screened for hantavirus IgG antibodies using commercial assays that represent the American and Eurasian hantavirus species. The overall seropositivity rate obtained was 10% and 6% for assays representing the Eurasia and America hantavirus species, respectively. The emergence of hantaviruses in Africa and their seroprevalence in the Cape region of South Africa as well as in our study warranted the development of a molecular assay to further investigate the presence of these viruses in southern Africa. In order to achieve this, a real-time RT-PCR was designed and optimized. The assay was designed by identifying in-house primers targeting the partial region of the S-segment of hantaviruses and hydrolysis probes targeting the inner region of the amplicon. The probes were based on nucleotide sequences targeting the Murinae-associated hantaviruses for the HNLS probe, Sigmodontinae- and Arvicolinae-associated hantaviruses for the ASPRB probe, as well as the SANGV probe for the African hantavirus Sangassou virus. The flavivirus RT-PCR targeted the NS5 region with a probe shown to successfully detect RNA samples that represent eight different flavivirus species. The hantavirus primers and probes were evaluated using RNA transcribed from synthetic genes representing the different hantaviral genotypes and subsequently reverse transcribed cDNA. The limit of detection was determined to range from ~160 to ~17 copies of DNA for the various hantaviral probes and flavivirus probe. In addition, a conventional multiplex PCR assay aimed at detecting CCHFV and flavivirus RNA in samples collected from undiagnosed patients presenting with a tick-bite and febrile illness was developed by using nested primers targeting the partial region of the genome of the S-segment of CCHFV and hemi-nested primers targeting the partial region of the NS5 gene of flaviviruses. When clinical samples from patients with known tick-bites, mild disease and no diagnosis were screened, a patient was restrospectively diagnosesd as having a CCHFV infection. This result highlights the need for awareness to arboviruses and viral hemorrhagic fevers in mild cases that may easy be overlooked but constitute a significant public health risk. Similarly, there needs to be an increase in awareness for travelers to South Africa at risk of returning to their country with an exotic viral haemorrhagic fever, highlighting the need for increased awareness and increased diagnostic capacity for arboviruses. Finally the current lack of registered human vaccines warrants continued investigation of the immunogenicity of selected viral proteins. The recombinant antigens developed for serological purposes were further employed in this study to determine the immunogenicity of the envelope domain III proteins of WNV and LGTV in a mouse model. Small molecule antigens or weakly immunogenic antigens frequently require an adjuvant to stimulate a stronger immune response. In addition, adjuvants can shift an immune response towards a Th1 or Th2 response as required based on immune correlates of protection. Groups of mice were immunized with purified r-WNVEDIII or r-LGTVEDIII protein alone, r-WNVEDIII or r-LGTVEDIII protein in combination with one of three adjuvants, including saponin, Titermax® gold and Alhydrogel® or one of the three adjuvants without a flavivirus protein. In the absence of any adjuvant the results from WNV protein alone were inconclusive whereas a strong IgG1 response was induced by LGTV EDIII. Briefly, protein alone or mixed with alum elicited a predominantly Th2 response whereas protein in combination with saponin or Titermax® gold induced a mixed Th1 and Th2 response. Mice immunized with r-WNVEDIII reacted against KUNV native antigen indicating that the protein was expressed in conformation exposing epitopes that are required to induce a detectable antibody response. The formulation of the WNV and LGTV proteins with different adjuvants produced similar results with a shift in response depending on the adjuvant. Despite an absence of being able to assess cell mediated responses using antigen stimulated splenocytes and profiling cytokine production as initially planned, the results do confirm that r-WNVEDIII and r-LGTVEDIII proteins are immunogenic in the absence of complete E protein, with ability to induce detactable antibody when formulated with adjuvant and that different adjuvants are able to have an immunomodulatory influence on the type of response induced.
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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.