COLLEGE OF BASIC AND APPLIED SCIENCES SCHOOL OF BIOLOGICAL SCIENCES CONSTRUCTION OF POTENT IMMUNOGENIC EPITOPES OF THE HAEMAGGLUTININS OF THE SEASONAL INFLUENZA A VIRUSES BY ERASMUS NIKOI KOTEY (10300632) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF PHD IN MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE. JANUARY 2021 University of Ghana http://ugspace.ug.edu.gh i DECLARATION This work was done under the guidance of Professors Osbourne Quaye, William Kwabena Ampofo and Munir Iqbal. I hereby declare that the results in this submission are from my research work and illustrations from work done by others are duly acknowledged. I declare also that the content of my work has not been submitted to any other academic institution. ………………………………… ………………………………… ERASMUS NIKOI KOTEY PROF OSBOURNE QUAYE (CANDIDATE) (PRINCIPAL SUPERVISOR) DATE: January 29, 2021 DATE: January 29, 2021 ………………………………… ………………………………… PROF WILLIAM AMPOFO PROF MUNIR IQBAL (SUPERVISOR) (SUPERVISOR) DATE: January 29, 2021 DATE: ……………………… University of Ghana http://ugspace.ug.edu.gh ii ABSTRACT Seasonal influenza viruses are renowned for recurring annual epidemics worldwide. The Influenza A subtypes H1 and H3 are the most dominant and prevalent in recent outbreaks in humans. As with other infectious diseases, vaccines are an important public health tool. However, influenza viruses continue to evolve evading pre-existing or transient vaccine- induced immunity in addition to antigenic pressures associated with antiviral drugs. For this reason, current seasonal influenza vaccines require annual review. Vaccine (immunogen) design, efficacy, and effectiveness presents a formidable seasonal influenza management issue. Passive immunotherapy has been proposed to offer tremendous protection when appropriately used in the management of influenza, either as a substitute or to complement vaccines. A well- designed immunogen that elicits a strong antibody response towards the conserved domains of the surface Haemagglutinin (HA) protein would be critical to avert virus evolution. A detailed analysis of the highly conserved regions spanning the fusion peptide, cleavage site, and the two heptad repeats for the HA gene in over 1000 and 21,000 H1 and H3 strains, respectively was therefore conducted. Chimeric haemagglutinins (cHAs) of these conserved regions were constructed by alignment with consensus sequences generated from exotic HAs (H5 and H9 for H1-based cHAs; H7 for H3-based cHA). These cHAs were successfully expressed in Drosophila S2 cell lines. Mice were then immunized with these cHAs to determine protection against lethal doses in virus challenges against H1 and H3 seasonal viruses. Serum from seroconverted mice applied in challenge experiments indicated the presence of anti-HA specific antibodies with broadly cross-reactive potential against H5 and H7 viruses for H1 and H3-based cHAs, respectively. University of Ghana http://ugspace.ug.edu.gh iii This study offers an alternative approach whereby multi-subtype or pan-group immunogens could be utilized for the design and generation of cross-reactive antibodies of potential therapeutic value for influenza in humans. University of Ghana http://ugspace.ug.edu.gh iv DEDICATION To all scientists contributing to the identification of novel therapeutics and universal influenza vaccine candidates. University of Ghana http://ugspace.ug.edu.gh v ACKNOWLEDGEMENTS Utmost thanks to God for blessings of strength and health to complete this academic journey. I would like to thank the faculty of Biochemistry, Cell, and Molecular Biology for this wonderful training opportunity, and especially the Quaye laboratory for bench space and mentorship. Sincere gratitude to the West African Centre for Cell Biology of Infectious Pathogens for fully sponsoring my PhD - tuition, stipend, and research costs. Heartfelt appreciation to my 3 supervisors for their dedicated guidance throughout PhD research thesis. I am most grateful to the wonderful team at the National Influenza Centre, Noguchi Memorial Institute for Medical Research for laboratory supplies, space, and equipment. This work would have not been possible without the tremendous support for cloning and expression experiments during a 6-months stay at the Avian Influenza Laboratory, The Pirbright Institute, Surrey, UK. Sincerest thanks to the team at the Centre for Plant Medicine Research, Mampong, for hosting the animal experiments with daily maintenance and monitoring of the study animals. I am also most grateful to my classmates who engaged me in inciteful discussions to address pressing questions that facilitated my work. Finally, I thank my family for their great patience and love whilst I was at distance on countless occasions due to my academic endeavour. University of Ghana http://ugspace.ug.edu.gh vi LIST OF ABBREVIATIONS AcNPV Autographa californica nuclear polyhedrosis virus ADCC Antibody-dependent cell-mediated cytotoxicity ADCL Antibody-dependent cell lysis ADCP Antibody-dependent cell-mediated phagocytosis APC Antigen-presenting cell AVMA American Veterinary Medical Association BCA Bicinchoninic acid BSA Bovine serum albumin CCL 28 Chemokine (C-C motif) ligand 28 CDC Centres for Disease Prevention and Control CD Cluster of differentiation cHA Chimeric haemagglutinin COBRA Computationally optimized broadly reactive antigen CPMR Centre for Plant Medicine Research, Mampong cRNA Complementary ribonucleic acid CTL Cytotoxic T lymphocyte CVV Candidate vaccine virus DNA Deoxyribonucleic acid ELISA Enzyme-linked immunosorbent assay University of Ghana http://ugspace.ug.edu.gh vii FCS Foetal calf serum FDA The Food and Drugs Authority FRO For research only GFP Green fluorescent protein GISAID Global initiative on sharing all influenza data GISRS Global Influenza Surveillance and Response Systems GPI Glycosyl phosphatidylinositol H1/HA1 Haemagglutinin type 1 H2/HA2 Haemagglutinin type 2 H3/HA3 Haemagglutinin type 3 H5/HA5 Haemagglutinin type 5 H7/HA7 Haemagglutinin type 7 H9/HA9 Haemagglutinin type 9 HA Haemagglutinin HBV Hepatitis B virus hCRM1 Human chromosome maintenance 1 protein HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEV Hepatitis E virus HI Haemagglutination inhibition HIV Human immunodeficiency virus University of Ghana http://ugspace.ug.edu.gh viii HPAI Highly pathogenic avian influenza viruses HPV Human papillomavirus HRP Horseradish peroxidase IAV Influenza A viruses IBV Influenza B viruses IEDb Immune-epitope database IFN ß Interferon Beta IFN γ Interferon Gamma IFV Influenza viruses IIV Inactivated Influenza Vaccine IRD Influenza research database IRR Influenza/ International reagents and resources I-TASSER Iterative threading assembly refinement LAIV Live-attenuated Influenza Vaccine LB Lysogeny broth M Matrix protein M1 Matrix protein 1 M2 Matrix protein 2 M2e Matrix protein 2 ectodomain MAFFT Multiple alignment using fast Fourier transform University of Ghana http://ugspace.ug.edu.gh ix MDCK Madin-Darby canine kidney cell line MHC II Major histocompatibility complex type 2 MHC Major histocompatibility complex mLD50 Mouse lethal dose 50 mRNA Messenger ribonucleic acid MVA Modified vaccinia virus Ankara N1 Neuraminidase type 1 N2 Neuraminidase type 2 N10 Neuraminidase type 10 N11 Neuraminidase type 11 NA Neuraminidase NCBI National Centre for Biotechnology Information NEP Nuclear export protein NIC National Influenza Centre NKC Natural killer cells NMIMR Noguchi Memorial Institute for Medical Research NP Nucleoprotein NS Non-structural protein NS1 Non-structural protein 1 NS2 Non-structural protein 2 University of Ghana http://ugspace.ug.edu.gh x PA Polymerase acidic PAGE Polyacrylamide gel electrophoresis PB Polymerase basic PB1 Polymerase basic 1 PB2 Polymerase basic 2 PBS Phosphate-buffered saline PBST 0.1% Tween-20 in Phosphate-buffered saline PCR Polymerase chain reaction PDB Protein Database PFU Plaque-forming unit PIV Parainfluenza viruses PLGA Polylactic-co-glycolic acid PMN Polymorphonuclear cells QIV Quadrivalent Influenza Vaccine RBS Receptor binding site RDE Receptor destroying enzyme RFLP Restriction-fragment length polymorphism RNA Ribonucleic acid RNP Ribonucleoprotein RSV Respiratory syncytial virus University of Ghana http://ugspace.ug.edu.gh xi S2 Schneider’s Drosophila Line 2 cells Sf Spodoptera frugiperda SDS Sodium dodecyl sulphate SIA Sialic acid SOC Super optimal broth with catabolite repression TGM Tris-glycine methanol Th 1 T-helper 1 T-cells Th 2 T-helper 2 T-cells TIV Trivalent Influenza Vaccine TLR Toll-like receptors TM Template modelling TPCK L-(tosylamido-2-phenyl) ethyl chloromethyl ketone UG-IACUC University of Ghana - Institutional Animal Care and Use Committee UK The United Kingdom US The United States UV Ultraviolet VLP Virus-like particle vRNP Viral Ribonucleoprotein WHO CC World Health Organization Collaboration Centre WHO Word Health Organization University of Ghana http://ugspace.ug.edu.gh xii TABLE OF CONTENTS DECLARATION ........................................................................................................................ i ABSTRACT ............................................................................................................................... ii DEDICATION .......................................................................................................................... iv ACKNOWLEDGEMENTS ....................................................................................................... v LIST OF ABBREVIATIONS ................................................................................................... vi TABLE OF CONTENTS ......................................................................................................... xii LIST OF FIGURES ............................................................................................................. xviii LIST OF TABLES ................................................................................................................... xx CHAPTER 1: INTRODUCTION, AIM, OBJECTIVES, AND RESEARCH QUESTIONS ... 1 Introduction ............................................................................................................................ 1 Aim: ........................................................................................................................................ 1 Objective 1: Generation of cHAs with intact conserved domains ......................................... 2 Objective 2: Expression of potentially immunogenic cHAs .................................................. 3 Objective 3: Assessment of cHAs to both induce broadly reactive anti-HA antibodies and confer protection to virus-challenged mice. ........................................................................... 5 CHAPTER 2: LITERATURE REVIEW ................................................................................... 7 Historical accounts on influenza as an ancient virus .......................................................... 7 Discovery and isolation of influenza A virus ..................................................................... 8 Isolation of Influenza B virus ........................................................................................... 10 University of Ghana http://ugspace.ug.edu.gh xiii Inception of influenza vaccines ........................................................................................ 11 Different aetiological agents underly influenza ................................................................ 14 Inception of coordination of global influenza .................................................................. 16 Second pandemic during the 20th Century: The Asian flu................................................ 17 Public acceptance of influenza vaccination in the US ...................................................... 17 The possibility that influenza viruses might have originated from migratory birds. ....... 18 Discovery of Amantadine: a potent inhibitor of cell entry by influenza A viruses .......... 19 Eruption of the 1968 pandemic Influenza: The Hong Kong flu ....................................... 22 Rimantadine – a more refined derivative of amantadine was generated. ......................... 24 Identification and licensing of new influenza antivirals: Zanamivir and Oseltamivir ..... 27 Systematic initiatives: the “one health” initiative, pandemic influenza operational plan, and full deployment of the PCR in the diagnosis of influenza ......................................... 32 Eruption of another pandemic influenza virus: The H1N1pdm09 ................................... 33 A new neuraminidase inhibitor - Peramivir ..................................................................... 35 Influenza viruses ............................................................................................................... 36 Influenza contraction, viral transmission, and pathogenesis ............................................ 38 Influenza virus structure and genome organization .......................................................... 39 Life cycle of influenza A virus ......................................................................................... 40 Influenza A virus haemagglutinin, the focus of many vaccine platforms ........................ 43 Characterization of influenza A virus HA ........................................................................ 45 Subtypes of influenza A viruses ....................................................................................... 46 Typical sequence conservations in the HA of influenza A viruses .................................. 46 University of Ghana http://ugspace.ug.edu.gh xiv Onward fight against influenza ......................................................................................... 48 CHAPTER 3: CURRENT AND NOVEL APPROACHES IN INFLUENZA MANAGEMENT .................................................................................................................................................. 49 Abstract ................................................................................................................................ 49 Introduction .............................................................................................................................. 50 Current influenza vaccines ....................................................................................................... 51 Use of the seasonal influenza vaccines. ............................................................................... 54 Novel influenza vaccine platforms ....................................................................................... 54 Virus-like particle (VLP) vaccines ................................................................................... 57 COBRA vaccines .............................................................................................................. 58 Synthetic influenza virus vaccines ................................................................................... 59 Epitope vaccines ............................................................................................................... 59 Antigen-presenting cell (APC) inducible vaccines .......................................................... 60 Nanoparticle-based influenza vaccines ............................................................................ 61 Viral - vectored vaccines .................................................................................................. 62 Current influenza managing antivirals ................................................................................. 63 Novel influenza management therapies ............................................................................... 63 Next-generation antivirals against influenza .................................................................... 63 Passive Immunotherapeutics for management of influenza ............................................. 69 Conclusions .......................................................................................................................... 77 CHAPTER 4: THE HAEMAGGLUTININS OF SEASONAL INFLUENZA A VIRUSES (H1 AND H3) POSSESS TARGETABLE CONSERVED DOMAINS ........................................ 78 University of Ghana http://ugspace.ug.edu.gh xv Abstract ................................................................................................................................ 78 Introduction .......................................................................................................................... 79 Results .................................................................................................................................. 81 H1 and H3 cHAs constructed. .......................................................................................... 81 cHAs are structurally and functionally like a typical HA by in silico prediction. ........... 89 cHA B-cell epitope and antigenic predictions .................................................................. 93 Discussion .......................................................................................................................... 101 Materials and Methods ....................................................................................................... 104 HA sequence selection and consensus sequence cHA construction in silico. ................ 104 cHA structural and functional predictions ...................................................................... 105 cHA B-cell epitope and antigenic predictions ................................................................ 105 CHAPTER 5: EXPRESSION OF CHIMERIC HAs IN S2 CELLS ..................................... 107 Abstract .............................................................................................................................. 107 Introduction ........................................................................................................................ 108 Results ................................................................................................................................ 109 Discussion .......................................................................................................................... 117 Materials and Methods ....................................................................................................... 119 cHA processing............................................................................................................... 119 cHAs Cloning and plasmid purification. ........................................................................ 120 Drosophila S2 cell culture preparation ........................................................................... 121 cHAs Expression in S2 cells ........................................................................................... 122 University of Ghana http://ugspace.ug.edu.gh xvi Transfection check by fluorescent microscopy or ELISA or Western blotting ............. 122 Purification of cHAs ....................................................................................................... 124 Expressed HA Haemagglutination activity assessment. ................................................. 124 CHAPTER 6: ASSESSING ANTI-cHAs ANTIBODY REACTIVITY AND PROTECTION OF INFLUENZA VIRUS-CHALLENGED MICE ............................................................... 125 Abstract .............................................................................................................................. 125 Introduction ........................................................................................................................ 126 Results ................................................................................................................................ 128 Mice selected for immunization and challenge experiments are pristine. ...................... 128 Stimulation of mice with cHAs or CVV HAs induced seroconversion. ........................ 130 Anti-cHAs antibodies cross-react with heterotypic HA viruses. .................................... 132 Anti-cHAs antibodies enabled weight rebound among cHAs-induced mice during virus challenge. ........................................................................................................................ 132 Discussion .......................................................................................................................... 138 Materials and Methods ....................................................................................................... 142 Serological assessment of mice for exposure to influenza A virus by the Haemagglutination inhibition (HI) assay ....................................................................... 142 Immunization regimen .................................................................................................... 142 The inhibitory capacity of resultant antibodies by Haemagglutination assay (HI) ........ 143 Challenge and outcomes ................................................................................................. 143 CHAPTER 7: GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATION 144 General Discussion ............................................................................................................. 144 University of Ghana http://ugspace.ug.edu.gh xvii Conclusion ............................................................................................................................. 150 Recommendation ................................................................................................................... 150 REFERENCES ...................................................................................................................... 151 APPENDICES ....................................................................................................................... 200 Appendix A: Full-length cHAs generated by consensus building of seasonal HAs and exotic HAs..................................................................................................................................... 200 Appendix B: Trimmed nucleic acid sequences used for the cloning experiment. ............. 205 Appendix C: Common/unique epitopes amongst the H3 HAs .......................................... 210 Appendix D: Common/Unique/Shared epitopes on the H1 HAs ....................................... 213 Appendix E: Quantitation of plasmids ............................................................................... 216 Appendix F: Haemagglutination assessment of cHAs and Candidate vaccine virus HAs 217 Appendix G: Baseline influenza A (H1N1) and (H3N2) exposure assessments. .............. 218 Appendix H: UG-IACUC clearance .................................................................................. 219 Appendix I: Primers for cloning experiments .................................................................... 220 University of Ghana http://ugspace.ug.edu.gh xviii LIST OF FIGURES Figure 1: Structure of the adamantane. ............................................................................... 19 Figure 2: Chemical synthesis of adamantane and structures of amantadine (1- adamantanamine)/ amantadine hydrochloride. .................................................................. 21 Figure 3: Structure of rimantadine. ..................................................................................... 26 Figure 4: Structure and synthesis of 4-guanidino-Neu5Ac2en. ......................................... 29 Figure 5: Structural resemblance of Zanamivir to GS 4104 (Oseltamivir). ..................... 31 Figure 6: Systematic reassortment of the H1N1pdm09...................................................... 34 Figure 7: The 20th-21st Century influenza pandemic timelines and persisting seasonal viruses...................................................................................................................................... 35 Figure 8: Structure of peramivir .......................................................................................... 36 Figure 9: Structural description of IAV. ............................................................................. 40 Figure 10: IAV replication cycle. .......................................................................................... 42 Figure 11: Structure of a Vietnamese mutant influenza A H5 HA. .................................. 44 Figure 12: Conserved sequence structure of selected influenza A viruses’ RBS. ............ 47 Figure 13: Mechanisms of antibody protection via passive immunization. ..................... 71 Figure 14: Diversity of selected HA sequences. ................................................................... 87 Figure 15: Visualization of predicted cHA structures in Pymol........................................ 91 Figure 16: B-cell epitopes and antigenicity predictions on the H1 HAs. .......................... 95 Figure 17: B-cell epitopes and antigenicity predictions on the H3 HAs. .......................... 98 Figure 18: Cloning of cHAs. ................................................................................................ 112 Figure 19: Rapid transfection checks on day 3 post-infection. ........................................ 116 Figure 20: Schematic mice immunization and challenge regimen. ................................. 129 Figure 21: Phylogenetic relationship amongst HAs of influenza A viruses. ................... 133 Figure 22: Weight monitoring of mice challenged with A/ARI-19-361/2019. ................ 134 University of Ghana http://ugspace.ug.edu.gh xix Figure 23: Lung viral load investigation in H3N2-challenged mice. ............................... 135 Figure 24: Weight monitoring of mice challenged with A/England/195/2009................ 136 Figure 25: Lung viral load investigation in H1N1-challenged mice. ............................... 137 University of Ghana http://ugspace.ug.edu.gh xx LIST OF TABLES Table 1: Summary of novel influenza virus vaccine platforms. ........................................ 56 Table 2: Influenza antiviral drugs approved or in clinical trials. ..................................... 65 Table 3: Antibodies undergoing clinical trials. ................................................................... 73 Table 4: Structural and functional predictions. .................................................................. 90 Table 5: Antigenic site substitutions on cHA-A. ................................................................. 92 Table 6: Unique and shared epitopes in the H1 HAs. ......................................................... 99 Table 7:Unique epitopes of the H3 HAs. ............................................................................ 100 Table 9: Assessment of murine seroconversion to cHAs. ................................................. 131 University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER 1: INTRODUCTION, AIM, OBJECTIVES, AND RESEARCH 1 QUESTIONS 2 Introduction 3 Influenza viruses cause a global disease called influenza that is associated with cold-like 4 symptoms. Unlike the common cold, influenza is associated with severer forms of coughs and 5 fever as cardinal signs (Monto, Gravenstein, Elliott, Colopy, & Schweinle, 2000). Annually, 6 the epidemics attributable to influenza is estimated to cause about half-a-million mortalities, 7 plus several millions of morbidities (Iuliano et al., 2018). However, based on previous 8 influenza pandemics, any future pandemics could cause over 200-300 % more mortalities or 9 morbidities. This makes it a disease of major public health concern and one that requires potent 10 means of intervention. 11 Vaccines and antivirals are available for the prevention or management of the disease; 12 however, vaccine efficacies are suboptimal and there also exists an arms race that continuously 13 renders the viruses resistant to a large number of the antivirals developed. A potent vaccine 14 with long-term protection is all it takes to efficiently lead to the reduction in influenza 15 epidemics and the prevention or delay of any unforeseen catastrophes. By this, a keen search 16 for a more diverse better-reacting vaccine is fervently desired as a game-changer. 17 Aim: 18 The main aim of this work was to ascertain the conserved domains of the haemagglutinins 19 (HAs) of the seasonal influenza A viruses and to exploit these domains in designing cross-20 reactive antibody-inducing immunogenic HA constructs. 21 22 University of Ghana http://ugspace.ug.edu.gh 2 Objective 1: Generation of cHAs with intact conserved domains 23 Active influenza vaccination regimens and platforms have driven -and are still driving- the 24 constant evolution of influenza A viruses and the many other platforms mostly being developed 25 are more likely to stagnate the problem. 26 Passive immunotherapy, using antibody administration, to control influenza infections, has 27 been proposed to be relevant in influenza management, in the absence of the desired candidate 28 vaccine (Kotey et al., 2019). Production of antibodies against conserved regions of the 29 influenza A virus HA will be an effective tool that will serve both to inhibit viral replication 30 and allow the immune system to develop an effective immunity to the presently infecting virus. 31 More specifically, designing immunogens that could experimentally induce such diverse 32 antibodies is necessary for this quest. 33 Therefore, this experiment was set up to employ in silico tools to construct seasonal HA 34 molecules that are representative of seasonal H1 and H3 viruses. 35 Hypothesis: The HAs of seasonal influenza A viruses possess conserved structures that are 36 potentially useful for the design of a recombinant immunogen. 37 Specific objectives: 38 1. To assess thousands of HAs of seasonal influenza A viruses and generate the 39 consensus conserved sequences. 40 2. To employ the conserved sequences in the construction of cHAs for 41 immunogenicity assessment. 42 43 University of Ghana http://ugspace.ug.edu.gh 3 Objective 2: Expression of potentially immunogenic cHAs 44 In recent times, a recombinant vaccine – the Flublok- exists which is expressed in the insect 45 fall armyworm (Spodoptera frugiperda, Sf) cells (Cox, Patriarca, & Treanor, 2008). The 46 specific cell type for this vaccine is referred to as ExpreSF+, derived from fall armyworm Sf9 47 cells. Protein expression in this system has been greatly aided by the Baculovirus, Autographa 48 californica nuclear polyhedrosis virus (AcNPV). However, it is thought that the Sf9 cells are 49 limited to the secretion of relatively smaller amounts of proteins that are expressed using the 50 system. Also, the cabbage looper (Trichoplusia ni) cell derivative BTI-TN-5B1-4 cells are 51 believed to express relatively higher amounts of proteins (Krammer et al., 2010; Palmberger et 52 al., 2011). Though the recombinant influenza vaccines expressed in the ExpreSF+ require 53 higher doses, about 3 times the dosage of the conventional egg-grown vaccines, their 54 immunogenicities are known to be decent (Treanor et al., 2007). 55 The choice of vaccine expression system has been mostly driven by user-preference and to 56 some extent, the robustness of the system: traditional diagnostic and screening methods 57 requiring the expression of proteins, for instance, are probably, more interested in systems 58 associated with both decent yields and physiologically native proteins. Similarly, 59 immunogenicity and cross-reactivity studies may tend to focus more on the physiologically 60 native version of an expressed protein; a good yield is often just an additional interest. 61 The Drosophila line 2 cells (S2) have also long been proposed as a versatile protein expression 62 system, showing comparable protein amounts to that produced by both the Trichoplusia ni and 63 Sf9 systems under the AcNPV transfection (Lee, Chen, Hsu, & Juang, 2000). They have also 64 expressed decent amounts of proteins when in ordinary plasmid-based transfections, without 65 the intermediation of a baculovirus (Iwaki and Castellino, 2008). More recently, other schools 66 of thought are that the mammalian cell systems are superior to insect-based systems, in terms 67 University of Ghana http://ugspace.ug.edu.gh 4 of similarities in protein modifications to the molecules on a mammalian-cell infecting virus, 68 hence, being immunologically superior to the insect cell counterparts (de Vries et al., 2012; 69 Ecker et al., 2020). 70 Unlike mammalian cells, like the HEK 293, that are associated with high-mannose 71 modification of expressed proteins, the Drosophila S2 cells are known to modify proteins with 72 paucimannose moieties and thereby depleting the requisite glycosylation. Furthermore, the 73 depletion of glycans to mono-glycosylation of influenza consensus cHAs has been shown to 74 increase the breadth of antibody-dependent responses, spanning cross-reactivity and infected 75 cell lysis (because of enhanced CD 8+ T-cell responses) (Liao et al., 2020). 76 Hence, for this work, the Drosophila S2 cells were used for expression of the in silico-designed 77 cHAs to enable exploration of their immunogenicity. 78 Hypothesis: cHAs expressed in S2 cells will allow for the exploration of their inherent 79 immunogenic properties. 80 Specific objectives: 81 3. To express high yields of cHAs. 82 4. To assess the haemagglutinating capacity of the cHAs. 83 84 85 University of Ghana http://ugspace.ug.edu.gh 5 Objective 3: Assessment of cHAs to both induce broadly reactive anti-HA antibodies and 86 confer protection to virus-challenged mice. 87 The head of the influenza A viruses’ HA as described, presents variable host immune-focussed 88 globular head regions that probably exert three main functions: facilitate virus-cell interaction 89 that aids viral infection; or by adapting to a new host; or as a means of escaping or evading 90 host immunity - the latter, being of critical interest in the development of several platforms that 91 harness the immunodominance of the HA in the synthesis of influenza vaccines. However, the 92 therapeutic use of discovered anti-HA antibodies recognizing relatively conserved domains of 93 the HA molecule is of great interest (Kotey et al., 2019). Of this antibody collection, the anti-94 HA stalk antibodies have received great attention (Corti et al., 2010; Margine et al., 2013a; 95 Nachbagauer et al., 2016; Sui et al., 2011; Wohlbold et al., 2015). Nevertheless, several other 96 antibodies have been discovered recently that interact with conserved domains on the globular 97 head of the HA (Benjamin et al., 2014; Boonsathorn et al., 2014; Krause et al., 2011; Yasuhara 98 et al., 2018; Yu et al., 2017). The major challenge, however, has to do with designing 99 immunogens that would achieve the induction of such protective antibodies. But noting the 100 relevance of these anti-HA antibodies targeting the globular head conserved domains in 101 inhibiting viral infection and transmission, therapeutic intervention or specific vaccine design 102 is likely achievable. As such, emphasis needs to be placed on the development of influenza A 103 virus-like HA antigens (or immunogens) that could have the potential of inducing a more 104 promiscuous collection of antibodies in diverse non-human primates. Perhaps, in this 105 collection, there will be polyclonal antibodies that could either inhibit conserved regions on the 106 head of the HA or the stalk. 107 108 University of Ghana http://ugspace.ug.edu.gh 6 Hypothesis: cHAs expressed in a system with limited post-translational carbohydrate 109 modification will induce antibodies directed to the exposed conserved domains 110 Specific objectives: 111 5. To assess seroconversion amongst mice immunized with the cHAs. 112 6. To assess the seroconverted serum to broadly-react with distant group-based HA-113 bearing viruses. 114 7. To assess the protection of mice immunized with the cHAs during a lethal virus 115 challenge. 116 117 118 University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER 2: LITERATURE REVIEW 119 Historical accounts on influenza as an ancient virus 120 History holds that influenza had long existed dating to as far back as before the 15th century. 121 However, it was until the 15th century when an epidemic broke out in Italy that the disease was 122 attributed to the “influence of stars” and thus named influenza. The Latin “influentia” meant 123 “to flow into” and during those days, it was believed that stars emitted intangible fluids that 124 could flow into and affect humans. By 1743, what the Italians called “influenza di catarro” 125 meaning “outbreak of the catarrhal fever” had spread gradually to Europe and was soon referred 126 to as Influenza in English. Since then, the name Influenza has often been abbreviated as flu, 127 which is more common lately (Francis, 1960). 128 By the end of the first world war in 1918, another outbreak occurred, recording up to about 500 129 million morbidities and at least 20 million mortalities worldwide. The outbreak was termed the 130 “Spanish flu” due to earlier thoughts that the virus had been spreading from Spain. It appears 131 that the very first person who was diagnosed with the virus responsible for the outbreak was a 132 military serviceman who was attended to in camp Funston located in Kansas. He was rushed 133 to Europe by fellow American Servicemen; here, it was believed that the virus evolved to a 134 highly virulent strain causing more havoc. This points to the notion that the outbreak had started 135 in the US and not Spain or Europe as thought. This premise was further corroborated by testing 136 of archived tissue samples in an American military hospital in South Carolina after almost 8 137 decades and realizing that this was then regarded as the swine flu (Kolata, 2001). 138 Amidst all the outbreaks that perhaps might have occurred centuries back, a lot of interest is 139 shown in influenza viruses and outbreaks that occurred spanning the 18th century until date. 140 The world war was almost subsiding when the unpropitious outbreak of influenza had occurred. 141 During the period, history described a lot of troop camping and movements, denoting closeness 142 University of Ghana http://ugspace.ug.edu.gh 8 in space among military personnel and thus fostering the rapid expansion of the infection. As 143 there were no vaccines or treatments, the outbreaks became a public health burden and numbers 144 of cases multiplied day-by-day. Other key factors included troop celebration of the truce and 145 because they were gradually disbanding, many more new contacts contracted the virus leading 146 to the resurgence in the numbers of cases firstly in the US. The infection turned sporadic, 147 involving the European continent (Erkoreka, 2010). 148 Discovery and isolation of influenza A virus 149 The infection was being referred to as Influenza or flu, but it was still not clear to 150 microbiological scientists what kind of pathogen caused this pandemic. Therefore, around 151 1933, a team of researchers attempted to identify the cause of the flu. Foremost, Smith and 152 colleagues 1933 played a crucial role in identifying that the cause of flu was a virus. They 153 arrived at this premise after they had obtained throat washes from persons who were acutely 154 infected with flu and subsequently filtered the washes with a collodion membrane with a pore 155 size of about 0.6 microns. Filtrates when instilled into the nares of ferrets, yielded disease 156 phenotypes indistinguishable from persons from which throat washes were obtained. As a 157 control, they realized that when the nares of a new batch of ferrets were instilled with throat 158 washes from healthy persons comprising 2 people who had recovered after a previous infection 159 and 2 others that did not have the infection, the disease phenotype was not established. Also, 160 filtered nasal wash from a patient suffering from a severe common cold could not cause disease 161 in ferrets. At this stage, the scientists were convinced that the pathogen that caused the flu was 162 filtrable providing a clue to suspect a virus. The study also confirmed the typical hallmark of 163 the contagious virus pathogen, which is its ability to spread from an infected ferret to an 164 uninfected when housed together within 24 hours or even when filtered materials from infected 165 tissues had been used to instil the nares of otherwise uninfected ferrets. Besides the human 166 strain of virus sourced from the throat washes, Smith and colleagues also realized that another 167 University of Ghana http://ugspace.ug.edu.gh 9 filtrable virus of the swine-origin yielded indistinguishable symptoms as that caused by the 168 viruses of the human origin when they had infected ferrets. The swine virus was previously 169 shown to cause a severer form of infection in swine in association with a haemophilic bacillus 170 - one that was not realized in their experiments on the ferrets; however, one striking observation 171 was the fact that ferrets that recovered from the swine virus challenge were protected from 172 challenge with the human viruses, but not the vice-versa (Smith, Andrewes, & Laidlaw, 1933). 173 Previous experiments had involved lightly etherized ferrets due to the strong reflex of the 174 animals to sneeze, compromising on the amount of infectious inoculum; Researcher Shope 175 obtained 2 swine flu viruses and designed a setup that required a complete knock-off of ferrets 176 using anaesthetics, to facilitate administration of a nearly appropriate dose of the inocula 177 created with these viruses. He also confirmed the transmissibility of the virus from infected 178 ferrets to uninfected ones, with retention of the classical symptoms. Several pathologies, 179 including ungroomed fur, inappetence, thumping, etc., were thoroughly described. There was 180 an earlier notion that the Haemophilus bacillus, H. influenzae suis exacerbated swine flu 181 infections in swine, and this was observed even when serially passaged swine flu virus in 182 ferrets, still showed the classical symptoms when pigs were infected. Also, amongst ferrets, 183 bacteriological assessments of the respiratory tracts of sick swine flu inoculated ferrets 184 confirmed the absence of bacteria and that, unlike the swine, ferrets did not require the presence 185 of a bacteria to exacerbate flu infection. This was further proved when H. influenzae suis only 186 or H. influenzae suis -spiked flu virus inoculum was used to challenge ferrets and observed that 187 the bacillus alone could not establish an infection and was indifferent in the case of the two 188 pathogens. Thus, in ferrets, the flu virus infection was independent of a bacillus as conversely 189 established for swine. Shope’s work debunked further the notion that the bacillus played a role 190 in the infectivity and severity of the swine flu infection when he observed that a pig that was 191 administered with both the bacillus and the virus at a certain passage developed only mild 192 University of Ghana http://ugspace.ug.edu.gh 10 symptoms. Shope demonstrated further that other than intranasal inoculation of ferrets with the 193 swine viruses, ferrets could not show the disease phenotype of the flu owing to subcutaneous 194 inoculation, whether an anaesthetic was used or not. This was used to clearly outline the route 195 of infection of the virus. Further, It was necessary to completely prove that the virus that was 196 used to induce disease in ferrets was also responsible for the disease in swine, using a cross-197 neutralization test; For this test, convalescent sera from ferrets were mixed (at different 198 proportions) with different proportions of viruses from ferret and the mixture was used to 199 intranasally inoculate pigs – the converse was also done, and it was observed that in both 200 instances, certain ferrets and pigs that received a favourable amount of sera mixed with either 201 the ferret/ swine-specific viruses were protected from infection compared with their controls 202 (Shope, 1934). 203 Isolation of Influenza B virus 204 By 1933, pandemic influenza A virus had been isolated in ferrets; however, it became 205 noticeable that not all the influenza disease had been caused by one virus. Thomas Francis 206 performed immunological experiments that confirmed that more than one type of virus could 207 cause influenza symptoms. During his investigations, he called this new virus the “Lee virus”. 208 He still could not clearly show that this new virus was different from the swine flu virus that 209 caused the earlier pandemic, until in 1940, when he could use neutralization assays to show 210 that a current virus causing an outbreak was similar to what he had detected before back in 211 1936 and that the virus that caused the 1918 pandemic and the current one was different. Thus, 212 to avoid confusion, this “Lee virus” was termed influenza B as the classical one was termed 213 the influenza A virus (Francis, 1940). 214 During the time when Thomas Francis had discovered the new virus (influenza B), Burnet who 215 had had an enormous amount of experience in isolating certain viruses in eggs, adopted this 216 University of Ghana http://ugspace.ug.edu.gh 11 process to attempt to isolate the influenza B virus so named. In his experiment, stock viruses 217 were prepared by inoculating ferrets and scraping off nares. Pathogenicity of these viruses was 218 assessed by the inoculation of mice closely followed by the analysis of mice lungs for 219 characteristic lesions. Subsequently, eggs were inoculated through several passages using 220 viruses harvested from both ferrets and mice. The end of the egg adaptation process also 221 indicated the characteristic lesions that led to the extent that the eggs were killed. For some of 222 these viruses, it was realized that they could no longer induce symptomatic disease in ferrets; 223 however, mice infections became milder, after inoculation with viruses severally passaged in 224 eggs. In most of the cases, viral passages in ferrets warranted virus infectivity in mice, and this 225 was retained even after egg passage, even though the egg-adapted viruses could only cause 226 milder infections in mice but not ferrets. This phenomenon was one of the interesting findings 227 that were observed during the passage in eggs. Noting that egg characteristics remained 228 constant throughout the experiments, it most definitely was something about the viruses that 229 caused them to gain high affinity and pathogenicity in eggs, after several egg passages. This 230 phenomenon led Burnet to posit the likelihood of using egg-adapted viruses as the source of 231 vaccine viruses for the vaccination of humans in the nearest future (Burnet, 1936). 232 Inception of influenza vaccines 233 Following Burnet’s posits, it became very necessary to first understand the immunity against 234 influenza viruses; But then, in several experiments, it was realized that working with one strain 235 of the virus, a cognate serum would often neutralize the virus, whereas things got more 236 complicated if several strains were employed. Insights were gained into this phenomenon when 237 Andrewes (1939) reckoned that two influenza-like infections of a person in a short period may 238 be underlined by different aetiological agents. Around this period, mind-boggling concepts 239 encircled a lot of scientific issues involving the epidemiological profitability of vaccination as 240 active immune responses were observed with matching strains but not with different strains in 241 University of Ghana http://ugspace.ug.edu.gh 12 a cross-neutralization test; 1. to what extent of protection can vaccines protect? 2. which is 242 more appropriate – inactivated virus or live virus vaccines? 3. what should the composition of 243 the vaccine be? (Andrewes, 1939; Smith & Andrewes, 1938). 244 Scientists feared that live viruses could revert to virulence, but attempts were still made using 245 mice and ferrets to advance the development of influenza vaccines. Some of these studies 246 principally identified subtle challenges stemming up from the right virus titres required for 247 inoculation, the animal source from which the vaccine candidates are generated, the inoculation 248 route, and even the state of the virus -whether living or dead. In one of these experiments, it 249 became clear that homotypic vaccines – whether living or dead – induced some level of 250 protection amongst mice or ferrets that were given the shots. However, vaccines from 251 heterologous species were poor in immunizing performance as they contained a lot of 252 interspecies material that interfered so much with immune responses against the minimal virus 253 content (Andrewes & Smith, 1939). 254 Experiments gradually extrapolated to humans when a group of scientists tried the egg-adapted 255 viruses as candidate vaccines to assess if that could induce some protective immunity amongst 256 vaccinees. The viruses – moderately virulent in ferrets and mice – were intranasally 257 administered to persons who had previously been immunized with a less virulent strain of the 258 influenza virus and had low serum antibodies against the virus. Subsequently, nasal exudates 259 collected from these persons were shown to exhibit viricidal activity against different strains 260 of the influenza viruses. This would, later, be realized that even exudates from normal human 261 nares exhibited a similar virucidal effect; hence, warranting further elucidating studies (Burnet, 262 Lush, & Jackson, 1939). 263 During these times, other experiments either had been done or were being done to ascertain 264 which form of vaccine was considerable. In most experiments, irradiated or heat-treated or 265 University of Ghana http://ugspace.ug.edu.gh 13 chemical-treated viruses had abolished viral infectivity and thus, no significant immunity could 266 be observed. However, one leading experiment was the one by Kidd (1938), showing that the 267 Shope papillomavirus could be inactivated by UV irradiation without affecting the antigenicity 268 of the virus. This was observed when the UV-irradiated virus cultures were administered 269 intraperitoneally. Most of the rabbits that received the shots were assessed both for the 270 formation of papilloma and for the induction of neutralizing antibodies. Using complement 271 fixation tests, neutralizing antibodies were detected even though no active viruses could be 272 extracted from the papilloma on the immunized rabbits. Kidd (1938) noted also that 273 inactivation of the papillomaviruses using strong acids and bases rendered the viruses both un-274 infectious and serologically unreactive. 275 Insights from Kidd’s findings spurred on the prospects of redesigning the inactivated influenza 276 vaccine employing UV irradiation as the tool for the inactivation. Therefore, Salk and 277 colleagues attempted an experiment on the abilities of unirradiated active influenza virus and 278 irradiated inactive virus to induce immunity via intraperitoneal inoculation of mice. They 279 employed virulent mice-infecting influenza viruses cultured on minced chicken embryos in the 280 presence of physiological salts. UV irradiations were conducted on viruses at different time 281 intervals just to ascertain the minimum effective time and irradiated viruses were further used 282 to immunize mice. Observations were that the optimal UV-irradiated inactive viruses had 283 nearly equal immunizing capacity as their unirradiated active viruses. Further quantitation of 284 the antigenicity of the irradiated viruses became necessary to authenticate the importance of 285 UV- irradiated influenza viruses as potential vaccine candidates; so, Salk and colleagues looked 286 at intraperitoneal immunization of mice with graded doses of the UV-irradiated doses of 287 influenza virus. After several days, each of these mice received an intranasal viral challenge. 288 Results were conclusive that optimal UV-irradiated influenza viruses maintained a decent level 289 University of Ghana http://ugspace.ug.edu.gh 14 of antigenicity, except that these viruses lose their immunizing capacity by about 100-fold 290 (Salk, Lavin, & Francis, 1940). 291 Different aetiological agents underly influenza 292 All the vaccine preparations during earlier works up until 1940-1941 have been influenza A 293 virus-based; however, these reports had given insights into the existence of yet another 294 aetiology which is dissimilar to the influenza A virus but could produce a similar disease 295 phenotype as does the influenza A viruses (Francis, 1940; Francis, 1940; Magill, 1940; Magill 296 & Tyndall, 1941). Studies by Lennette and colleagues rather consolidated the presence of 297 distinct viruses causing influenza in each of the outbreaks from which virus-infected samples 298 were obtained. This experiment also showed to an appreciable degree, the presence of other 299 uncharacterized viruses (Lennette, Rickard, Hirst, & Horsfall, 1941). Thus, by 1942, 300 experiments were carried out to assess the importance of an influenza B vaccine. 301 Eaton and colleagues performed several mice inoculations and passages of an influenza B virus 302 strain, and then the virus was subsequently inoculated on eggs, within either the embryo’s 303 amnion or the chorioallantoic fluids. Isolated viruses were then treated with formaldehyde, 304 based on earlier experiments and the notion that the influenza A vaccine formulated inactive 305 using formaldehyde reduced the incidence of the infection by about half (Brown, Eaton, 306 Meiklejohn, Lagen, & Kerr, 1941; Horsfall, Lennette, Rickard, & Hirst, 1941; Martin & Eaton, 307 1941). Thus, in the experiments by Eaton & Martin (1942), the chorioallantoic fluids were 308 examined for both bacterial and neurotropic virus contamination. One mouse, during the 309 assessment of the presence of a neurotropic agent by intracerebral inoculation, died showing 310 marked lesions in the brain. Wondering what may have caused this, it was not farfetched that 311 the influenza B viruses, other than any neurotropic virus, may have been the cause of the brain 312 destruction and the subsequent death. It, therefore, became indispensable to experiment on 313 University of Ghana http://ugspace.ug.edu.gh 15 active influenza B virus as a vaccine candidate, except to inactivate it before administration to 314 animals or human subjects. Hence, inactivating all vaccine lots by treatment with 315 formaldehyde. Tests for viral activity in mice were performed by either intranasal or 316 intracerebral inoculation. This time, there were no records of lesions in the brains and 317 subsequently, other mice receiving the shot intraperitoneally were protected against an 318 intranasal virus challenge. This experiment paved the way to begin experimentation on human 319 subjects combining both influenza A and B vaccine preparations. Both the new influenza B 320 vaccine formula and the previous complex influenza A-distemper virus vaccine earlier reported 321 by Horsfall and colleagues was administered subcutaneously on the left and right arms, 322 respectively (Horsfall et al., 1941). Blood was collected from the participants before and 2 323 weeks post-inoculation for neutralization experiments. Sera from these blood specimens were 324 heat-inactivated, serially diluted and mixed with active viruses, and intranasally inoculated into 325 mice. Eaton & Martin (1942) observed that vaccination with inactivated influenza B vaccine 326 yielded an appreciable amount of both neutralizing antibodies, as well as complement-fixing 327 antibodies. 328 Several investigative experiments with human subjects begun to spring up. A typical one was 329 by Bodily and Eaton, who studied the specificity of antibodies by either neutralization and 330 agglutination experiments on sera (from acute or convalescent cases), in response to specific 331 influenza virus -type A and B- strains due to either natural infection or vaccination (Bodily & 332 Eaton, 1942). 333 An influenza epidemic broke out around 1947 post-World War II and then, the current vaccine 334 formula that had been developed failed to immunize recipients due to some sort of antigenic 335 changes on the epidemic-causing influenza virus. The vaccine failure was described as one of 336 the principal causes of the spread amongst military personnel in the UK, US, France, etc., 337 University of Ghana http://ugspace.ug.edu.gh 16 albeit, relatively less virulent to cause a pandemic (Chu, Andrewes, and Gledhill, 1950; 338 Rasmussen, Stokes, & Smadel, 1948; Sartwell & Long, 1948). 339 Inception of coordination of global influenza 340 This current and other earlier influenza-related epidemic/ pandemic experiences drove the 341 World Health Organization (WHO) to establish a World Influenza Centre, stationed in the 342 laboratories of the National Institute for Medical Research in London. Subsequently, Regional 343 and National level laboratories were established to collaborate with the World Influenza 344 Centre. Other setups were established to enable the sharing of influenza-related information 345 with both the WHO headquarters in Geneva and the Influenza information Centre in the US 346 (Chu et al., 1950). 347 Around 1952, the Global Influenza Surveillance and Response System (GISRS) was set up by 348 the WHO, to coordinate national laboratories’ surveillance activities and monitor the gradual 349 evolution of influenza viruses. Their network of surveillance and reporting laboratories has 350 increased steadily from 26 to about 152 institutions in 113 countries at present (Zhang & Wood, 351 2018). Four years after the setup of the GISRS, the influenza studies facility of the CDC 352 laboratories in the Atlanta, US, became designated as a WHO Collaborating Centre (CC) for 353 Surveillance, Epidemiology, and Control of influenza. The WHO CC was tasked with 354 influenza-related work such as the assessment of circulating viruses genetically, 355 immunologically, and susceptibility to antivirals. Currently, there are a total of 7 WHO CCs: 2 356 in the US; 1 in Australia; 1 in the UK; 1 in China; 1 in Japan; and 1 in Russia. Each WHO CC 357 has been involved in the global selection of candidate vaccines to produce the annual influenza 358 vaccines. They also act to offer both technical assistance and diagnostic reagent support to all 359 members of the GISRS networks. Lastly, they serve as a repository of influenza virus 360 University of Ghana http://ugspace.ug.edu.gh 17 specimens and isolates obtained due to global surveillance by the NICs (CDC, 2019; WHO, 361 2022). 362 Second pandemic during the 20th Century: The Asian flu 363 Just about a year after a WHO CC had been set up in Atlanta, another influenza pandemic had 364 sprouted up. A collection of reports in the US indicated that there was an ongoing outbreak of 365 influenza-like illness (Anderson, 1957; Jensen, 1957; Reyes, Serfling, & Tayback, 1957). The 366 influenza virus causing the ongoing outbreak had been first identified as a type of influenza A 367 virus in China during the earlier part of 1957. It appeared that the causative virus had spread 368 progressively from China to Singapore and then to the US. Thus, all the occurrences in the 369 cities of the US were confirmed to emanate from the Far East (Langmuir, 1961; Mc, 1958). 370 Subsequently, virus isolation and classification attempts confirmed that the Far East infecting 371 virus originated from avian as it had 3 proteins [including Haemagglutinin type 2 (H2) and 372 Neuraminidase type 2 (N2)] that originated from avian, that had reassorted with other human-373 infecting influenza virus proteins (Meyer, Hilleman, Miesse, Crawford, & Bankhead, 1957). 374 Before the year ended, the outbreak had affected almost all 6 continents of the globe (Mc, 375 1958). Recently modelled experiments indicated that even though the virus had spread rapidly, 376 the mortality rate was moderate compared with the 1918 pandemic (Cécile Viboud et al., 2016). 377 Public acceptance of influenza vaccination in the US 378 Observations made from influenza-related outbreaks include higher morbidities and mortalities 379 amongst persons with underlying chronic conditions, as is usually observed with the aged who 380 are mostly associated with some of these diseases. Such immunocompromised persons are 381 deemed worthy of enormous attention. In those days of experiencing the 1957 outbreak, in the 382 US, the Surgeon General had advocated for active vaccination regimens for the public with an 383 emphasis on the immunocompromised persons (persons with chronic diseases such as diabetes 384 University of Ghana http://ugspace.ug.edu.gh 18 mellitus, chronic bronchopulmonary diseases, cardiovascular diseases, renal diseases, 385 Addison’s disease), including pregnant women. Decisions put forward by the Advisory 386 Committee for Immunization, culminated in the public acceptance of influenza vaccination as 387 an adopted practice in the US. But the vaccination would not end up for this high-risk group of 388 people only: subtle doses were also recommended for everybody else spanning children (3 389 months old into pre-school and 6 to 12 years old) to adults of ages 13 and beyond (ACIP, 1961; 390 Rosenstock, 1961). 391 The possibility that influenza viruses might have originated from migratory birds. 392 In 1961, there were records of dying terns at the shores of South Africa, in 4 main coastal areas. 393 Terns were known as migratory birds that occasionally frequent the shores of South Africa to 394 breed. However, during the expectant breeding period, there was a massive decline in the 395 number of birds that were present. Also observed was that over 1000 dead birds at the four 396 main focal points at the shores of South Africa. This alarming decline in the numbers of the 397 birds had warranted for the Veterinary services to engage in active research to identify the 398 aetiology of the catastrophe. Utilizing electron microscopy, it appeared that the Tern virus was 399 morphologically like an influenza A virus (Becker, 1963). Down the line, it became very 400 necessary to isolate and characterize this tern virus. Becker collected some influenza A virus 401 strains: one isolated from a Tern in South Africa; another isolated from a chicken in Scotland; 402 a third one isolated from Cape Town; an Influenza B virus; an influenza C virus; and a 403 Newcastle disease virus. The Scotland strain had previously been shown to be morphologically 404 similar to the tern virus and so in the experiment, mice infection and cross-neutralization 405 experiments with all the viruses used confirmed that only the two viruses might be variants of 406 the same strain of the virus. The tern virus present in dead European terns were subsequently 407 the cause of the influenza infection among the common terns, switching ideas to the perspective 408 University of Ghana http://ugspace.ug.edu.gh 19 that these migratory birds may be responsible for the inter-continental transmission of influenza 409 viruses amongst local birds that they encounter (Becker, 1966). 410 Discovery of Amantadine: a potent inhibitor of cell entry by influenza A viruses 411 Whilst influenza research was becoming more prominent, concerns began to ensue about not 412 just prevention using vaccines, but also the management of persons that get infected. 413 Researchers initiated active searches for compounds of therapeutic value, one of which was 414 called 1-Adamantanamine (Amantadine). Adamantane is the most stable member with the 415 molecular formula, C10H16, and its name stems from the fact that it is made up of 3 cyclohexane 416 rings that assume the shape -in terms of the spatial arrangement of the carbon atoms- of a 417 diamond; hence, regarded as close to diamond in Greek as “adamantinos” (Senning, 2006). 418 Adamantane was first discovered in petroleum in 1933, but synthesis of the compound was a 419 big challenge even though the structure was proposed (Figure 1) (Landa & Macháček, 1933). 420 421 Image adopted from Landa & Macháček (1933). 422 Figure 1: Structure of the adamantane. 423 424 After several attempts had been made, Schleyer was successful at proposing a chemical 425 equation (Figure 2) that would aid the easy synthesis of an appreciable amount of adamantane 426 (Schleyer, 1957). 427 428 University of Ghana http://ugspace.ug.edu.gh 20 429 University of Ghana http://ugspace.ug.edu.gh 21 A. 430 431 Image A adapted from Schleyer (1957) 432 433 Image B adopted from Davies et al. (1964) 434 435 436 Image C adopted from Kirschbaum (1983) 437 Figure 2: Chemical synthesis of adamantane and structures of amantadine (1-438 adamantanamine)/ amantadine hydrochloride. 439 University of Ghana http://ugspace.ug.edu.gh 22 A. Adamantane was appreciably synthesized by the hydrogenation of the dicyclopentadiene in the presence of 440 Platinum oxide (in ether) and further cyclised in the presence of aluminium chloride (a Lewis acid). B and C. The 441 amantadine was notably administered as the amantadine hydrochloride. 442 443 Strides had been made in the synthesis of adamantanes and chemical synthetic methods have 444 been applied to increase yields drastically, but how this became applicable as a drug for the 445 treatment of influenza is still intriguing and unclear. Nevertheless, later, amantadine (1-446 adamantanamine) had been shown to exert therapeutic efficacy against influenza A viruses in 447 a dose-dependent manner (Davies et al., 1964). Subsequent studies showed that treatment with 448 Amantadine hydrochloride had caused retention of infectious influenza A viruses at the surface 449 of an infected cell, indicating drug interference with the cell entry process by influenza A 450 viruses (Hoffmann, Neumayer, Haff, & Goldsby, 1965). 451 Around 1966, the National Institutes of Allergy and Infectious Diseases (NIAID) 452 recommended the use of Amantadine both to treat and prevent influenza A virus infections 453 following approval (based on the susceptibility of the Asian flu viral strains to the drug) by the 454 US Food and Drugs Authority (FDA) (Maugh, 1979). The drug had a minimal-to-no inhibitory 455 effect on influenza B viruses and so was solely applied for the treatment of influenza A virus 456 infections. 457 Eruption of the 1968 pandemic Influenza: The Hong Kong flu 458 Not too long after the introduction of the use of amantadine for the treatment and prevention 459 of influenza A virus infection, another pandemic broke out: The Hong Kong flu pandemic. 460 This time, it was a new virus subtype that had a new haemagglutinin (H3) and its neuraminidase 461 gene was of the virus of the previous Asian flu pandemic (in 1957)- that is, the N2 (Ghendon, 462 1994). This influenza A (H3N2) virus was first noticeable in the Americas where it had been 463 University of Ghana http://ugspace.ug.edu.gh 23 reported to have caused over a hundred thousand deaths and then worldwide, over a million. 464 History noted two typical waves: the first one which was quite benign -in mostly the Americas- 465 and affected mostly aged people as was hypothesized that persons exposed to the Asian flu of 466 1957 might have had N2-specific antibodies that conferred some level of protection; and then 467 a second wave that badly affected mostly Europe and Asia due to drifts on the N2 gene (C. 468 Viboud, Grais, Lafont, Miller, & Simonsen, 2005). 469 Several reforms had been made following the successful containment of the A (H3N2) virus 470 but as if that were not enough - by 1976, in the US, there were reports about a respiratory 471 disease outbreak amongst military recruits living in Fort Dix, New Jersey. At first, it was 472 thought to be an adenoviral infection -as confirmed by the Walter Reed Army Laboratory - 473 during an earlier outbreak in the year in Fort Meade, Maryland. For a timely intervention, the 474 causative agent needed to be identified. This warranted the collection and shipment of throat 475 washes from infected recruits to a New Jersey Laboratory. In New Jersey, the specimens 476 collected were shown to harbour influenza A viruses and this would be confirmed later (in the 477 same year, though) in the CDC laboratories as influenza A viruses of the swine-origin 478 (Neustadt, 1978). 479 Reformative approaches, such as vaccination, were necessary for consideration and so during 480 this period, representative viruses were shared with Kilbourne’s laboratory for virus growth 481 and isolation attempts. In his laboratory, challenges were revolving around the growth of these 482 viruses, leading to the resort of the tried and tested recombination approach. In this approach, 483 Kilbourne’s lab team mated the rapidly growing laboratory isolate PR8 (A/PR8/34) strain and 484 one of the viruses from New Jersey (A/NJ/11/76), in the presence of antibodies that inhibited 485 the HA and NA of the PR8 strain. Resultant recombinant (termed X-53) bore the internal genes 486 of the PR8 strain and the surface proteins (HA and NA) of the New Jersey virus. Selected 487 University of Ghana http://ugspace.ug.edu.gh 24 recombinants were, therefore, employed in the development of vaccines against the swine 488 influenza outbreak (Palese, Ritchey, Schulman, & Kilbourne, 1976). 489 The swine influenza outbreak had led to the full activation of reformative measures, chiefly, 490 vaccination based on Kilbourne’s X-53 virus. An approximated 25% of the US population (i.e., 491 about 48 million people) were vaccinated by the 10th month upon the inception of the outbreak. 492 However, a typical setback was the resurrection of a neurologic disease called the Guillain-493 Barre Syndrome, a condition that was often experienced with vaccination regimens, but this 494 time, was more than what was expected. Seeing this disease creep into the homes of some 495 vaccine recipients triggered a halt on the vaccination programme. But, overall, some 496 appreciable dimension of vaccine-induced protection was observed (Neustadt, 1978). 497 Rimantadine – a more refined derivative of amantadine was generated. 498 One of the therapeutic milestones was the remodification of amantadine into an analogue drug 499 called rimantadine. The remodification became necessary due to growing body of knowledge 500 depicting that influenza A viruses had begun to develop some resistance to the amantadine. For 501 instance, investigations by Hay and colleagues both buttressed other earlier posits that both the 502 HA and Matrix protein 2 (M2) played a crucial role in the viruses that developed resistance to 503 the amantadine. Findings from his team further pinpointed locations of mutations that led to 504 resistance mainly on the hydrophobic amino acids that span a region of the M2 protein (i.e., 505 amino acids between positions 25-43) and thereby positing the mechanism of action of the 506 amantadine hydrochloride to be on the membrane-spanning domains. Roping of the HA into 507 the mechanism of resistance was further explained to be due to the apparent functions of the 508 M2 acting with other viral proteins during syntheses and assembly. Thus, its inhibition by the 509 amantadine mostly led to the reduction in the numbers of the HA that were expressed by the 510 virus and these HA proteins even suffered a consequence of an altered structure as this affected 511 University of Ghana http://ugspace.ug.edu.gh 25 reactivity with antibodies (Hay, Wolstenholme, Skehel, & Smith, 1985). Further studies by 512 Hay and colleagues yielded informative results detailing mutations in the HA gene amongst 513 resistance viruses as occurring less frequently and thus, specific mutations in the M2 protein 514 may be responsible for the resistance to the amantadine (Hay, Zambon, Wolstenholme, Skehel, 515 & Smith, 1986). 516 Rimantadine, another derivative of adamantanes was developed sometime to serve as a more 517 refined pharmacokinetically favourable drug to either complement the use of amantadine or 518 better still, as a substitute drug that would circumvent some adverse reactions amongst 519 amantadine recipients. Just like amantadine, rimantadine (1-adamantane-methylamine 520 hydrochloride) inhibits the in vitro infection of influenza A viruses. However, it was thought 521 to be superior to amantadine (Koff & Knight, 1979): In cell culture, where studies by Wallbank 522 (1969) showed that a relatively lesser amount of rimantadine (than amantadine) was required 523 to inhibit 50% of Rous and Esh Sarcoma Viruses (Wallbank, 1969); and in animals, where the 524 amount of rimantadine (similar to amantadine) lead to relatively lower titres of influenza A 525 viruses in pulmonary lesions and lungs, in addition to relatively lower time-dependent HI titres, 526 with the subsequent observation of a more reduced transmission of viruses from treated 527 infected mice to non-treated sterile contact mice (Schulman, 1968). Conversely, in a clinical 528 trial involving the management of a naturally occurring influenza infection during an outbreak 529 in a US penitentiary, both amantadine and rimantadine showed comparable efficacies, even 530 though in most instances, rimantadine (which was one-third dose more than amantadine) 531 mainly performed relatively better: an indication that perhaps the apparent difference was due 532 to the dissimilarity in drug concentration (Wingfield, Pollack, & Grunert, 1969). 533 In a study to investigate the prophylactic efficacy of rimantadine and amantadine, it was 534 observed that an appreciable number of persons receiving amantadine had dropped out due to 535 the development of side effects on their central nervous system; thus, selecting rimantadine as 536 University of Ghana http://ugspace.ug.edu.gh 26 the more preferred drug choice for the prophylactic or therapeutic management of influenza 537 (Dolin et al., 1982). 538 Rimantadine (Figure 3) gradually gained some attention in the management of influenza: By 539 1986, it was used to treat against uncomplicated influenza A (H3N2) with the realization of a 540 satisfactory efficacy, in terms of the reduction in rates of nasal secretions and the accelerated 541 times for abatement of fever and symptoms (Hayden & Monto, 1986); 542 543 Image adopted from Suzuki et al. (2016). 544 Figure 3: Structure of rimantadine. 545 546 A more interesting finding in the then Union of Soviet Socialist Republics (USSR) buttressed 547 the point that rimantadine is one of several influenza antivirals that had withstood the test of 548 over 20 decades of use, and that in all these years, efficacy did not depreciate amidst the 549 emergence of resistant strains (Kubar, Brjantseva, Nikitina, & Zlydnikov, 1989). Following 550 several studies, rimantadine had received the US FDA approval for use as either prophylaxis 551 or treatment for influenza A virus infections. However, due to adverse effects and the 552 emergence of resistance strains (that may easily be transmissible), the use of both the 553 amantadine and the rimantadine is, thus, subject to stringent guidelines, considering different 554 age categories, persons with chronic illnesses, regulated dosages, and frequency of drug use 555 (Arden, Cox, & Schonberger, 1994). 556 University of Ghana http://ugspace.ug.edu.gh 27 Identification and licensing of new influenza antivirals: Zanamivir and Oseltamivir 557 Influenza virus emergence and re-emergence had continued. All such continuing cases leading 558 to the explorative discovery and licensing of new anti-influenza compounds: The Oseltamivir 559 and Zanamivir. In addition to influenza virus emergence or re-emergence, influenza B viruses 560 have not received much attention with the use of the M2 protein inhibitors (the amantadine and 561 rimantadine). The identification of zanamivir and oseltamivir proved very useful, as this drug 562 had a broad-inhibitory capacity against all influenza Viruses discovered as of then. 563 First, the rational design of zanamivir followed closely after the unravelling of the crystal 564 structures of the haemagglutinin (HA) and neuraminidase/ sialidase (NA)- the two surfaces 565 proteins of the influenza viruses (Colman, Varghese, & Laver, 1983; Varghese, Laver, & 566 Colman, 1983; I. A. Wilson, Skehel, & Wiley, 1981). The identification of the sialidase 567 (neuraminidase) notably generated a lot of attention as many of such sialidase-possessing 568 microbes had been shown to exert some pathogenicity in man (Schauer, 1983, 1985). Several 569 studies, following on, supported a previous posit that the influenza sialidase played a critical 570 role in the disaggregation of new virions after successful cycles of replication in an infected 571 cell (Palese, Tobita, Ueda, & Compans, 1974). Subsequently, it made sense to think of the 572 influenza viruses as able to use the sialidase in the process of dissemination in the mucus lining 573 respiratory tracts, which is known to be rich with lots of sialic acids (Colman & Ward, 1985; 574 Klenk & Rott, 1988; Schulman & Palese, 1977). But more importantly, the known crystal 575 structure of the influenza sialidase/ neuraminidase lead Itzstein and colleagues to apply the 576 rational computer-aided drug design to design two analogues of sialic acids – the 4-Guanidino-577 2,4-Dideoxy-2,3-Dehydro-N-Acetylneuraminic and 4-amino-2,4-Dideoxy-2,3-Dehydro-N-578 Acetylneuraminic, with the former showing a relatively higher fold of inhibition (against both 579 influenza A and B viruses in both in vitro and in vivo) than the amantadine (von Itzstein et al., 580 1993). The 4-Guanidino-2,4-Dideoxy-2,3-Dehydro-N-Acetylneuraminic (also known as, 4-581 University of Ghana http://ugspace.ug.edu.gh 28 guanidino-Neu5Ac2en) would later be selected for being superior to the inhibition of a breadth 582 of influenza viruses (Woods et al., 1993). 583 By 1994, a workup for the synthesis of the 4-guanidino-Neu5Ac2en had been proposed (Figure 584 4) (von Itzstein, Wu, & Jin, 1994), and several other studies had verified the inhibitory capacity 585 of the drug in diverse experimental designs (Hayden, Rollins, & Madren, 1994; Ryan, 586 Ticehurst, Dempsey, & Penn, 1994; Thomas, Forsyth, Penn, & McCauley, 1994). 587 University of Ghana http://ugspace.ug.edu.gh 29 588 Image adopted from von Itzstein et al. (1994) 589 Figure 4: Structure and synthesis of 4-guanidino-Neu5Ac2en. 590 591 University of Ghana http://ugspace.ug.edu.gh 30 The drug had gained the name, Zanamivir by the time the first efficacy and safety studies were 592 being carried out, and here, Hayden and colleagues had seen great success in terms of the drug 593 intervention during the clinical trials (Hayden et al., 1997), receiving US FDA approval in 1999 594 for the treatment of influenza caused by the types A and B viruses (Oxford, 2000). 595 Oseltamivir, another sialidase/ neuraminidase-inhibitor was also being developed concurrently, 596 and this was because zanamivir had a very poor bioavailability via the oral route of 597 administration and was highly efficacious when administered via the intranasal inhalation 598 route. Oseltamivir (then called RO 64-0796 or GS4104) was designed based on the structure 599 of zanamivir and therefore exhibit great resemblance to each other (Figure 5), except that a 600 more stable carbocyclic template was used in place of the Neu5Ac2en dihydropyran ring such 601 that Oseltamivir would have better bioavailability and rapid excretion when orally 602 administered, in addition to desirable efficacy against influenza viruses (Kim et al., 1997). 603 604 University of Ghana http://ugspace.ug.edu.gh 31 605 Image adopted from Kim et al. (1998) 606 Figure 5: Structural resemblance of Zanamivir to GS 4104 (Oseltamivir). 607 Oseltamivir (GS 4104) is the resultant metabolite of the GS 4071. 608 609 Oseltamivir was designed as a pro-drug that unleashes its active anti-influenza metabolite that 610 subsequently exhibited acceptable pharmacokinetic values, besides tolerability and efficacy in 611 adult subjects during clinical trials (Hayden et al., 1999; Massarella & Nieforth). With such 612 appreciable efficacy against both influenza A and B viruses, the Oseltamivir was also approved 613 for use by the US FDA in 1999, just a few months after the Zanamivir was approved (CDC, 614 1999). 615 University of Ghana http://ugspace.ug.edu.gh 32 It would not take too long for the use of the adamantanes to be discouraged for the treatment 616 of influenza A virus infections. This was around 2006 when the US CDC gathered that there 617 was an outrageous spike of adamantane-resistant influenza A viruses as captured by 618 surveillance activities (Bright, Shay, Shu, Cox, & Klimov, 2006). The discovery of the anti-619 influenza properties of the neuraminidase inhibitors - zanamivir and oseltamivir- came in just 620 handy to replace the adamantanes in the fight against the influenza viruses. 621 622 Systematic initiatives: the “one health” initiative, pandemic influenza operational plan, and 623 full deployment of the PCR in the diagnosis of influenza 624 Soon, the fight against influenza viruses would turn robust, as all possible arms of health 625 security reforms had been deployed: 626 The “one health/ one medicine initiative” in 2007 was of interest to the American Veterinary 627 Medical Association (AVMA) with reasons being that there’s a constant human-interface in all 628 facets of human life and that these interactions could foster zoonotic disease “spill-overs” in 629 humans. This initiative strategized to involve all arms of Veterinarians, Physicians, public 630 health, food and agriculturists, and environmentalists, to draw attention to the public health 631 importance of human-animal interactions which could be crucial in the eruption of either a 632 zoonotic disease amongst humans or even, an anthroponotic disease amongst animals (Kahn, 633 Kaplan, & Steele, 2007). This was going to benefit a lot in the global management of influenza 634 due to the story about the origin of influenza viruses (i.e., from birds) and the fact that pigs 635 acted as mixing vessels in the brewing of reassortants which could have a propensity to cause 636 future pandemics. 637 The pandemic influenza operational plan, an initiative to give credence to the ability of newly 638 isolated avian influenza virus amongst birds and humans to be the next likely cause of an 639 University of Ghana http://ugspace.ug.edu.gh 33 influenza virus-related pandemic, was birthed. The isolated virus was characterized to be 640 highly pathogenic to birds and caused fatal infections in humans, thus, creating the scare that 641 the next pandemic could be of avian influenza origin. The plan was, therefore, established both 642 to evaluate national and global healthcare capacities in the detection and containment of any 643 pandemic influenza if one should arise (Ortu, Mounier-Jack, & Coker, 2008). 644 At the time, one of the documented challenges was the heterogeneity in laboratory methods for 645 influenza diagnoses – they were mostly less sensitive and undermined the burden of influenza. 646 The pandemic influenza operational plan also advocated the adoption of the PCR system to 647 enhance the sensitivity and specificity of detection so that such high-fidelity diagnosis would 648 aid in the rapid containment of any influenza if detected at all (Simmerman & Uyeki, 2008). 649 Not too long, the CDC would set up the Influenza Reagents and Resources (IRR) to support 650 the national influenza centres in the PCR-diagnosis of influenza. 651 Eruption of another pandemic influenza virus: The H1N1pdm09 652 Interestingly, all these preparations in anticipation of a future pandemic were applied in 2009, 653 when a triple reassortant (as illustrated in Figure 6) led to a new pandemic (Neumann, Noda, 654 & Kawaoka, 2009). Before the end of 2009, a monovalent H1N1 vaccine was produced to 655 combat the new influenza strain, 6 months post inception (CDC, 2009a, 2009b). 656 657 University of Ghana http://ugspace.ug.edu.gh 34 658 Image adopted from Neumann et al. (2009) 659 Figure 6: Systematic reassortment of the H1N1pdm09. 660 The report by Neumann and colleagues reveals the nature of the present virus as a reassortant of a Eurasian avian-661 like swine influenza virus and a previous triple reassortant virus (one comprising genetic segments of classic 662 swine, North American avian, and human H3N2 influenza viruses). 663 664 The last influenza pandemic observed is the one caused by the H1N1pdm09 in 2009. Though, 665 scientists fear occurrence of another influenza outbreak, it is still not quite certain whether there 666 is going to be another pandemic anytime in the nearest future (Figure 7). 667 University of Ghana http://ugspace.ug.edu.gh 35 668 Figure 7: The 20th-21st Century influenza pandemic timelines and persisting seasonal 669 viruses. 670 The chart here outlines the pandemics caused as a result of influenza viruses. There is still a lot of anxiety about 671 a possible forthcoming influenza-related pandemic and thus, warranting the continuous search for more potent 672 interventions spanning the discovery of universal influenza vaccine candidates and novel therapeutics. 673 674 A new neuraminidase inhibitor - Peramivir 675 Both zanamivir and oseltamivir have been useful in the management of influenza viruses even 676 amidst the challenges with resistance. Peramivir (also known as, Rapivab) [Figure 8] is another 677 drug that was developed based on the rationally designed previous drugs. But this would 678 subsequently be administered intravenously to exert an almost similar or better efficacy against 679 influenza A and B viruses and that warranted the FDA approval in 2014 (Hata, Akashi-Ueda, 680 Takamatsu, & Matsumura, 2014). 681 University of Ghana http://ugspace.ug.edu.gh 36 682 Image adopted from McLaughlin, Skoglund, & Ison (2015) 683 Figure 8: Structure of peramivir 684 685 Influenza viruses 686 Influenza is a contagious disease caused by orthomyxoviruses that comprise the genera 687 influenza- A, B, C, and D -viruses. Influenza presents mostly as an acute upper respiratory tract 688 infection that exerts significantly nearly a million morbidities, plus about half-a-million 689 mortalities, all-year-round (WHO, 2014; Bouvier & Palese, 2008; Kimura et al., 1997; Su, Fu, 690 Li, Kerlin, & Veit, 2017). Broadly, influenza viruses are known to infect both mammals 691 (human, swine, canine, equine, etc.) and avian (such as ducks, chickens, etc.). Influenza A 692 viruses (IAV) mainly exhibit a broad host range -infecting both mammals and avian; and each 693 of Influenza B, C, and D viruses by far infect at least one mammal. As indicated, IAVs 694 (modelled in Figure 9A) are of tremendous public health importance as they have been the 695 main focal viruses responsible for all historical pandemics and many other epidemics recorded 696 among both mammals and avian (Glezen & Couch, 1997; Stuart-Harris & Schild, 1976). 697 Nevertheless, influenza caused by the influenza B viruses (IBV) also poses a significant burden 698 University of Ghana http://ugspace.ug.edu.gh 37 on public health systems (Paul Glezen, Schmier, Kuehn, Ryan, & Oxford, 2013). Hence, the 699 categorization of seasonal influenza viruses comprises both the IAVs and IBVs that mainly 700 infect humans. Two main influenza A virus subtypes that are currently co-circulating globally 701 are the A(H1N1) pdm09 and the A(H3N2), whereas two lineages of the IBVs (i.e., Yamagata 702 and Victoria) are equally co-circulating (Rota et al., 1990). 703 Cases of influenza establishment have been recorded in all ages of individuals (Dosseh, Ndiaye, 704 Spiegel, Sagna, & Mathiot, 2000), however, the high-risk population encompasses persons 705 with compromised immunity due to persisting chronic diseases, children less than 5 years, 706 pregnant women, caretakers of old people’s homes, and the aged (Cheng, To, Tse, Hung, & 707 Yuen, 2012; Harper, Fukuda, Uyeki, Cox, & Bridges, 2005; Nair et al., 2011; W. W. Thompson 708 et al., 2009). Influenza has been managed mainly with antivirals and vaccines: common 709 antivirals being the neuraminidase inhibitors and the matrix 2 protein (ion channel) inhibitors 710 (Gubareva, Kaiser, & Hayden, 2000; McKimm-Breschkin, 2002; Van Voris, Betts, Hayden, 711 Christmas, & Douglas, 1981). The use of vaccines has also been relied on as both a preventive 712 and severity reduction measure. However, these antivirals exhibit either time-restricted 713 efficacy – must be administered within a very narrow period (about 48 hours) between onset 714 and diagnosis, or strain-type restricted efficacy - works against only molecularly susceptible 715 strains (Gubareva et al., 2000; McKimm-Breschkin, 2002). Matrix protein 2 inhibitor class of 716 compounds, also known as adamantanes has been eliminated due to both drug-associated side 717 effects and extensive resistance to seasonal IAVs (Deyde et al., 2007). Therefore, more-or-less 718 rapid emergency warrants for many other drug classes such as the influenza virus polymerase 719 inhibitors (i.e., Favipiravir, Baloxavir marboxil, and Pimodivir), the viral glycoprotein 720 haemagglutinin inhibitors (e.g., Arbidol and Nitazoxanide), the host-targeting sialidase (Das 721 181), and the inhibition of viral release due to drug interference with the influenza viral 722 nucleoprotein (e.g., Ingavirin) (Kotey et al., 2019). The use of conventional vaccines have also 723 University of Ghana http://ugspace.ug.edu.gh 38 had a major impact on the control of influenza (Pebody et al., 2018; Pepin et al., 2019; Sah, 724 Medlock, Fitzpatrick, Singer, & Galvani, 2018; Shim et al., 2018); however, its suboptimal 725 efficacy is a challenge that has been closely associated with “antigenic imprinting”, waning of 726 immunity, vaccine efficacy decline due to repeated vaccination and antigenic-drift or-shift 727 driven mismatch (Belongia et al., 2015; Ferdinands et al., 2017; Gostic, Ambrose, Worobey, 728 & Lloyd-Smith, 2016; Paules, Marston, Eisinger, Baltimore, & Fauci, 2017; Ramsay et al., 729 2019; Shim et al., 2018; Yewdell, Webster, & Gerhard, 1979). Given that influenza is a global 730 burden and the active influenza vaccination platforms are currently limiting, it is important to 731 explore unconventional vaccine development approaches. 732 Influenza contraction, viral transmission, and pathogenesis 733 Influenza is established when any of the viruses infect mucosal surfaces on the respiratory 734 airways, due to aerosol-transmission from an infected individual to a non-infected individual. 735 Notably, wild birds are the reservoirs of the avian IAVs and are responsible for virus shedding 736 throughout the globe, via both air- and waterways (Stallknecht & Brown, 2016). Seasonal 737 influenza viruses, especially, the IAVs on the other hand, have been reported to persist due to 738 both local and external seeding of epidemic strains (Nelson & Holmes, 2007; Nelson, 739 Simonsen, Viboud, Miller, & Holmes, 2007; Nelson et al., 2006; Russell et al., 2008a, 2008b) 740 The viruses are characterized by glycoproteins -the Haemagglutinins - that both define their 741 host specificity and partly play pathogenic roles: in that, a model influenza virus contacts a 742 specific host by the interaction of its haemagglutinins (HA) with specific sialylated receptors 743 on cells lining the airways (Klenk & Rott, 1988). Subsequently, aided by another surface 744 glycoprotein - the Neuraminidase- virions replicate from the initial virus are dispersed to infect 745 neighbouring cells (Liu, Eichelberger, Compans, & Air, 1995). 746 University of Ghana http://ugspace.ug.edu.gh 39 The influenza viruses are highly infectious and require a relatively small amount of inoculum 747 to establish an infection in an exposed susceptible individual, with symptoms acutely 748 manifesting within 3 days post-contraction. Affected individuals largely present with 749 obstructed upper respiratory airways, coughs, high fever, fatigue, and myalgia amongst many 750 other symptoms. Symptoms are estimated to prevail for up to about a week, but the situation 751 may worsen in certain instances where affected persons fall under the high-risk category of 752 persons (Cheng et al., 2012). 753 Influenza virus structure and genome organization 754 A model influenza A virus (Figure 1a) is either a spherical or filamentous particle with a size 755 between 80 and 120 nm in diameter (Lamb & Parks, 2007; Noda et al., 2006). The structure 756 takes shape due to reinforcement of the outer viral lipid envelope by the matrix protein 1 (M1). 757 Embedded within the envelope is the HA, NA, and matrix protein 2 (M2) in declining order of 758 abundance (Nayak, Balogun, Yamada, Zhou, & Barman, 2009). Within the core of the M1 are 759 eight ribonucleoprotein complexes, each of which is made up of three polymerase subunits 760 [i.e., Polymerase basic 1 and 2 (PB1 and PB2) and Polymerase acidic (PA)] and nucleoproteins 761 (NP), all of which are closely associated with a negative-sense RNA segment (Area et al., 762 2004). An RNP particle assumes the structure of a returning twisted rod that has further been 763 coiled to form a helical structure (Compans, Content, & Duesberg, 1972) (Figure 9B) and by 764 function, is responsible for the transcription and overall replication of the influenza virus 765 (Coloma et al., 2009). The size of the RNA segments is estimated to fall within 890 and 2,341 766 nucleotides in length (Lamb & Parks, 2007; Noda et al., 2006). More importantly, each 767 component of the RNP is a prerequisite for a virus to thrive, in terms of infecting, replicating, 768 and re-infecting neighbouring cells. 769 770 University of Ghana http://ugspace.ug.edu.gh 40 771 772 773 774 775 776 777 Image adapted and modified from Kotey et al. (2019) 778 779 780 Figure 9: Structural description of IAV. 781 A: Basic structure of IAV; B: Ribonucleoprotein complex 782 783 Life cycle of influenza A virus 784 Using the HA, the virus first makes contact and adsorb to a susceptible cell via interaction with 785 sialylated receptors, which triggers the mechanism of endocytosis (Wenjie Zheng & Tao, 786 2013). Endosomal maturation by acidification is an immune process that aids the destruction 787 of endocytosed pathogens; however, this is favourable for the influenza virus, in that the M2 -788 A B HA NA M2 University of Ghana http://ugspace.ug.edu.gh 41 an ion channel- conducts protons which leads to the activation of the fusion domain of the HA 789 and subsequently facilitating fusion of the viral envelope with the endosomal membrane, 790 thereby dissociating the vRNPs from the M1 (Das, Aramini, Ma, Krug, & Arnold, 2010). Viral 791 RNPs (vRNPs) are then released and shuttled directly into the nucleus due to the recognition 792 of nuclear localization signals (NLSs) on the nucleoproteins (Wu & Panté, 2009). Within the 793 nucleus, two main viral replication pathways occur: in one pathway, the viral polymerase 794 complex initiates the synthesis of a positive-sense strand complementary RNA (+cRNA) for 795 each of the 8 negative-sense strand viral genomic segments, and these are required to further 796 make more genomic segments (the viral RNP, vRNP) th