Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection (2014)

In nature, proteins with weak binding affinity often use a multivalency approach to\nenhance protein affinity via an avidity effect. Interested in this multivalency approach,\nwe have isolated a carbohydrate binding module (CBM) that recognises sialic acid\n(known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcus\npneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from them\nusing molecular biology. Multivalent CBM40 constructs were designed either using a\ntandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBM\nand Vc4CBM, respectively, or through the addition of a trimerization domain derived\nfrom Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms of\nproteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due to\nthe position and flexibility of the linker between the trimerization domain and the\nCBM40 domain, site directed mutagenesis was employed to introduce a disulphide\nbond between the monomers at positions S164C and T83C of the CBM40 domain in\norder to promote a stable orientation of the binding site for easier access of sialic\nacids.\nData from isothermal titration calorimetry (ITC) reveals that interaction of multivalent\nCBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropy\ncontributing unfavorably to the interaction suggesting that these proteins establish a\nstrong binding affinity to their ligand minimizing dissociation to produce stable\nmultivalent molecules. However, using surface plasmon resonance (SPR), a mixed\nbalance of entropy and enthalpy contributions was found with all constructs as\ndetermined by Van’t Hoff plots. This proved that binding does not occur through a\nsimple protein-ligand interaction but through disruption of hydrophobic and/or ionic\nhydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization.\nHowever, little is known about their detailed structure when in multivalent form, as\nattempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due to\nlinker and domain flexibility. Only the trimerization domain (TD) part from\nPseudomonas aeruginosa pseudaminidase was successfully crystallized and\nstructure was determined to 3.0 Å without its CBM40 domain attached.\nIn this thesis, we have also reported on the potential anti-influenza and anti-\nparainfluenza properties of these proteins, which were found to block attachment and\ninhibit infection of several influenza A and parainfluenza virus strains in vitro. As\nwidely mentioned in literature, terminal sialic acids on the cell surface of mammalian\nhost tissue provide a target for various pathogenic organisms to bind. Levels of viral\ninhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBM\nconstructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most of\nthe multivalent proteins tested were also effective against A/WSN/33 H1N1 and\nA/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V.\ncholerae sialidase CBM40 domain showed great effect in inhibiting virus infection\nduring cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT)\nfollowed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which was\nagainst hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S.\npneumoniae sialidase known as Sp-CBMTD showed negligible effect on cell\nprotection. All constructs were further tested for cytotoxicity in mammalian cell culture\nas well as undergoing an inhibition study on viral replication proteins. For the in vivo\nstudy, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats and\nmice from hPIV3 and Streptococcus pneumoniae infections, when given intranasally\nin advance or on the day of infection. Therefore, these novel multivalent proteins\ncould be promising candidates as broad-spectrum inhibitors or as a prophylactic\ntreatment for both influenza and parainfluenza associated diseases.