Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus...

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MonoclonalAntibodiestotheHaemagglutininHAISubunitofthePandemicInfluenzaA/H1N12009VirusandPotentialApplicationtoSerodiagnosis

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Monoclonal antibodies to the Haemagglutinin HA1 subunit of the pandemic influenza A / H1N1 2009 virus and

potential application to serodiagnosis

Journal: Journal of Medical Virology

Manuscript ID: JMV-10-1858.R2

Wiley - Manuscript type: Research Article

Date Submitted by the Author:

23-Sep-2010

Complete List of Authors: Samuel, Dhan; Health Protection Agency, Centre for Infections,

Virus Reference Department Warrener, Lenesha; Health Protection Agency, Centre for Infections, Virus Reference Department Hoschler, Katja; Health Protection Agency, Centre for Infections, Virus Reference Department

Keywords: Influenza A/ H1N12009, Monoclonal antibodies, HA1 subuint

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Monoclonal antibodies to the Haemagglutinin HA1 subunit of the pandemic influenza A / H1N1 2009 1

virus and potential application to serodiagnosis 2

Dhanraj Samuel*, Lenesha Warrener, Katja Hoschler 3

Virus Reference Department, Health Protection Agency, Centre for Infections 4

*Corresponding Author: dhan.samuel@hpa.org.uk 12

Tel: 44 (0) 20 8327 6254 13

Fax: 44 (0) 20 8205 8195 14

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ABSTRACT: 15

In order to provide specific serological reagents for pandemic influenza A/H1N1 2009 virus, 16

monoclonal antibodies (Mabs) to recombinant haemagglutinin component HA1 (rHA1) were 17

generated after fusing spleen cells from a mouse immunised with rHA1 protein derived from influenza 18

strain A/California/06/09 H1N1 with a mouse myeloma cell line. 19

Five hybridoma clones secreting Mabs specific for the rHA1 protein derived from pandemic influenza 20

A/H1N1 2009 and not for rHA1 from seasonal H1N1 influenza strains A/Brisbane/59/07 and 21

A/Solomon Islands/03/06 were identified by EIA. Mabs 7H4, 9A4 and 9E12 were reactive in Western 22

blots with full length rHA and/or rHA1 subunit derived from A/California/06/09 strain. Only Mab 1F5 23

inhibited haemagglutination of turkey red blood cells with recombinant NIBRG-121 virus derived from 24

A/California/07/09, but did not react in Western blots. Immunostaining of MDCK cells infected with 25

NIBRG-121 was localised to the membrane/cytoplasm for four of the reactive Mabs. The differing 26

reactivity of the Mabs in Western blots, immunostaining, EIA and haemagglutination inhibition assay 27

suggest that at least four of the five Mabs recognise different epitopes on HA1 of the pandemic 28

influenza A/ H1N1 2009 virus. 29

Ferret antisera to pandemic influenza A /H1N1 2009 (A/England/195/09 and A/California/07/09 30

strains) and sera from human subjects vaccinated with Influenza A (H1N1) 2009 Monovalent Vaccine 31

(CELTURA ®, Novartis Vaccines, Germany), inhibit binding of 1F5-HRP to biotinylated rHA1 derived 32

from A/California/06/09 in a competitive EIA, suggesting that the epitope recognised by this Mab also 33

evokes an antibody response in infected ferrets and vaccinated humans. 34

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INTRODUCTION. 36

Influenza A viruses are negative sense, single-stranded RNA viruses that belong to the genus 37

Influenzavirus A in the family Orthomyxoviridae. The haemagglutinin (HA) of influenza resides on the 38

surface of the virus and initiates the viral infectious cycle by attaching to sialic acid residues in host 39

cells and inducing fusion (Skehel and Wiley, 2000). Depending on the virus strain, host cell type and 40

growth conditions used, the HA can exist in its uncleaved form (HA0 MW ~76 KDa) or in its cleaved 41

form, consisting of two di-sulphide linked chains HA1 and HA2 (Klenk et al., 1975; Lazarowitz and 42

Choppin ,1975). HA is the major viral antigen against which neutralising antibodies are produced, and 43

influenza epidemics are associated with changes in its antigenic structure (Lamb and Krug, 1996). 44

Influenza pandemics occur when an influenza virus with a HA, against which there is little or no 45

existing immunity, emerges in human populations and transmits efficiently from human to human 46

(Garten et al., 2009). Between March and April 2009, a previously unknown variant of influenza A/ 47

H1N1 caused sporadic infections and epidemics in North America and Mexico, resulting in a global 48

pandemic (MMWR 2009a; MMWR 2009b , Hancock et al., 2009; Miller et al., 2010 and Ikonen et al., 49

2010.). Antigenic and genetic characterisation of the 2009 pandemic influenza A/ H1N1 virus revealed 50

it to be very different from seasonal H1N1 viruses (Garten et al., 2009). Structural analysis revealed 51

up to 27% difference between the amino acid sequence of HA1 of the pandemic virus influenza A/ 52

H1N1 2009 and the 2007 seasonal influenza A H1N1 virus, with the majority of the changes in the 53

globular, antigenic sites of the HA1 protein (Ikonen et al., 2010). Consequently, serological reagents 54

specific for seasonal influenza H1N1 cannot be used for detection of the pandemic influenza A/ H1N1 55

2009 virus. 56

The aim of the present study was to generate monoclonal antibodies specific for the HA1 of pandemic 57

influenza A/ H1N1 2009 strain and to characterise these antibodies in terms of specificity, potential 58

utility in research and serological investigations of the influenza A/ H1N1 2009 pandemic. 59

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MATERIALS AND METHODS 61

Viral proteins: The following purified recombinant proteins, all of which were 6 x His tagged and 62

expressed in 293 cells, were obtained from e-Enzyme LLC (Montgomery Village, MD, USA): 63

haemagglutinin HA1 (A California/06/09 H1N1; amino acids 18-344), haemagglutinin HA (A/ 64

California/06/09 H1N1; amino acids 18-530), haemagglutinin HA1 (A/Brisbane/59/07 H1N1; amino 65

acids 18-343), haemagglutinin HA (A/Brisbane/59/07 H1N1; amino acids 18-530), haemagglutinin 66

HA1 (A/Solomon Islands/3/06 H1N1; amino acids 18-344), haemagglutinin HA (A/Solomon 67

Islands/3/06 H1N1; amino acids 18-530), haemagglutinin HA1 (A/New Caledonia/20/99 H1N1; amino 68

acids 18-345), haemagglutinin HA (A/New Caledonia/20/99 H1N1; amino acids 18-530), HA 69

(B/Malaysia/2506/04; amino acids 12-547), haemagglutinin HA1 (A/Vietnam/1203/04 H5N1; amino 70

acids 1-345) and haemagglutinin HA (A/Vietnam/1203/04 H5N1; amino acids 18-530). 71

Biotinylation of proteins: Approximately 50µG of the viral proteins and 6mG of bovine serum albumin 72

(BSA) were biotinylated using the EZ-Link™NHS-Biotin from Pierce (Rockford, IL, USA) essentially as 73

described in the supplied instructions. 74

Streptavidin coated microtitre plates: 12 x 8 U-well strip microplates, (Greiner Bio-one, Stonehouse, 75

UK) were first coated with 100µL/well of a 50µG/mL solution of BSA-biotin in 0.05M carbonate buffer, 76

pH 9.5 at 2-8ºC overnight. The plates were washed with PBS containing Tween-20 (0.05% v/v, PBST) 77

and then blocked with 200µL/well of a blocking buffer (Microimmune Ltd, Hounslow, UK) for 2h at 78

37ºC. To the BSA-biotin plates was added 500nG/mL streptavidin (Prozyme, Inc, Hayward, CA, USA) 79

in the blocking buffer for 1h at 37ºC and the wells aspirated and stored at 2-8ºC until used. 80

Monoclonal antibody production: A BALB/c mouse was immunised subcutaneously at fortnightly 81

intervals on four separate occasions with a mixture containing 40µG of rHA1 (A/California/06/09) 82

protein in 90µL of PBS containing 50µG of N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (MDP, 83

Sigma -Aldrich Co. Ltd, Poole, UK) and an equal volume of Freund’s Incomplete Adjuvant (Sigma -84

Aldrich Co. Ltd, Poole, UK). A test bleed was taken one week after the last immunisation. Three days 85

prior to splenectomy, the mouse was again immunised subcutaneously as before, except that 25µG of 86

rHA1 was used. 87

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The splenocytes from the immunised mouse were fused with the NS1 mouse myeloma cell line by 88

standard procedures described previously (Kohler and Milstein, 1976; Tedder et al., 1982) and the 89

fused cells distributed into 10 x 96-well tissue culture plates containing selective hypoxanthine, 90

aminopterin and thymidine medium. The serum obtained from the test bleed was used to develop the 91

enzyme immunoassays (EIAs) used for sceening hybridoma supernatants described below. 92

Hybridomas found to be positive in the EIA were expanded and cloned by limiting dilution. 93

EIA for screening of hybridoma supernatants: Separate microtitre plates (Greiner Bio-one, 94

Stonehouse, UK) were coated with rHA1 subunit (A/California/06/09) and whole rHA 95

(A/California/06/09) in carbonate coating buffer as described for streptavidin coated plates. In 96

addition, biotin-rHA1 plates were prepared by adding 100µL/well of biotinylated rHA1 (200nG/mL) in 97

3% (w/v) BSA in PBST to the streptavidin coated plates and incubating at 2-8ºC overnight. After 98

washing with PBST, these wells were also used to screen hybridoma supernatants in the EIA 99

procedure described below. 100

To 100µL of hybridoma supernatants was added 250µL of 1% (w/v) milk in PBST containing 0.05% 101

(w/v) Bronidox-L (Henkel, Germany, PBSTBx). 100µL of the diluted hybridoma supernatant was 102

added to wells of rHA1 (A/California/06/09), rHA (A/California/06/09) and biotin-rHA1 103

(A/California/06/09) coated plates and incubated for 1h at 37ºC. The plates were washed four times 104

with PBST and 100µL/well of rabbit anti-mouse IgG-horseradish peroxidase (HRP) conjugate (Dako 105

UK Ltd, Ely, UK), diluted 1/4000 in 1% (w/v) milk in PBSTBx, was added and incubated for 30min at 106

37ºC. The plates were washed and incubated with 100µL/well of TMB substrate (Microimmune, 107

Hounslow, UK) until colour developed. The colour development was terminated by the addition of 108

100µL/well of 0.5M hydrochloric acid and the optical density readings at 450/620nm in each well 109

recorded. 110

Isotyping of monoclonal antibodies: Goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3 and IgM were 111

obtained from Jackson Immunoresearch Laboratories (Stratech Scientific Ltd, Newmarket, UK). 112

Tissue-culture supernatants (100µL) from cloned hybridomas were added to immobilised biotin-rHA1 113

(A/California/06/09) wells as described above and incubated for 1h at 37ºC. After washing the wells 114

as before in PBST, 100µL of a 1/2000 dilution of each of the goat-anti-mouse isotyping reagents in 115

1% (w/v) milk in PBSTBx was added and incubated for 30min at 37ºC. The wells were washed as 116

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before and incubated with 100µL of a 1/20000 rabbit anti-goat IgG-HRP conjugate (Sigma -Aldrich 117

Co. Ltd, Poole, UK) in 1% (w/v) milk in PBSTBx and incubated for 30min at 37ºC. The bound HRP 118

was detected with TMB substrate as described above. 119

EIA for the quantitation of IgG in culture supernatants: Microtitre plates were coated with 100µL/well 121

of rabbit anti-mouse IgG (5µG/mL, Sigma, Dorset, UK) in 0.05M carbonate buffer, pH9.5 overnight at 122

2-8 ºC. The wells were washed and 100µL of dilutions of IgG (derived from clone 1F5) in 1% (w/v) 123

milk in PBSTBx was added to the wells in triplicate for construction of a standard curve. 124

Simultaneously 100µL of Mab culture supernatants, diluted in the same buffer, were added in 125

triplicate to separate wells and incubated at 37 ºC for 1h. The plates were washed as before and 126

100µL of a 1/4000 dilution of rabbit anti-mouse IgG-HRP conjugate was added. After washing the 127

wells four times with PBST, the bound HRP was detected with TMB substrate. 128

SDS PAGE and Western Blotting: Recombinant viral proteins were separated electrophoretically 129

under reducing conditions on a NuPAGE 10% Bis-Tris gel (Invitrogen Ltd, Paisley, UK) using MOPS 130

running buffer and the electrophoresed proteins transferred onto nitrocellulose membranes essentially 131

as described by Towbin et al., (1979) and detailed in the product literature from Invitrogen Ltd. The 132

membranes were then incubated with a 1/20 dilution of supernatants derived from each of the cloned 133

Mab producing cell lines in 1% (w/v) milk in PBST or with a 1/100 dilution of serum from the mouse 134

test bleed in the same buffer solution for 1h at room temperature (RT). The membranes were then 135

washed and incubated with a 1/20000 dilution of a rabbit anti-mouse IgG-alkaline phosphatase 136

conjugate (Stratech Scientific Ltd, Newmarket, UK). After washing, the bound Mabs were detected 137

with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium (NBT/BCIP) as substrate (Leary 138

et al., 1983). 139

Haemagglutination Inhibition (HAI): The ability of the Mabs to inhibit agglutination of 0.5% turkey red 140

blood cells (RBC) by two recombinant influenza A virus strains NIBRG-121 and NIBRG-122, derived 141

from A/California/07/09 and A/England/195/09 respectively and supplied by the National Institute for 142

Biological Standards and Controls (NIBSC), UK, was tested essentially as described earlier (Ellis and 143

Zambon, 1997). Briefly, supernatants were diluted twofold in PBS, starting at a 1:8 dilution, and 4 144

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haemagglutination units of virus were added, followed by incubation for one hour at RT, after which 145

the turkey RBC were added. Results were interpreted after 30min incubation at RT. 146

Cell-ELISA and Immunostaining of virus infected MDCK cells: Madine-Darby canine kidney (MDCK) 147

cells were infected with recombinant virus NIBRG-121 at a multiplicity of infection of 0.05 particles per 148

cell and dispensed into wells of 96-well tissue culture plates using standard procedures (Rowe et al., 149

1999). One hundred microlitres of diluted Mab supernatants in 1% (w/v) milk PBSTBx were added to 150

the wells and incubated for 1h at RT. The wells were washed four times by gentle aspiration of the 151

antibody solution and washing with PBST. The wells were incubated with 100µL rabbit anti-mouse 152

IgG-alkaline phosphatase conjugate diluted 1/20000 in 1% (w/v) milk PBSTBx or with 100µL of rabbit 153

anti-mouse IgG-HRP conjugate diluted 1/4000 in the same buffer. The wells were washed four times 154

with PBST as before and further washed twice with water before immunostaining. The HRP 155

conjugates were revealed by the addition of 100µL of a soluble TMB substrate for the cell-ELISA and 156

stopping the reaction after colour development with 100µL 0.5M HCl and reading the optical density at 157

450/620nm. For localisation of Mab staining, the HRP conjugates were visualised after addition of 158

100µL of a precipitating substrate, TrueBlue™, (KPL, Gaithersburg, MD, USA) and the alkaline 159

phosphatase conjugates were revealed with the addition of 100µL/well of NBT/BCIP substrate, as 160

described for Western blotting above. 161

Competitive EIA: 163

Supernatant from Mab 1F5- hybridoma cell-culture was harvested and IgG purified using protein A 164

column and labelled with horseradish peroxidase (HRP) using standard procedures (Ishikawa et al., 165

1983 and Nilsson et al., 1981). The purified conjugate contained 1.44 moles of enzyme per mole of 166

IgG and the antibody concentration was approximately 2mG/mL. To streptavidin coated plates (see 167

above) 100µL/well of a 100nG/mL biotin-rHA1 from A/California/06/09 in 1% (w/v) milk PBSTBx was 168

added and incubated at 37ºC for 1h. The plates were then washed four times in PBST. 169

An equal volume of HRP conjugated 1F5 Mab (1F5-HRP) diluted 1/1000 was mixed with an equal 170

volume of competing antibody. The competing antibodies were undiluted Mab supernatants, or 1/5 171

dilutions of ferret antisera to influenza A virus strains (see Figure 3a for strains) and serum samples 172

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from humans pre and post immunisation with Celtura ® vaccine (containing the inactivated surface 173

antigen preparation of the reassortant NYMC X-179A virus) in 1% (w/v) milk PBSTBx. One hundred 174

microlitres per well of this was then added in duplicate to streptavidin-biotin-rHA1 wells for 1h at 37ºC. 175

After washing, the plates were incubated with TMB substrate (Microimmune Ltd, Hounslow, UK) at 176

room temperature for 20min. Known concentration of unconjugated antibody from clone 1F5 was 177

used as the competing antibody to generate a standard curve. Four or eight replicate control wells, in 178

the absence of competing culture supernatant or sera from ferrets and human vaccinees respectively, 179

were used to calculate the mean OD450/620nm of binding by 1F5-HRP. The % inhibition was calculated 180

using the equation: (mean OD450/620nm 1F5-HRP – mean OD450/620nm competing supernatant or serum 181

sample) / mean OD450/620nm 1F5-HRP x100. Where competing serum samples gave OD450/620nm values 182

greater than the control in the absence of serum, the result was designated a value of zero % 183

inhibition. 184

Experimental Ethics: All experimental work involving animals were performed in compliance with the 185

Animals (Scientific Procedures) Act1986 under licence and in accordance with ethical standards of 186

the Declaration of Helsinki. 187

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RESULTS 188

EIA screening of hybridoma colonies: Fourteen days post fusion virtually all of the 960 wells from the 189

fusion plates had colonies of hybridoma growth and consequently, supernatant from all the wells were 190

screened by EIA. Three strategies were used for the screening procedure. Reactivity of the 191

hybridoma supernatant to rHA or rHA1 derived from H1N1 (A/California/06/09) coated directly on to 192

microtitre wells or to biotinylated rHA1 derived from H1N1 (A/California/06/09), immobilised using 193

streptavidin coated plate. The serum derived from the test bleed of the immunised mouse gave strong 194

EIA reactivity in all three assays. The five monoclonals taken forward all reacted with biotin-rHA1 195

coated wells. Only one of these Mabs reacted with rHA1 coated directly onto microtitre wells. The EIA 196

positive hybridoma cells were cloned by limiting dilution. 197

EIA for the estimation of IgG concentration culture supernatants: From a standard curve constructed 198

using purified mouse IgG1, the concentration of IgG in the culture supernatants used in the 199

characterisation of Mabs was estimated. The concentrations of antibody in the supernatant for Mabs 200

1F5, 9A4 and 7H4 were between 15-18µG/mL, and for supernatant 9E12, was much higher 201

(26.0µG/mL). The antibody concentration in the supernatant of Mab 1G10 was only 1.0µG/mL (see 202

Table I). 203

Characterisation of the monoclonal antibodies: Since all five clones reacted with biotinylated rHA1 204

subunit derived from A/California/06/09, we assessed their reactivity with biotinylated rHA 205

A/California/06/09 and with biotinylated rHA1 subunit derived from A/Brisbane/07/09 H1N1 and A/ 206

Solomon Islands/03/06 H1N1 in EIA using streptavidin to capture the biotinylated proteins. All five 207

clones were specific for rHA and rHA1 derived from A/California/06/09 and did not react in EIA with 208

the biotinylated recombinant proteins from A/Brisbane/07/09 or with A/Solomon Islands/03/06. Serum 209

from the test bleed reacted with all four biotinylated proteins. Isotyping of the Mab supernatants 210

showed that all five clones secreted IgG1 antibodies (Table I). 211

In Western blots, serum from the test bleed of the immunised mouse, used to generate the Mabs, 212

stained all the recombinant haemagglutinin proteins derived from H1N1 influenza strains, but not the 213

recombinant haemagglutinin form H5N1 and influenza B (Figure 1a). Mabs from clones 1F5 and 214

1G10 did not react with any of the proteins in Western blots, although the lack of staining observed for 215

1G10 may be explained by its low antibody concentration in culture supernatants. Mab 7H4 gave 216

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intense immunostaining with rHA and rHA1 from A/California/06/09, but did not stain any other 217

recombinant protein (Figure 1b). Mab 9A4 gave intense reaction with rHA from A/California/06/09, but 218

only weak staining with rHA1 from A/California/06/09 (Figure 1c). Mab 9E12 showed weak reaction 219

with rHA derived from A/California/06/09 only (Figure 1d). 220

In a cell-ELISA, all five Mabs reacted with the recombinant virus NIBRG-121, with differing intensities 221

(See Table 1). Mab 1F5 and 9A4 gave the highest OD signals using TMB substrate. Diffuse 222

cytoplasmic/membrane staining of infected cell was observed with Mabs 1F5 and 9A4 (Figure 2a and 223

2b) using both the insoluble substrates, NBT/BCIP substrate (with alkaline phosphatase conjugate) 224

and TrueBlueTM (with HRP conjugate). This was different from the weak cytoplasmic/membrane 225

staining pattern observed with 9E12 (Figure 2c). The weak staining of infected cells by 1G10 may be 226

the result of the low concentration of antibody in the culture supernatant used (Figure 2d). 227

Only Mab 1F5 inhibited haemagglutination of turkey RBC by recombinant virus NIBRG-121 (derived 228

from A/California/07/09) and recombinant NIBRG-122 (derived from A/England/195/09). The HAI titre 229

of 1F5 with NIBRG-121 was eight fold higher than with NIBRG-122 (Table 1). 230

In competitive EIA, only serum from the test bleed and the unconjugated tissue culture supernatant 231

from clone 1F5 competed effectively with 1F5-HRP labelled antibody. A standard curve for the 232

competitive EIA using purified, unconjugated 1F5 antibody revealed that the detection limit was 233

75nG/mL. Partial inhibition of 1F5-HRP binding in EIA was observed with tissue culture supernatants 234

Mab 9A4 (38% inhibition) and 9E12 (12%). No inhibition was observed with supernatants from Mab 235

7H4 and 1G10. 236

The inhibition of 1F5-HRP with ferret sera in the competitive EIA is shown in Figure 3a and the 238

corresponding HAI results with NIBRG-121 are shown over the bars in the graph where available. 239

Two ferrets immunised with the same, recent European swine-lineage influenza strain 240

(A/Aragon/3218/08; provided by the Director of the National Centre for Microbiology, Instituto de 241

Salud Carlos III, Madrid, Spain) showed between 10 and 16% inhibition of 1F5-HRP in the 242

competitive EIA, whereas ferrets immunised with the 2009 pandemic influenza strain 243

A/California/07/09 H1N1 and influenza A/England/195/09 H1N1 showed approximately 55% and 30% 244

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inhibition of conjugate binding respectively. Of the ferret antisera to recent seasonal influenza 245

A/H1N1, only the A/Brisbane/59/07 showed any significant inhibition of 1F5-HRP binding (7.7%) in the 246

competitive EIA. All other ferret antisera failed to inhibit 1F5-HRP binding. The ferret antisera to the 247

two pandemic influenza A / H1N1 2009 virus strains had high HAI titres with NIBRG-121. Significant 248

but lower HAI titres were obtained for ferret antisera to the recent swine-lineage virus 249

A/Aragon/3218/08 and to antisera to the strain A/swine/England/117316/86. 250

The inhibition of 1F5-HRP binding obtained with sera from 15 subjects pre and post vaccination with 252

Celtura ® adjuvanted pandemic H1N1 vaccine is shown in Figure 3b and the HAI titres obtained for 253

each of the specimens is given on the bar chart. Twelve of the 15 sera taken on the day the vaccine 254

was administered (day 0) did not inhibit 1F5-HRP binding. This included five sera with HAI titres of 16 255

or greater on day 0. Serum from one subject in particular (subject 12) had an HAI titre of 256 on day 256

0, but did not show any inhibition of 1F5-HRP in the competitive EIA. Of the remaining three sera 257

collected on day 0, two had HAI titres of <8 and one of 16 (subjects 7, 11 and 15) and these inhibited 258

1F5-HRP (inhibition of 23%, 24% and 29% respectively). Most subjects had seroconverted by HAI 259

and also produced antibodies that inhibited 1F5-HRP binding in the competitive EIA by Day 21, with 260

two exceptions, subjects 3 and 4. Subject 3 showed seroconversion in the competitive EIA on day 42, 261

whereas subject 4 remained seronegative by competitive EIA at this time and maintained an HAI titre 262

of 64, as observed on day 21. In general, sera from subjects giving high % inhibition of 1F5-HRP in 263

the competitive EIA also had high HAI titres, although there were some exceptions (see subjects 12 264

and 6 taken on day 21). The correlation between HAI titres and % inhibition in the competitive EIA 265

was poor (R2 <0.3). 266

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DISCUSSION 268

An operational consequence of the emergence of the pandemic influenza A / H1N1 2009 was that the 269

pathogen and specimens from suspected cases needed to be handled at a higher biological 270

containment level than for seasonal influenza viruses. This restricted the propagation of virus in cell 271

culture to laboratories with high containment facilities and further restricted availability of the virus or 272

viral proteins for developing serological reagents. However, within three months of the first cases of 273

the pandemic influenza A / H1N1 2009 virus being reported in Mexico and the USA, rHA proteins 274

derived from the pandemic strain in purified form became available commercially. This prompted us to 275

generate monoclonal antibodies to rHA in order to provide additional serological research tools for 276

studying the pandemic influenza A / H1N1 2009 virus strains. 277

Recombinant HA1 derived from A/California/06/09 H1N1 proved to be immunogenic, since serum 278

taken from the test bleed of the immunised mouse showed reactivity with rHA1 subunit and whole rHA 279

(derived from the A/California/06/09 strain) coated wells and with biotinylated rHA1 from the same 280

strain suspended from streptavidin coated wells in EIA. The majority of hybridomas screened did not 281

react with the recombinant proteins coated directly onto polystyrene wells. This may have occurred as 282

a result of failure in the fusion process to fuse splenocytes with specificity for the proteins. However, 283

success with using biotinylated rHA1 suspended from streptavidin coated plates for identifying 284

reactive hybridomas suggested that conformational presentation of the rHA protein was important. It 285

may be that the rHA1 and rHA undergo changes in conformation on binding to polystyrene wells, as 286

was shown for HA binding to polyvinyl by Yewdell (2010). Yewdell (2010) demonstrated recently that 287

some Mabs to discontinuous epitopes on influenza A HA can induce refolding of denatured proteins 288

bound to polyvinyl plates over a two day incubation period. Thus it is possible that with extended 289

incubation of the hybridoma supernatant during the screening process, more hybridoma clones 290

reactive to conformational epitopes in rHA1 may have been identified. 291

Four of the five Mabs to the pandemic H1N1 influenza A/California/ 06/09 generated in this study 292

appeared to recognise different epitopes as demonstrated by the differing pattern of reactivity 293

observed in Western blots, staining of virus infected cells, HAI and in competitive EIA. The 294

concentration of IgG in the culture supernatant of Mab 1G10 was ten times lower than that found for 295

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other Mabs and it was therefore difficult to establish if this Mab recognised a unique epitope. All Mabs 296

were specific for the pandemic influenza A/ H1N1 2009 strain and did not cross-react with pre-2009 297

seasonal H1N1 strains. Unlike the Mabs used in this work, which proved to be specific for 298

A/California/06/09 in EIA and Western blots, the polyclonal serum from the test bleed mouse showed 299

cross-reactions with the HA1 of influenza A/Brisbane/59/07 and influenza A/Solomon Islands/03/06 300

and A/New Caledonia/2/99. This is not surprising since there is ~70% amino acid sequence homology 301

between the HA1 of A/California/06/09 and seasonal influenza A/ H1N1 (Ikonen et al., 2010). 302

There are only three amino acid changes in the haemagglutinin between A/California/07/09 and 304

A/England/195/09 (L49I, P91S and I323V) and this was sufficient to result in an eight fold difference in 305

HAI titres of 1F5 seen with the two recombinant viruses. Furthermore, in competitive EIA there was 306

greater inhibition of 1F5-HRP binding by ferret antiserum to A/California/06/09 compared to 307

A/England/195/09. Although the observed differences with the animal sera may be due to differing 308

responses to infection in individual ferrets, the combined HAI and competitive EIA data suggest that 309

the amino acid changes in the two haemagglutinins result in a significant change in the epitope 310

recognised by the 1F5 antibody. If there are significant changes in the IF5-epitope in other 2009/10 311

virus isolates, then Mab IF5 could prove useful for tracking antigenic drift in the pandemic influenza 312

A/H1N1 2009 virus strains. 313

In the competitive EIA, inhibition of 1F5-HRP binding to the biotinylated rHA1 of A/California/06/09 314

would only be expected if competing antibodies either compete directly for the epitope recognised by 315

Mab 1F5 or if the competing antibodies bind to other epitopes on the rHA1 that are close enough to 316

interfere sterically with 1F5-HRP binding. Since Mab 1F5 inhibits haemagglutination of turkey RBC by 317

NIBRG-121, it would follow that any antibodies that inhibit 1F5-HRP in the competitive EIA as a result 318

of steric interference will inhibit haemagglutination. Although this proved to be the case for most of the 319

ferret antisera and post vaccination sera tested (Figures 3a and 3b), the correlation between the HAI 320

titres and % inhibition observed in the competitive EIA was poor. This is not surprising, since HAI 321

measures a polyclonal antibody response directed towards a number of antigenic epitopes on the 322

haemagglutinin receptor binding site of the virus, whereas the competitive EIA only measures 323

antibodies in the competing serum that inhibit binding of a single epitope on haemagglutinin of the 324

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virus. Although 1F5 inhibits haemagglutination of NIBRG-121, it does so through binding to a single 325

epitope on the virus, albeit represented multiple times on the virion. Ferret antisera and immune 326

human sera effect haemagglutination by binding to multiple epitopes, represented multiple times on 327

the virion. 328

An alternative explanation for the poor correlation between HAI and the competitive EIA may be that 329

antibodies which do not inhibit HAI may nevertheless bind to distal sites on the biotinylated rHA1 and 330

cause conformational changes. These conformational changes may have two opposing effects in the 331

competitive EIA. It may improve presentation of the 1F5 epitope, augmenting the signal obtained in 332

the competitive EIA as a result of increased binding of 1F5-HRP, as was observed with some of the 333

sera tested (data not shown). Conversely, an induced conformational change may have an adverse 334

affect on the 1F5 epitope structure, inhibiting binding of 1F5-HRP to the biotinylated rHA1. 335

Our data on a limited number of subjects investigated in this work indicates that vaccination with 336

Celtura ® vaccine induces an immune response to the haemagglutinin and specifically to an epitope 337

shared by 1F5 antibody or to epitopes close to this site. It would be of interest to determine whether 338

individuals infected with the pandemic influenza A/ H1N1 2009 virus also mount a significant antibody 339

response to the 1F5-epitope or epitopes close to this site. If further studies show this to be the case 340

then the competitive EIA described here could provide a potentially useful and specific sero-341

epidemiological tool for studying exposure to infection with the pandemic virus as well as to determine 342

efficacy of vaccination. The competitive EIA has the added benefit that it is simple, safe and not as 343

work-intensive as the haemagglutination inhibition and microneutralisation assays. 344

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ACKNOWLEDGEMENTS 345

The authors would like to thank Dr Joanna Ellis and Dr Monica Galliano for providing information on 346

amino acid sequence data for the HA1 of 2009 pandemic and seasonal influenza A/ H1N1 viruses. 347

The authors would also like to acknowledge Surita Gangar, Janice Baldevarona and Aram Afsar for 348

technical assistance with haemagglutination inhibition and culture of MDCK cells. 349

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REFERENCES 351

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two children - Southern California, March-April 2009. MMWR Morb Mortal Wkly Rep 58: 400-2. 353

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Donis R, Katz J, Finelli L, Bridges CB, Shaw M, Jernigan DB, Uyeki TM, Smith DJ, Klimov AI, Cox NJ. 364

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in humans. Science. 325: 197-201 366

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pandemic H1N1 influenza virus. N Engl J Med. 361: 1945-52. 369

Ikonen N, Strengell M, Kinnunen L, Österlund P, Pirhonen J, Broman M, Davidkin I, Ziegler T, 370

Julkunen I. 2010. High frequency of cross-reacting antibodies against 2009 pandemic influenza 371

A(H1N1) virus among the elderly in Finland. Euro Surveill.15: pii: 19478. Available online: 372

http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19478 373

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Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels 402

to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350- 4. 403

Yewdell JW. 2010. Monoclonal antibodies specific for discontinuous epitopes direct refolding of 404

influenza A virus hemagglutinin. Mol Immunol 47: 1132- 6. 405

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LEGENDS 406

Table I. Characterisation of monoclonal antibodies to the Haemagglutinin HA1 subunit of the 407

pandemic influenza A/ H1N1 2009 virus. 408

Isotyping of Mabs and their reactivity in EIA, Cell-ELISA, haemagglutination inhibition (HAI) 409

and Western blots are shown in the table. NIBRG-121 and NIBRG-122, are recombinant 410

viruses derived from influenza A/California/07/09 and A/England/195/09 strains, respectively. 411

Intensity of staining of MDCK cells infected with NIBRG-121 virus using insoluble substrates, 412

NBT-BCIP (for alkaline phosphatase conjugate) and True-Blue (for HRP conjugate), by 413

each Mab supernatant was scored as strong (+++), moderate (+), weak, (+/-) or not detected 414

(-). Intensity of staining of whole rHA or rHA1 subunit, derived from influenza 415

A/California/06/09 H1N1 virus, in Western blot was scored as for immunostaining of MDCK 416

cells infected with NIBRG-121. 417

Figure 1. Western blot analysis of test bleed and monoclonal anti-HA1 antibodies. 418

Recombinant HA proteins derived from influenza strains were analysed using SDS-PAGE 419

under reducing conditions, transferred to nitrocellulose and immunostained with (A) serum 420

from test bleed and Mabs (B) 7H4 (C) 9A4 and (D) 9E12. Lanes 1 and 12: SeeBlue® Plus2 421

molecular weight standard, Lane 2 and 13: SeeBlue® molecular weight standard, Lane 3: 422

HA (A/New Caledonia/20/99 H1N1), Lane 4: HA (A/California/06/09 H1N1), Lane 5: HA1 423

(A/California/06/09 H1N1), Lane 6: HA (A/Solomon Islands/3/06 H1N1), Lane 7: HA 424

(A/Brisbane/59/07 H1N1), Lane 8: HA (A/Vietnam/1203/04 H5N1), Lane 9: HA1 425

(A/Vietnam/1203/04 H5N1), Lane 10: HA (B/Malaysia/2506/04), Lane 11: Blank. 426

Figure 2: MDCK cells infected with recombinant NIBRG-121 virus immunostained using 428

monoclonal antibody and anti-mouse alkaline phosphatase conjugate. Staining of infected 429

cells with (A) Mab 1F5 (B) Mab 9A4 (C) Mab 9E12 and (D) Mab 1G10 is shown. 430

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Figure 3a: Inhibition by ferret antisera of Mab 1F5-HRP binding in a competitive EIA. The 432

antisera to the influenza A strains are identified on the abscissa and the % inhibition of 1F5-433

HRP binding for each of these is shown on the ordinate. The numbers shown above the bars 434

in the graph are the corresponding HAI titres obtained for each antiserum with NIBRG-121. 435

Figure 3b: Inhibition by pre and post vaccination sera of Mab 1F5-HRP binding in a 437

competitive EIA. The HAI titres for each subject pre and post vaccination with Celtura ® 438

vaccine are shown above or at the base of the individual bars in the graph. 439

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Table I

Monoclonal Antibody Reactivity in EIA (OD 450/620nm) HAI titre Mab reactivity with MDCK cells

infected with NIBRG-121 virus in:

Reactivity in

Western blot

Designation

Isotype Conc.

µG/mL

Biotin-rHA1 subunit

A/California/06/09 Biotin-Whole rHA

A/California/06/09

Biotin rHA1 subunit

A/Solomon Is./03/06 Biotin rHA1

subunit

A/Brisbane/59/07

Cell-ELISA

HRP:TMB

OD 450/620nm

NBT-BCIP True-

Subunit

1F5 IgG1 16.1 0.555 0.357 0.017 0.018 128 16 1.018 +++ +++ - -

1G10 IgG1 1.0 0.056 0.050 0.011 0.013 <8 <8 0.177 +/- + - -

7H4 IgG1 17.7 0.248 0.098 0.014 0.018 <8 <8 0.095 - - +++ +++

9A4 IgG1 15.2 1.138 0.569 0.013 0.016 <8 <8 0.718 +++ +++ +++ +/-

9E12 IgG1 26.0 0.597 0.108 0.009 0.011 <8 <8 0.170 +/- + - +

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Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus...

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