Performing Department
Veterinary Population Medicine
Non Technical Summary
Control of influenza viruses in domestic swine through killed vaccine administration assumes that the critical antigenic sites needed for elimination or reduction of the virus within the pig are the same as known for humans. The fact that these assumptions are incorrect is reinforced by the fact that there has only been a marginal reduction of viral shedding and control of the virus within the swine population of North American and the European Union. The key antigenic sites of the influenza hemagglutinin (HA) for swine are unknown. To map the antigenic epitopes of the swine influenza virus (SIV) HA, swine-specific anti-HA monoclonal antibodies (mAb) are needed. Currently, swine mAbs are not available and need to be generated. The purpose of this study is to generate swine specific anti-HA influenza mAbs by hyperimmunizing pathogen-free swine with purified HA (recombinant; rH3) from a current antigenic drift variant of H3N2 SIV, a virus similar to that which swept the Canadian swine industry beginning in 2005 and resulted in several human cases of the disease during that period. Once swine-specific mAbs are generated, their binding affinities and neutralization rates will be compared to swine-specific anti-HA polyclonal antibodies collected from the same immunized pigs and to human-derived anti-HA human-derived mAbs from St. Jude Library. Finally, a map of the swine H3 HA molecule including localization and structure of the antigenic sites compared to that of human H3 HA will be made. By use of H3N2 SIV, direct comparison of actual swine immune responses may be made with the extensive data available for H3N2 infection of man. Knowledge of the antigenic sites of H3 SIV will allow researchers to determine the mutations of the influenza virus that make it possible to escape vaccinal immunity, to predict cross-protection between vaccine strains and field strains by detecting changes in the identified key antigenic sites of immunity, and possibly identify new targets for SIV vaccine design. Control of influenza virus in swine is prudent and feasible given they are potential intermediate hosts for interspecies transmission and have been implicated as mixing vessels capable of creating viruses of pandemic potential. To date, we have completed all animal studies and have generated and sequenced out full length single chain fragment variable antibody (scFvs) against SIV-specific rH3 and H3N2 using the yeast surface display system (isolated by the yeast c-myc tag and rH3 reactivity). We are seeking funds to complete epitope mapping and characterization of scFvs.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The goals of this project are to: 1) Confirm single chain fragment variable antibody (scFvs) binding to recombinant swine hemagglutinin 3(rH3) and determine influenza virus binding and affinity ranges. Specific Aim 2: Create a swine specific H3 epitope map using scFvs against rH3 and swine H3N2. Upon completion of goals 1 and 2, we expect to confirm scFvs binding to rH3 and to swine H3N2 and to identify cross-reactivity with other hemagglutinins and influenza viruses from various hosts, if any. Furthermore, linear, conformational and discontinuous epitope sites will be identified and a 3D rendering of scFv-rH3 will visualize binding sites (e.g. binding site is in a pocket, masked by other viral features). These studies have the potential for high impact in creating and characterizing novel monoclonal antibodies (scFvs) with high affinity to target hemagglutinin and influenza viruses that may aid in current diagnostics. scFvs will be used by influenza surveillance programs worldwide and detect emerging and reemerging influenza infections in swine, a population of animals that is currently neglected for surveillance and may serve as mixing vessels for potential human influenza cases.
Project Methods
Plasmids from scFvs E. coli transformants will be extracted, restricted and cloned into pNL9 (yeast secretion vector). The newly created plasmid will be transfected into yeast cells. Secreted scFvs will be purified and confirmed by mass spectrometry. rH3 and H3N2 binding to scFvs will be confirmed by Western blot analysis. In order to determine scFv specificity, other hemagglutinin proteins from human, avian and swine sources will be included. Affinity binding will be determined by flow cytometry of scFvs yeast clones and rH3 binding. The equilibrium dissociation constant (Kd) will be calculated using the following equation: y = m1 + m2* m0/(m3 + m0) , where y = MFI at a given rH3 concentration, m0 = rH3 concentration, m1 = MFI of no rH3 control, m2 = MFI at saturation, and m3 = Kd. Generated R square values of 0.998 and greater will give accurate Kd values within 30 per cent. Overlapping epitopes will be determined by competitive binding assays. The following antibodies with known binding sites to rH3 will be biotin labeled: mouse anti-influenza A virus hemagglutinin H3 monoclonal antibodies clones B263M and lnA227 and those provided by Dr. Richard Webby's laboratory. Yeast cells displaying individual scFvs reactive to swine rH3 will be labeled with mouse anti-c-myc-FITC mAb. Yeast cells will be resuspended in PBS containing 0.05% BSA and 100 nM of unlabeled rH3 for 1 h at room temperature. Unbound antigen will be removed by PBS washes and yeast will be resuspended and equally divided into tubes for each biotin labeled antibody. Bound rH3 will be detected by incubating yeast cells with biotin labeled antibodies (known binding sites to H3) separately for 30 min on ice and unbound mAbs will be removed by washing with PBS. rH3-mAbs complexes will be detected by the addition of streptavidin-PE. Yeast cells will be analyzed using flow cytometry by gating the FITC+/PE+ population. A positive PE population indicates that yeast scFv surface displayed cells reactive to rH3 bound to an epitope that is non-overlapping and non-competing with biotinylated mAbs. Non-overlapping/non-competing scFvs will be further analyzed for novel conformational, discontinuous or linear epitopes. Conformational epitopes will be determined by mass spectrometry of scFv bound to rH3. Spectra will be recorded at a 25 kV acceleration voltage with a deflection pulse of 1 micro s. Epitope sequences will be analyzed using X!Tandem and Scaffold software. In order to confirm linear, discontinuous and conformational epitopes, scFvs binding to rH3 PepScan and CLIPS arrays will be conducted. Arrays will be designed to include 15 mer peptides (in triplicate) with 4 overlapping amino acids. scFvs binding will be determined by optical density will be recorded on an ELISA plate reader and software. scFV binding to peptides will be determined by the colorimetric shift compared to the no scFv control. Identified scFv paratopes will be visualized graphically using Schrodinger suite. A 3D rendering of swine H3 will be created by stringing the amino acid sequence through the available human H3 model available through the Influenza Research Database.