Medical Microbiology

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Browsing Medical Microbiology by Subject "Antigens"

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    Identification of antigen-specific serological cross-reactivity among survivors of Crimean-Congo Haemorrhagic fever
    (University of the Free State, 2013) Rangunwala, Azeeza; Burt, F. J.
    Abstract not available
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    ItemOpen Access
    Identification of antigenic regions and linear B cell epitopes on yellow fever virus
    (University of the Free State, 2013-02) Smouse, Shannon Lucrecia; Burt, F. J.
    English: Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.
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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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    Preparation of recombinant antigens for demonstrating antibody responses in patients with Crimean-Congo haemorrhagic fever virus infections
    (University of the Free State, 2011-06) Samudzi, Rudo Ruth; Burt, F. J.
    Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia, Russia and the Balkans. The causative agent, CCHF virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Cases of CCHF are diagnosed annually in southern Africa. Increasing numbers of cases are seen in regions of Asia and in the past ten years CCHFV has emerged in several countries in the Balkans and re-emergence in south-western regions of the Russian Federation. Diagnosis of CCHFV infections during the acute phase is based on isolation of the virus or amplification of viral RNA. Patients that survive the infection have a demonstrable IgG and IgM antibody response, usually from day 5 to 7 after onset of illness. Current serological diagnostic assays based on ELISA or IF use inactivated virus which requires biosafety level 4 facilities for culturing the virus and therefore limits the number of laboratories that can prepare suitable reagents. Preparation of recombinant antigens would enable laboratories to perform serological diagnosis of CCHFV infections and surveillance studies. The purpose of this study was to prepare a recombinant CCHFV nucleoprotein using a bacterial expression system, to determine if the protein was immunogenic and to determine if the protein was able to detect IgG antibodies in survivors of CCHFV infection. The complete open reading frame of the gene encoding the NP of CCHFV was amplified by RT-PCR using primers specifically designed with restriction sites engineered to the primers to facilitate cloning. The amplicon was cloned into pGEM® T Easy vector using T/A cloning and the gene sequenced to confirm that the correct gene had been amplified and cloned into the vector for downstream cloning and expression applications. Initially we aimed to express the native gene using a bacterial expression system and the NP gene was rescued from the recombinant plasmid and cloned into pQE-80L vector using the BamH1 and Pst1 restriction sites present in the multiple cloning site on the vector. Various attempts were made to express the CCHFV NP protein however no protein was detectable using SDS PAGE methods or Western blot. The nucleotide sequence that we had determined for the open reading frame of our gene encoding the NP was analysed using the Rare Codon Analysis Tool software and we elected to codon optimize the gene for expression in E. coli. The optimized gene was synthesized by GenScript and supplied cloned in the multiple cloning site of pUC57. The optimized gene was excised from pUC57 and cloned into pColdTF bacterial expression vector. A 106 kDa protein was expressed from the construct likely representing the HIS tagged TF chaperone protein fused to the CCHFV NP protein and confirmed by Western blot analysis. A higher yield of the protein was present in the insoluble phase and as optimization of the growth and induction conditions did not significantly alter the insoluble to soluble ratio of the expressed protein, the protein was harvested from the insoluble phase by denaturing, purification and refolding of the protein. The biological activity of the recombinant protein was confirmed using immunoassays and by immunizing mice to determine if the antibodies induced by the recombinant protein could be detected using an antigen prepared from the whole virus. Four of five mice immunized with the recombinant NP had a detectable antibody response using an immunofluorescent assay. Serum samples from acute and convalescent patients collected at varying stages after onset of illness were reacted in a Western blot with the recombinant CCHFV NP protein. The recombinant antigen was able to detect IgG antibody in all the convalescent patient sera except two sera collected on days 14 and 15 during the acute phase. In contrast all the samples were detected using the recombinant antigen in an ELISA. Due to the potential biohazardous nature of samples only samples collected two weeks after onset of illness were tested. The results showed 100% concordance with the results obtained in an ELISA using mouse brain derived antigen. The assay was shown to be reproducible and stability studies showed that four months after preparation the protein was still active. A full validation of the protein using a large panel of serum samples from confirmed CCHF patients is now required. The results suggest that bacterially expressed proteins lacking post translational modifications and folding that occur with mammalian and baculovirus expression can be used in ELISA to detect IgG antibody against CCHFV in human sera which finds application in diagnostics, epidemiologic and surveillance studies.