IMMUNO- CHEMOTHERAPEUTIC INTERACTIONS IN BOVINE ONCHOCERCIASIS Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Germanus Soh Bah July 2018 Summary Immuno-chemotherapeutic interactions in bovine onchocerciasis Germanus Soh Bah Onchocerciasis or river blindness is a debilitating skin disease caused by Onchocerca volvulus, with 15.5 million people afflicted in Africa. The adult stage of the causative agent, a filarial worm, resides in collagenous nodules and has a lifespan of 10 - 15 years. The control of onchocerciasis using annual mass drug administration with ivermectin targets the first-stage larvae (microfilariae, Mf). However, ivermectin fails to kill adult worms, and the treatment is contraindicated in individuals co-infected with another filarial nematode (Loa loa) due to the risk of severe adverse events. Whereas antibiotics of the tetracycline class can be adulticidal, treatment protocols are too long for widespread use. Tetracyclines target the obligate endobacterial symbiont of O. volvulus (Wolbachia), but the precise mechanism of worm killing is not fully defined. Previous data obtained from a bovine model of onchocerciasis (Onchocerca ochengi) indicate that worm death is associated with an immunological shift in the nodule from a neutrophil-dominated response to a local eosinophilia. We attempted to boost eosinophil responses against adult worms by vaccination preceding sub-lethal antibiotic therapy (SLT) in cattle. We assessed for the first time the effect of immunisation against an immunomodulatory antigen, onchocystatin, either alone or in combination with SLT, on skin Mf load, worm viability and intradermal nodule size at six time-points over one year. Systemic antibody and tissue eosinophil responses to vaccination were also evaluated. The effects of oxytetracycline on the female worm proteome and nodular transcriptome were analysed with reference to the O. ochengi, Wolbachia and bovine genomes. Immunochemotherapy was neither micro- nor macrofilaricidal, although significant levels of serum IgG and nodule eosinophilia were stimulated. “Gold standard” prolonged adulticidal therapy (ADT) induced a 60% reduction in adult worm viability after 52 weeks, while SLT was ineffective. Using label-free proteomics, 50% of 2,548 identified proteins were quantifiable between antibiotic-treated and control female worm samples. Wolbachia constituted <1% of these proteins, while the remainder were distributed between O. ochengi (30%) and bovine proteins on the worm surface (70%). The majority of the 203 differentially-regulated proteins of bovine origin were significantly enriched for cathelicidin, annexin, serpin, S- 100/ICaBP calcium binding, and Ras domains. Those associated with neutrophils were downregulated, whereas an eosinophil major basic protein homologue was upregulated, by the ADT regimen. Worm cuticle proteins were upregulated and all Wolbachia proteins were I downregulated by 36 weeks post-treatment. Applying RNA-seq to whole nodules, transcripts were assigned to each organism (68% worm, 28% bovine, 4% Wolbachia). In the ADT group, genes regulating apoptosis in bovine mitochondria (VDAC1) and worm neurons (CES-1 and CES-2) were highly expressed at early time-points. Filarial heat-shock proteins and innate immunity receptors were also upregulated, but there was reduced expression of the bovine NKG2D ligand 4 in the ADT group. Conversely, SLT upregulated the production of this ligand and other elements of the bovine immune response. Finally, Wolbachia initially upregulated stress-induced morphogen bolA and response regulator pleD alongside energy metabolism; but the latter was downregulated by the end of the experiment. In conclusion, prior immunisation failed to increase the efficacy of SLT in the O. ochengi model. Killing of adult worms following the ADT regimen was associated with decreases in neutrophil antimicrobial proteins, upregulation of an eosinophil granule protein and apoptotic events in both bovine cells and the worm. Disruption of Wolbachia energy metabolism and dysregulation of filarial innate immunity may also underlie the fatal effects of antibiotic treatment on O. ochengi. II Acknowledgements I am very grateful to Dr Vincent N. Tanya who recruited me into the filarial research consortium to fill in a gap created by Dr Emmanuel Niba who emigrated to the USA in January 2007. Originally lackadaisical, l accepted the offer, but worked without reserve towards the success of EU projects SCOOTT 2 and 3. I only became interested in filarial research when Dr Benjamin L. Makepeace, my primary supervisor and Dr Vincent N. Tanya, my co-supervisor encouraged me to enrol in a part-time PhD program at the University of Liverpool to be supported with funds from the EU project EPIAF project. Ever since then, my research ambition changed from Veterinary Theriogenology to Parasitology and things have never been the same again. I am also very thankful to Prof. Jonathan M. Wastling, my secondary supervisor, but would have wished to have had more time with him. He facilitated my enrolment into the programme and empowered Ben to guide me throughout this study. I am very grateful to the University of Liverpool for putting in place an advisory panel to accompany and independently evaluate my research performance. Prof. Diana Williams and Prof. Alan Radford were members of my Independent Progress Assessment Panel. They showed understanding of my problems and advised me on time management, as well as enlightening me on my responsibilities. For this, I am very grateful. Working in the labs both in the UK and back in Cameroon has been very challenging. Dr Stuart D. Armstrong mentored me on proteomics while Drs Alistair Darby, Dong Xia and Ritesh Krishna assisted and guided me through the processing and quantification of transcriptomics data. For these, l am very grateful to them. The first postdoc researcher I had to share laboratory bench space with at the University of Liverpool was Dr Krystyna Cwiklinski, with whom I attended optional courses on bioinformatics and post genomic technology. She was always keeping an eye on what l was doing and would not hesitate to remind me of the rules while in the laboratory. Consequently, I caused no accident while in the lab; neither was there any serious III contaminations of my samples or our bench space. I am grateful for encouragement received from Paul Gilmore in the Vet. Parasitology and Dr Mariwan Mohammed in the proteomics laboratory. Above all, I am very grateful to the laboratory boss, Catherine Hartley, for providing me with all the safety tips and technical support needed to sail through. She also coordinated the reception and processed all shipment of my samples from Cameroon. In Cameroon l was supported in the lab by Mme Wachong Kum Ngangyung Hendrietta, Mr Mfopit Youssouf and Mr David Ekale. I am very grateful for their help in the evaluation of worm viability, skin microfilarial count and measurements of nodule diameter. The administrative staff of IRAD Wakwa was led by Dr Messine Ombionyo (2010 – 2013) and late Dr. Abubakar Danjouma (2013 – 2015). They facilitated my fieldwork and endorsed my travel documents. Dr Venasius Lenzemo, the Chief of IRAD Bambui (2015 - 2018) showed a lot of support as l was finalising my studies. Field studies were carried out with the support of Mr Nana Soulemanou (night guard), Mr. Aminou Djida (of blessed memory); the herdsman of the experimental herd. Herdsmen of the IRAD Wakwa Centre, Mr Bobbo Ahmadou, Mr Laminou Mohamadou and Aminou assisted in the tedious exercise of animal restrain during sampling. Mr Hamman Saidou contributed in negotiating the purchase and transportation of the research cattle. Above all, I am particularly grateful to the Director General of IRAD, Dr Woin Noé, for his support towards the completion of this thesis. My family was in the greatest financial needs while undertaking this study. My junior Brother, Eng. Ernest Forkwa in Nigeria had to miss an academic year because we could not pay his fees while Dr Peter Nji completed his medical course in Accra thanks to support in form of financial loan from one of his university professors. I am grateful to God for enabling them to successfully complete their courses and most importantly for keeping us united as a family thereafter. Without this show of solidarity, particularly on the part of my wife (Mah Nancy Akam); Dad (Mr Bah John Nduh); Mum (Ma Odilia Tifuh Nduh); brothers (Eng. Gerald Ngati Bah and wife Monique, Mr Vitalis Ngwa Bah and wife, Eng., Ernest Forkwa Bah and wife, Mr. Paul IV Chick Bah and wife and Dr Peter Nji Bah and wife); sisters (Mrs Mandeng Regina Ngum and husband, Mrs Fonjong Immaculate Anwi and husband and Miss Emmanuella Muyang Bah); and my children (Tifuh Joy Soh, Akohnwi Pedro Akam Soh, Florence-Nightingale Akwi Soh and Amalia Anwi Soh) l could not have made it. Special thanks go to the Makepeace family (Jenny the wife, and kids Jude and Lois) for the programmed shared meals at the beginning and end of each of my visits to Liverpool. Jenny would come pick me up from my residence and return me to my home at the end. My Cameroonian friends were barrister Gabriel Achunkwe and wife Emeline Mufo; Mme Evelyn Falloh; Michael Ngando alias Locoman; Dr Willy Mbafor and wife Frida; the larger Cameroonian community in Liverpool and Dr Gerald Tata, transporter of extra house luggage back to Cameroon. I am indebted to my Landlord (J Moss Ltd) for the immense support in facilitating the processing of my travel documents and provision of accommodation. I cannot end this page without listing some of the students with whom l schooled. Drs Patrick Craig, Elsa-Gayle Zekeng, Nicola Beesley, Alison Howell, Walsh Tessa, Lisa Luu and John Graham-Brown, etc. This study was sponsored by a European Union collaborative research project known by the acronym EPIAF (Enhanced Protective Immunity against Filariasis). I also received financial support from the Bill and Milanda Gates Foundation via the biomarker (BMGF1) project and NIH grants to cover part of my extended study period.