Isolation of broadly cross reactive human antibodies against dengue virus using a human non immunized fab phage display library

Introduction 1.1 Epidemiology Dengue virus (DENV) is an arthropod-borne virus belonging to the Flaviviridae family Consisting of four related but antigenically distinct serotypes (DENV1-4), they rely on mosquito vectors that live in close association with human for effective transmission DENVs are of immense global clinical importance; approximately 50-100 million cases of DENV infections worldwide and about 500,000 cases of the life-threatening form of severe dengue – dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur annually (WHO, 1997a) Dengue is now considered to be the most rapidly spreading vector-borne disease, posing a serious public health threat (Pinheiro et al., 1997) The World Health Organization (WHO) estimates that at least 100 countries are endemic and about 40% of the world’s population is at risk of infection each year (Tan, 1997) Its global emergence and re-emergence may be a result of multiple factors, including unplanned and uncontrolled rapid urbanization combined with inadequate wastewater management, a lack of effective mosquito control leading to the increased distribution and density of vector and global climate change; all of which combined to result in increased spread of the virus (Kyle et al., 2008; Pinheiro et al., 1997) Increased movement of people among population centres via modern transportation may also contribute to the resurgence of dengue (Gubler, 2002b) Additionally, some studies have suggested that the resurgence of dengue infections may be due to microevolution of the virus, resulting in more virulent strains replacing the less virulent genotypes (Klungthong et al., 2008; Steel et al., 2010; Weaver et al., 2009) Substantial economic, political and social effects associated with major dengue urban epidemics, such as those seen in Cuba (1977-79, 1997) (Guzman et al., 2008), Delhi (1996) (Kabra et al., 1999), Taiwan (2002) (Teng et al., 2007) and Brazil (2008) (Honorio et al., 2009) all add to the growing concern of the disease Dengue is also a leading cause of morbidity in tourists and military personnel who travel to dengue endemic areas (Gubler, 2002a; Webster et al., 2009) With no approved vaccine or cure, dengue thus continues to pose as a major public health problem Although significant effort and resources had been applied towards DENV vaccine development over the last decades and several candidates are now entering late phase clinical trials, a safe and efficacious vaccine is not likely to be ready in the near future It is thus vital to improve our understanding of the pathogenesis and life cycle of DENV to reveal potential targets which may be of use in the development of a successful vaccine and be potentially of use in the development of chemotherapy 1.2 Mosquito vectors Transmission of dengue is dependent on mosquitoes belonging to the genus Aedes A albopictus and A polynesiensis can sustain transmission, but the primary and most important vector for DENVs is A aegypti, whose behavior and bionomics contributed to the highly efficient inter-human transmission of dengue This mosquito is highly adapted to the urban environment and prefers artificial water containers such as flower vases or coconut shells as larval habitats; its eggs are able to survive for a long period, withstanding desiccation, therefore able to exploit increased breeding sites provided by uncontrolled and unplanned urbanization (Halstead, 2008) Adult A aegypti uses human habitations as resting and host-seeking habitat, where the female mosquito feed on human hosts for blood as the source of protein for oogenesis and energy for flight, maximizing human-vector contact and minimizing exposure to insecticides sprayed outdoor (Weaver et al., 2010) In addition, A aegypti is a nervous feeder which often feeds on multiple human hosts in one single meal, increasing the number of hosts and the probability of becoming infected Recently, studies have reported the increasingly similar breeding behaviour of A albopitus to A aegypti, shifting from an outdoor to indoor domestic environment, causing a greater risk of dengue transmission (Dieng et al., 2010; Rao et al., 2010) The environment also has a part to play as studies have shown that dengue epidemics tend to coincide with rainy seasons, corresponding to observations of significant increase in mosquito larval populations (Rappole et al., 2000) Furthermore, the rate of viral propagation in mosquitoes is influenced by ambient temperature and relative humidity, where a rise in environmental temperature shortens the extrinsic incubation period, ie the time needed for a female mosquito to becoming infective after acquiring an infectious bloodmeal (Halstead, 2008) Various groups have reported on meteorological influences on the abundance of the adult dengue vector, and efforts to develop predictive models to instigate pre-emptive vector control operations have been reported (Chadee et al., 2007; Dibo et al., 2008; Favier et al., 2006) While positive relationships between rainfall, temperature, humidity and dengue vector abundance have been reported by several groups, the relative significance of each of these factors varied among the different reports These conflicting observations may be due to the differing local ecology of the areas under study, with these three variables impacting A aegypti populations in slightly different ways; highlighting the need to investigate the diversity of relationships between entomological and meteorological indices at local ecological scales (Azil et al., 2010) Community-based programs aimed at vector control through the elimination of A aegypti breeding sites or by application of larvicides have been carried out in the Americas (1940s – 1970s) (Soper, 1963; Tsikarishvili et al., 1964), Cuba (1980s) (Kouri et al., 1989) and Singapore (1960s -1980s) (Tan, 1997) and were found to be highly effective While these efforts in Singapore and Cuba continue, those in the Americas were not sustainable as various factors eroded their effectiveness including development of pesticide resistance, an awareness of the side-effects of insecticide, decrease in government funding for public health services and the failure of horizontal programs integrating education and community to motivate community participation (Knudsen et al., 1992) As a result, A aegypti rapidly re- colonised the Americas after the cessation of the A aegypti eradication program and dengue reappeared Peculiarly, Singapore experienced a surge of dengue incidences despite low premises index after 15 years of low dengue incidence This may be due to a combination of factors, including lowered herd immunity as a result of reduced dengue transmission as well as a shift of dengue transmission profile, whereby there was increasing virus transmission away from home, consequently, cases in adults predominated as opposed to children as in other parts of Southeast Asia (Ooi et al., 2006) Most dengue infections, particularly in young children, are mild or silent, whereas adults are more likely to be clinically overt, and may contribute to a resurgence of dengue incidence (Ooi et al., 2009; Ooi et al., 2003; Seet et al., 2005) 1.3 Clinical manifestation DENV infections cause a spectrum of clinical outcomes, ranging from asymptomatic to mild flu-like illness to classical dengue fever (DF) and the severe dengue hemorrhagic fever (DHF) DF is a self-limiting febrile illness associated with fever, headache, myalgia, nausea and vomiting, accompanied by joint pains, weakness, rash and thrombocytopenia The fever may last for to days, with a saddleback pattern, characterized by a drop in fever after a few days only to rebound 12 to 24hr later DHF often follow secondary dengue infections, but may sometimes occur in primary infections, especially in infants (Dietz et al., 1996; Halstead et al., 2002) DHF is characterized by high fever, haemorrhagic manifestations and circulatory failure as a result of plasma leakage Increased vascular permeability may lead to shock or dengue shock syndrome (DSS), which is associated with a very high mortality rate if not managed properly Although less common, severe dengue infection may give rise to complications such as encephalitis, hepatitis, myocarditis and renal dysfunction (Pancharoen et al., 2002) According to the WHO DHF case definition (WHO, 1997a) developed in the 1970s, a patient with acute sudden onset of high fever for 2-7 days with platelet count of less than 100 x 109/L and signs of plasma leakage as well as haemorrhagic tendency is classified as having DHF (WHO, 1997b) However, atypical clinical manifestations of dengue infection which can also lead to poor clinical outcomes are increasingly reported, challenging the current definitions (Deen et al., 2006) The Increasing trend of adult dengue has caused a review of the WHO classification scheme, useful in case management of paediatric patients, as it may underestimate severe dengue in adults; the course of dengue infection in adults may be complicated by differences in physiological state compared to children, as well as the greater prevalence of morbid conditions, such as asthma, hypertension, cardiovascular diseases and diabetes (Chen et al., 2004; Guzman et al., 1992; Limonta et al., 2008) In the light of these observations, the WHO has recently reviewed the dengue guidelines and published a different set classification scheme (WHO, 2009) 1.4 Dengue virus 1.4.1 General virology DENVs consist of a single-stranded positive-sense RNA genome of about 11kb with a cap structure at the 5’ end but lacks a poly(A) tail at the 3’ end; a single long open reading frame (ORF) is flanked by 5’ and 3’ untranslated regions which are important cis-acting elements for replication, transcription and translation The ORF is translated as one polyprotein which is co- or post-translationally processed by host and viral proteins in distinct cellular compartments into three structural proteins, the capsid (C), precursor membrane (prM) and envelope (E) as well as seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Zhang et al., 2003) (Fig 1) The three structural proteins are associated with the membrane of the rough endoplasmic reticulum (rER) by C-terminal membrane-spanning regions While host signalase in the rER lumen is responsible in cleavages distal to the membrane-spanning regions of C, prM and E; a cleavage proximal to the membrane-spanning region of C is carried out by viral protease in the cytoplasm, both of which are essential for viral particle assembly (Mukhopadhyay et al., 2005) Upon cleavage, the newly generated C-terminal of prM, which contains the transmembrane domain, is anchored to the membrane and serves as the signal sequence for the translocation of E The prM is able to fold into its native structure independently of E; however, several studies have shown that E requires the co-expression of prM to acquire its native conformation, suggesting a chaperone-like role of prM in the folding of E (Chang et al., 2003; Kim et al., 2008) Partially assembled flavivirus nucleocapsid, which consists of the RNA genome organized with 180 copies of C protein, buds from the endoplasmic reticulum (ER), becoming enveloped by the host-derived lipid bilayer that carries with it the two viral surface proteins: prM and E, which interact to form prM-E heterodimers, forming immature virus particles Shortly before the virus is released from the cell, the 91 residue precursor portion (pr) of prM in the N-terminal region is cleaved off, leaving the C-terminal portion, the mature membrane protein (M) anchored in the membrane Eventually, soluble pr peptide and mature virus are released into the extracellular environment (Yamshchikov et al., 1993; Yu et al., 2009; Yu A B Fig Schemetic diagram of DENV genome and polyprotein (Adapted from (Perera et al., 2008)) (A) The single-stranded positive sense RNA viral genome is ~11 kb in length with a capped 5’ end (B) Membrane topology of the polyprotein The viral RNA is translated as a single polyprotein and processed by cellular and viral proteases (denoted by arrows) to give structural proteins: capsid (C), pre-membrane/membrane protein (prM/M) and envelope (E); and non-structual proteins: NS1, NS2A/B, NS3, NS4A/B and NS5 Signalase cleavage in the ER release prM and E from the polyprotein, but they remain anchored on the luminal side of the membrane C is also anchored in the ER membrane (on the cytoplasmic side) by a conserved hydrophobic signal sequence at its C-terminal end This signal sequence is cleaved by the viral NS2B–NS3 protease prM is cleaved into pr peptide and M by host furin during virus maturation in the TGN The non-structural proteins are processed mainly by the viral protease NS2B–NS3 in the cytoplasm with the exception of NS1, which is released from NS2A by a yet unidentified protease in the lumen of the ER NS2A/2B and NS4A/4B are anchored in the ER as transmembrane proteins et al., 2008) The cleavage site of prM occurs immediately after the amino acid (aa) sequence Arg-X-Arg/Lys-Agr (X is variable), which corresponds to the consensus sequence of the protease furin and this cleavage reaction probably takes place in the acidic compartment of the TGN; this is supported by studies showing inhibition of cleavage by raising the pH of the acidic intracellular compartments using acidotrophs or by furin inhibitors, suggesting that this cleavage reaction involves furin and is pH dependent (Guirakhoo et al., 1992; Junjhon et al., 2008; Randolph et al., 1990; Yu et al., 2008) It is believed that during the transport of the immature virion through the ER and Golgi apparatus to the cell surface, prM protects E from undergoing the lowpH induced conformational changes which are required for membrane fusion and infectivity, thus preventing premature fusion with host membrane, as immature virions with uncleaved prM are non-infectious, being unable to fuse with cells even at acidic conditions (Elshuber et al., 2003; Kim et al., 2008) 1.4.2 Structure and maturation Fusion of viral and host cell membrane is an obligatory step of entry and subsequent infection for enveloped viruses Viral surface proteins are the critical agents involved, primed to facilitate fusion of the lipid bilayers and they are usually produced as inactive precursors which require proteolytic cleavage to achieve their fusogenic potential The best-studied example is the influenza virus haemaglutinin (HA), which belongs to the class I fusion proteins It is synthesized as a single-chain precursor and requires cleaveage into two chains, HA1 and HA2, before the trimeric HA can become fusion competent (Gething et al., 1986) HA1 is responsible for receptor binding which leads to endocytic uptake of the virus; acidification of the endosomes triggers conformational rearrangement of HA2, resulting in the exposure of the N-terminal fusion peptide which inserts into the endocytic membrane, further conformational change results in fusion of viral and host cell membrane (Harrison, 2008) All class I fusion proteins are two-chain products of a cleaved, single-chain precursor with a hydrophobic N-terminal fusion peptide that is liberated by the cleavage Flavivirus fusion proteins on the other hand, belong to an architecturally and evolutionarily distinct class of fusion proteins, known as the class II fusion proteins These proteins associate with a second ‘protector’ protein, whereby cleavage primes the fusion protein to respond to acidic pH (Elshuber et al., 2003; Kielian, 2006; Modis et al., 2004) For flaviviruses, the E protein binds cell surface receptors and is involved in virus entry by envelope fusion with host cellular membrane It is a glycoprotein of approximately 55kDa with twelve strictly conserved cysteine residues forming six disulphide bridges (Roehrig et al., 2004; Stiasny et al., 2006a) X-ray crystallographic analysis of the structure 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Virus pr-E Protein Interactions PLos Path 6(10): e1001157 142 Appendix I Table of amino acids nomenclature from www.sigmaaldrich.com) 143 (Adapted Reagents: Media and agar for Humanyx phage display library 16g/L Tryptone (Bacto, BD) + 10g/L Yeast 2TY Extract (Bacto, BD) + 5g NaCl + deionized water 2TY + 100μg/ml Ampicillin + 50μg/ml 2TYAK Kanamycin 2TY + 15g/L Agar (Bacto, BD) + 100μg/ml 2TYAK agar Ampicillin + 50μg/ml Kanamycin 2TY + 15g/L Agar (Bacto, BD) + 100μg/ml 2T AG agar Ampicillin + 20% Glucose (v/v) For preparation of SDS-PAGE gel 4x Sep rating buffer 1.5mM Tris-HCl, pH 8.8 4x Stacking buffer 0.5mM Tris-HCl, pH 6.8 1.0g Ammonium persulphate 10ml deionized water 0.225M Tris-HCl, pH 6.8 + 0.05% Bromophenol Blue + 50% Glycerol + 5% SDS 0.225M Tris-HCl, pH 6.8 + 0.05% Bromophenol Blue + 50% Glycerol + 5% SDS + 0.25M DTT 10% Ammonium persulphate 5x non-reducing SDS-PAGE sample buffer 5x reducing SDS-PAGE sample buffer Solution and media for PPh.D-12 random dodecapeptide library (NEB) 0.1M NaHCO3, pH8.6 + 5mg/ml BSA + 0.02% Blocking buffer NaN3 (Filter sterilized) Coating buffer 0.1M NaHCO3, pH8.6 50mM Tris-HCl, pH 7.5 + 150mM NaCl + 0.1% 0.1% TBST Tween 20 50mM Tris-HCl, pH 7.5 + 150mM NaCl + 0.5% 0.5% TBST Tween 21 Elution buffer 0.2 M Glycine, pH 2.2 + 1mg/ml BSA Neutralization buffer M Tris-HCl, pH LB broth 20g/L LB Broth, Lennox (Difco, BD) 35g/L LB Agar, Lennox (Difco, BD) + 0.01µM IPTG + 0.005% Xgal 10g Tryptone + 5g Yeast extract + 5g NaCl + 7g Agar 10mM Tris-HCl, pH8.0 + 1mM EDTA + 4M sodium iodide 10mM Tris-HCL, pH 7.5 + 1mM EDTA LB agar for phate tittering Top agar Iodide buffer TE buffer Buffers for IFA Block buffer Wash buffer 5mg/ml BSA + 0.1% Saponin + 0.01% NaN3 in PBS 1mg/ml BSA + 0.1% Saponin + 0.01% NaN3 in PBS 144 ... screening all serotypes of DENV sequentially using a human non -dengue immunized phage display library (Humanxy library) and to convert the Fab fragments isolated into full-length human IgG1 in a mammalian... human phage library such as the Humanyx Fab phage display library was constructed by amplifying the V-genes from the IgM mRNA of B cells of non- immunized individuals This procedure provides antibodies. .. RT and washed with water Plaques were counted with naked eye and plaque forming unit (pfu)/ml was calculated 43 2.5 Panning for human Fab from a phage display library Human Fab screening was