PREPARATION OF RECOMBINANT ANTIGENS FOR DEMONSTRATING ANTIBODY RESPONSES IN PATIENTS WITH CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS INFECTIONS Rudo Ruth Samudzi 2011 University of the Free State Bloemfontein Campus PREPARATION OF RECOMBINANT ANTIGENS FOR DEMONSTRATING ANTIBODY RESPONSES IN PATIENTS WITH CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS INFECTIONS Rudo Ruth Samudzi B.Med.Sc Dissertation submitted in fulfilment of the requirements for the degree Master of Medical Science at the University of the Free State Promoter: Prof F.J. Burt Department of Medical Mircobiology and Virology, Faculty of Health Sciences, University of the Free State University of the Free State, Bloemfontein Campus June 2011 TABLE OF CONTENTS   DECLARATION ....................................................................................................................................................... v  ACKNOWLEDGEMENTS ..................................................................................................................................... vi  ABSTRACT .............................................................................................................................................................. 1  CHAPTER 1 ............................................................................................................................................................. 3  LITERATURE REVIEW .......................................................................................................................................... 3  1.1 Introduction and history ............................................................................................................................ 3  1.2 Virus classification, characteristics and biology ................................................................................... 3  1.3 CCHFV reservoirs and vectors .............................................................................................................. 7  1.4 Epidemiology ........................................................................................................................................... 10  1.5 Clinical manifestations............................................................................................................................ 13  1.6 Clinical pathology and pathogenesis .................................................................................................. 14  1.7 Laboratory diagnosis of CCHF .............................................................................................................. 16  1.8 Differential diagnosis .............................................................................................................................. 19  1.9 Prevention, treatment and control ........................................................................................................ 21  1. 10 Problem identification, aims and objectives..................................................................................... 22  CHAPTER 2 ........................................................................................................................................................... 25  CLONING AND SEQUENCE ANALYSIS OF THE GENE ENCODING THE NUCLEOPROTEIN OF CCHFV SPU415/85 .................................................................................................... 25  2.1 Introduction ............................................................................................................................................. 25  2.2 Materials and Methods .......................................................................................................................... 25  2.2.1 Viral RNA ............................................................................................................................................. 25  2.2.2 Primers ................................................................................................................................................. 26  2.2.3 One step Reverse Transcriptase Polymerase Chain Reaction ................................................... 26  2.2.4 Agarose gel electrophoresis ............................................................................................................. 27  2.2.5 DNA purification .................................................................................................................................. 27  2.2.6 Concentration of DNA ......................................................................................................................... 28  2.2.7 Cloning of gene encoding NP into pGEM® T Easy bacterial vector using TA cloning ............. 28  2.2.8 Preparation of chemically competent cells ...................................................................................... 31  2.2.9 Ligation reactions ................................................................................................................................ 31  2.2.10 Transformation of chemically competent JM109 cells ................................................................. 32  i 2.2.11 Plasmid purification ........................................................................................................................... 33  2.2.12 Restriction enzyme digests .............................................................................................................. 34  2.2.13 DNA sequencing of CCHFV NP gene in pGEM®T Easy vector ................................................ 35  2.3 Results ...................................................................................................................................................... 36  2.3.1 One step RT-PCR for amplification of the gene encoding the NP of CCHFV SPU 415/85 ..... 36  2.3.2 A/T cloning of the CCHFV SPU415/85 NP amplicon into pGEM® T Easy vector .................... 38  2.3.3 Sequencing and sequence analysis of the gene encoding CCHFV SPU415/85 in pGEM®T Easy vector ......................................................................................................................... 40  2.4 Summary .................................................................................................................................................. 44  CHAPTER 3 ........................................................................................................................................................... 45  BACTERIAL EXPRESSION OF RECOMBINANT CCHFV NP FROM NATIVE AND CODON OPTIMIZED GENES ............................................................................................................................................. 45  3.1 Introduction .............................................................................................................................................. 45  3.2 Materials and methods ........................................................................................................................... 47  3.2.1 Cloning of CCHFV NP gene into pQE-80L bacterial expression vector using restriction sites ....................................................................................................................................................... 47  3.2.2 DNA sequencing of CCHFV NP gene in pQE-80L vector ............................................................. 49  3.2.3 Bacterial expression of pQE-80L-CCHFV NP using IPTG induction ......................................... 50  3.2.4 SDS-Polyacrylamide gel electrophoresis ........................................................................................ 51  3.2.5 Western blot analysis of His tagged CCHFV NP ............................................................................ 52  3.2.6 Codon optimization of CCHFV NP gene .......................................................................................... 53  3.2.7 Cloning of gene encoding codon optimized CCHFV NP from pUC57 into pCold TF bacterial expression vector ................................................................................................................ 54 3.2.8 Plasmid purification ............................................................................................................................. 57  3.2.9 DNA sequencing of pColdTF-opCCHFV NP ................................................................................... 58  3.2.10 Bacterial expression of pColdTF-opCCHFV NP using IPTG induction .................................... 58  3.2.11 Protein solubility ................................................................................................................................ 59  3.2.12 Denaturation, purification and refolding of recombinant His-tagged proteins from the insoluble phase .................................................................................................................................. 60  3.2.13 Concentration of proteins ................................................................................................................. 61  3.2.14 Characterization of expressed His tagged opCCHFV NP by Western blot analysis .............. 62  3.3 Results ...................................................................................................................................................... 63  3.3.1 Cloning of the CCHFV nucleoprotein gene in pQE-80L bacterial expression vector ............... 63  3.3.2 Sequencing and sequence analysis of the gene encoding CCHFV SPU415/85 in pQE-80L vector ..................................................................................................................................................... 64  ii 3.3.3 Bacterial expression of pQE-80L-CCHFV NP using IPTG induction .......................................... 67  3.3.4 Codon optimization of CCHFV NP gene .......................................................................................... 68  3.3.5 Cloning of gene encoding the codon optimized NP from pUC57 into pCold TF bacterial expression vector ................................................................................................................................ 77  3.3.6 Sequencing and sequence analysis of the gene encoding CCHFV SPU415/85 in pCold TF vector ..................................................................................................................................................... 79  3.3.7 Bacterial expression of pColdTF-opCCHFV NP using IPTG induction ....................................... 82  3.3.8 Solubility, purification and characterization of His-tagged proteins ............................................. 83  3.4 Summary .................................................................................................................................................. 87  CHAPTER 4 ......................................................................................................................................................... 88  FUNCTIONAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT CCHFV NP USING A CODON OPTIMIZED GENE AND EVALUATION OF THE ANTIGEN FOR DETECTION OF ANTI-CCHFV IgG ANTIBODY.................................................................................. 88  4.1 Introduction .............................................................................................................................................. 88  4.2 Materials and Methods ........................................................................................................................... 91  4.2.1 Human serum samples ....................................................................................................................... 91  4.2.2 Western blot analysis for detection of anti-CCHFV IgG in human sera ...................................... 92  4.2.3 Immunization of mice with recombinant CCHFV NP protein ........................................................ 92  4.2.4 Detection of anti-CCHFV IgG antibody in immunized mice by IFA ............................................. 93  4.2.5 Enzyme-linked immunoassays .......................................................................................................... 94  4.2.5.1 IgG ELISA using recombinant antigen .......................................................................................... 94  4.2.5.2. IgG ELISA using whole virus mouse brain derived antigen ..................................................... 95  4.2.6 Repeatability ......................................................................................................................................... 96  4.2.7. Selection of cut-off values ................................................................................................................. 96  4.2.8 Determination of antibody titers against CCHFV in convalescent sera ...................................... 96  4.2.9 Sensitivity of IgG ELISA using recombinant antigen ..................................................................... 97  4.2.10 Stability of bacterially expressed pColdTF-opCCHFV NP .......................................................... 98  4.2.11. Statistical analysis of data .............................................................................................................. 98  Normalization of data .................................................................................................................................... 98  4.3 Results ...................................................................................................................................................... 99  4.3.1 Induction of humoral antibody response using recombinant CCHFV NP ................................... 99  4.3.2 Detection anti-CCHFV IgG antibody in human sera ...................................................................... 99  4.3.2.1. Western blot analysis ..................................................................................................................... 99  4.3.2.2 IgG ELISA using recombinant antigen ........................................................................................ 101  4.3.2.2.1 Selection of cut off ...................................................................................................................... 102  4.3.2.2.2. Stability of bacterially expressed recombinant NP ............................................................... 103  iii 4.3.2.2.3. Interassay variability of recombinant antigen ELISA ............................................................ 104  4.3.2.2.4. Detection of IgG antibodies in human sera using ELISA .................................................... 105  4.3.2.2.5. Sensitivity of the recombinant NP antigen ............................................................................ 108  4.4 Summary ................................................................................................................................................ 109  CHAPTER 5 ....................................................................................................................................................... 110  DISCUSSION .................................................................................................................................................... 110  REFERENCES .................................................................................................................................................. 116  APPENDIX 1 ........................................................................................................................................................ 126  ELISA RAW DATA ...................................................................................................................................... 126  APPENDIX 2 ........................................................................................................................................................ 130  LIST OF FIGURES ...................................................................................................................................... 130  LIST OF TABLES ........................................................................................................................................ 133  LIST OF ABREVIATIONS .......................................................................................................................... 135  APPENDIX 3 ..................................................................................................................................................... 136  Title and abstract of paper to be submitted to Journal of Virological Methods for review ................ 136  APPENDIX 4 ........................................................................................................................................................ 137  Title and abstract of presentation at the South African Society for Biochemistry and Molecular Biology (SASBMB), 18-20th January 2010, Bloemfontein ................................................................... 137  APPENDIX 5 ........................................................................................................................................................ 139  Title and abstract of poster presented at the 13th International Congress on Infectious Diseases (ICID) 9 – 12th March 2010, Miami, USA.................................................................................................. 139  APPENDIX 6 ........................................................................................................................................................ 140  Title and abstract of presentation at the Faculty of Health Sciences AstraZeneca, Research Forum 27-28th August 2010, University of the Free State, Bloemfontein .......................................... 140       iv DECLARATION I declare that the dissertation hereby submitted by me for the M.Med.Sc (Virology) degree at the University of the Free State, Bloemfontein, is my own independent work and has not been previously submitted by me at another institution/faculty. I further more cede copyright in favour of the University of the Free State. Rudo Ruth Samudzi 08/06/2011       v ACKNOWLEDGEMENTS I would like to thank the following persons and institutions: • Prof Felicity. J. Burt for her wonderful work in supervising this project. Thank you for your willingness to help me whenever I encountered problems. Thank you for your encouragement and scientific knowledge that added quality to the project. I have really learned a lot and am looking forward to learning more. • The Department of Medical Microbiology and Virology, Faculty of Health Sciences for providing the facilities that enabled me to complete this project. • The Polio Research Foundation for financial assistance throughout the period of my study. • My colleagues and friends for support not only academically but also on a personal level. Thank you to Lehlohonolo Mathengtheng, Kulsum Kondiah, Shannon Smouse, Mitta Mamabolo, Manie Hanekom, special thanks to Azeeza Rangunwala for lending me your laptop to type up, to Carina Combrinck and Arina Jansen for assistance with editing, my childhood friends Beatrice Kyambadde, Eriva Kyambadde, Grace Mukasa, Rita Buanyomi for their support. To all those I have not mentioned, you’re not forgotten. Thank you. • My parents and siblings for their prayers and faith in me. I will always appreciate you. • My boyfriend, Vernon Mwazi for always being there for me even at the most stressful moments. Your motivation, love and faith in me has made this possible. • God, my Saviour and Lord who has carried me through this challenging time and blessed me in all aspects of my life. vi ABSTRACT 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. 1 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. 2