JVI Accepts, published online ahead of print on 3 March...

1

Influenza H1N1 A/Solomon Island/3/06 virus receptor binding specificity correlates with

virus pathogenicity, antigenicity and immunogenicity in ferrets

Qi Xu#, Weijia Wang

#, Xing Cheng, James Zengel and Hong Jin

*

MedImmune, 319 North Bernardo Ave, Mountain View, CA 94043

#Both authors contributed to the work equally

Key words: Influenza H1N1 virus, receptor binding specificity, pathogenicity,

antigenicity and immunogenicity in ferrets

*Corresponding author:

Hong Jin, Ph.D

MedImmune

319 North Bernardo Ave

Mountain View, CA 94043

Tel: 650 603 2367

Fax: 650-603 3367

Email: [email protected]

Abstract: 234 words

Number of text pages: 31

Number of Figures: 5

Number of Tables: 4

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.02489-09 JVI Accepts, published online ahead of print on 3 March 2010

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Abstract

Influenza viruses attach to cells via a sialic acid moiety (sialic acid receptor) that

is α2-3 linked, or α2-6 linked, to galactose (α2-3SAL or α2-6SAL); sialic acid acts as a

receptor for the virus. By lectin staining, we demonstrated that the α2-6SAL

configuration is predominant in the respiratory tract of ferret including trachea, bronchi,

and lung alveoli tissues. Recombinant wild type (rWt) influenza A/Solomon Island/3/06

(SI06) (H1N1) viruses were constructed to assess the impact of the hemagglutinin (HA)

variations (amino acids 190 or 226) identified in natural variants on virus replication in

the upper and lower respiratory tract of ferrets, virus antigenicity, and immunogenicity. A

single amino acid change at residue 226 (from Gln to Arg) in the HA of SI06 resulted in

the complete loss of binding to α2-6SAL, and concomitant loss of the virus’s ability to

replicate in the lower respiratory tract of ferrets. In contrast, the virus with Gln226 in the

HA protein has receptor binding preference for α2-6SAL and replicates efficiently in the

lungs. There was a good correlation between viral replication in the lungs of ferrets and

disease symptoms. In addition, we also showed that the 190 and 226 residues impacted

viral antigenicity and immunogenicity. Our data emphasizes the necessity of thoroughly

assessing wild type influenza viruses for their suitability as reference strains and for

carefully selecting the HA antigen for vaccine production during annual influenza

vaccine evaluation processes.


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Introduction

Influenza viruses cause recurring, highly contagious respiratory disease in

humans, resulting in approximately 36,000 deaths (primarily in the elderly) in the United

States annually. Influenza viruses continually undergo genetic changes as they replicate

in humans, resulting in antigenic drift. Infection with seasonal influenza virus is mainly

manifested by respiratory tract infection, with high morbidity and mortality in individuals

who have poor pre-existing immunity to influenza viruses, mainly the very young, and in

people with inadequate immune responses such as the elderly. Introduction of new

hemagglutinin (HA) subtypes such as the 1918-1919 “Spanish” influenza virus (H1N1),

the 1957 (H2N2), and 1968 (H3N2) viruses caused three pandemics in the last century, as

well as the influenza pandemic of 2009 caused by 2009 swine-origin H1N1 influenza

virus.

Virus pathogenicity is determined by the interplay between virus and host.

Multiple factors determine virus virulence, such as the ability of the virus to trigger

innate immunity (38), the ability to induce degradation of host RNA polymerase II

enzyme (31), apoptosis mediated by the PB1-F2 protein (5, 7), contributions of the viral

RNA polymerase proteins, the NS1 and M2 proteins (17, 36, 42), and viral receptor

binding preference (19, 27).

The binding of influenza viruses to their target cells is mediated by the viral HA

protein which recognizes cell surface glycoconjugate receptors that terminate in sialic

acid residues; the sialic acid residues may be α2-3 linked (α2-3SAL) or α2-6 linked (α2-

6SAL). Human influenza viruses readily bind to the α2-6SAL receptor on the human


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respiratory tract epithelium (9, 21). The lack of sustained human-to-human transmission

of avian H5N1 viruses is likely due to their α2-3SAL binding preference (34, 40). Ferrets

have been proven to be a suitable model to study influenza viruses for virus

pathogenicity, transmissibility, attenuation, immunogenicity, and protective efficacy of

the vaccine strains, and for evaluation of antiviral drugs. The animal is a permissive host

for all influenza A and B viruses and develops febrile illness and symptoms that are

similar to those of humans. Ferrets also have marked similarities to humans in lung

physiology, airway morphology, and cell types present in the respiratory tract, including

the distribution of α2-6SAL receptor for human influenza viruses (26, 41).

During annual influenza vaccine production processes, the HA and neuraminidase

(NA) gene segments of Wt influenza viruses are reassorted with a vaccine donor strain

that donates the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS)

required for virus replication. Cold adapted (ca) A/Ann Arbor/6/60 is used as the master

donor virus for live attenuated influenza A vaccine strains that have the characteristic

cold adapted, temperature sensitive, and attenuation phenotypes. The A/Puerto Rico/8/34

(H1N1) is normally used as the donor virus for inactivated influenza A vaccines. Wt

influenza viruses are routinely used as reference strains for strain surveillance and for

vaccine strain evaluation to ensue those influenza vaccines are immunogenic and

antigenically similar to Wt viruses. During the egg expansion process, the HA gene of Wt

influenza viruses may mutate, allowing the viruses to replicate more efficiently in eggs.

The egg-adaptation mutations normally result in amino acid changes in the receptor

binding sites to allow virus to bind to α2-3SAL better but the egg adaptation mutations

may also affect virus antigenicity (3, 10, 20).


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In this study, we found that Wt A/Solomon Island/3/07 (Wt SI06) virus expanded

in eggs contained amino acid variations in HA residues 190 and 226. To evaluate the

impact of these amino acid variations on virus receptor binding preference, virus

replication in the animal host, immunogenicity, and antigenicity, we constructed

recombinant A/Solomon Island/3/07 (rWt SI06) viruses containing single or double

amino acid substitutions. We found that a single amino acid mutation at residue 226 of

the SI06 HA protein affects not only virus receptor binding preference but also the ability

of the virus to replicate in the lower respiratory tract of ferrets. By lectin staining, we

showed that the respiratory tract of ferrets contains predominantly α2-6SAL, and the

viruses with the strong affinity for the α2-6SAL replicated efficiently in the lungs,

whereas viruses that bound exclusively to the α2-3SAL were unable to replicate in the

lungs. Our data indicate that virus receptor binding preference plays an important role in

viral pathogenicity, immunogenicity, and antigenicity.


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Materials and Methods

Generation of recombinant viruses

The biologically derived Wt SI06 (bWt SI06) received from the Centers for

Disease Control (CDC) was previously amplified three times in embryonated chicken

eggs. The 8 gene segments of bWt SI06 were amplified by RT-PCR and cloned into the

expression vector pAD3000 (15). Each cDNA clone was completely sequenced and

compared with the sequence obtained from sequencing of RT-PCR amplified cDNA

using vRNA as template. Mutagenesis of plasmid was performed by using the

QuikChange Site-Directed Mutagenesis kit (Agilent, La Jolla, CA) and sequence changes

were confirmed by sequencing. Recombinant viruses were rescued by transfection of

cocultures of 293T cells and Madin-Darby Canine Kidney (MDCK) cells with eight

plasmids encoding the 8 genomic cDNAs of Wt SI06, and the rescued virus was

designated rWt SI06. All of the viruses were propagated in the allantoic cavities of 10- to

11-day-old embryonated chicken eggs. The genomic sequence of the recombinant viruses

was verified by cDNA sequencing.

Receptor-binding assays using sialic acid-specific red blood cells

Chicken red blood cells (cRBCs) (HEMA Resource and Supply Inc.) were re-

sialylated as previously described (25). One hundred microliters 10% cRBC was

incubated with 50 mU Vibrio cholera neuraminidase (Sigma, St. Louis, MO) at 37°C for

1 hr to remove sialic acid from cRBC. Complete removal of sialic acid from cRBCs was

confirmed by the lack of hemagglutination. Subsequently, desialylated cRBCs were

incubated with 2.5 mU of α2-3(N)-sialyltransferase (Calbiochem, La Jolla, CA) or 2 mU


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of α2-6(N)-sialyltransferase (Calbiochem, La Jolla, CA) and 1.5 mM cytidine-5’-

monophospho-N-aceylneuramine acid (Sigma, St. Louis, MO) for 1.5 hr at 37°C. The

resialylated cRBC was suspended (0.5%, v/v) in phosphate-buffered saline (PBS, pH 7.2-

7.6). Hemagglutination assays were performed in V-bottomed microtiter plates. Fifty

microliters 2-fold serially diluted virus was incubated at room temperature for 1 hr with

50 µl of 0.5% cRBC that had been resialylated (α2-3, or α2-6). The hemagglutination

titer was defined as the reciprocal of highest virus dilution that hemagglutinated cRBC.

Ferret studies

Eight to ten week-old male and female ferrets (n = 3/group) from Simonsen

Laboratories (Gilroy, CA) or Triple F Farms (Sayre, PA) were used to assess virus

replication in the respiratory tract and to evaluate virus immunogenicity. Ferrets were

housed individually and inoculated intranasally with 7.0 log10PFU of virus per 0.2 ml

dose. Three days after infection, ferrets were euthanized, and the lungs and nasal

turbinates were harvested. Virus titers in the lungs and nasal turbinates were determined

by the EID50 assay and expressed as 50% egg infectious dose per gram of tissue

(log10EID50/g). Virus infected ferrets were monitored for body temperature per rectum

using Welch Allyn’s Sure Temp thermometer (Welch Allyn, Skaneateles Falls, NY) and

observed for clinical symptoms including nasal symptoms, stool changes and activities

twice daily. Ferret’s body weight was measured daily. Nasal symptoms were defined by

scores 0-3 (0: no symptoms were observed; 1: nasal rattling; 2: nasal discharge on

external nares; 3: mouth breathing) as described previously (28). Stool change scores

were defined by scores 0-3 (0: firmed/normal; 1: soft/watery; 2: mucous/yellow/green; 3:

bloody or absent). Activity scores were also defined by scores 0-3 (0: fully playful; 1:


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only respond to play overtures and did not initiate any play activity; 2: alert but not at all

playful; 3: neither playful nor alert). Ferrets assigned for the immunogenicity studies

were bled on Days 14 and 28 post-infection and sera collected on Day 14 were assayed

for antibody titers by the hemagglutination inhibition (HAI) and microneutralization

assays.

Serum antibody detection by hemagglutination inhibition and microneutralization

assays

Serum antibody levels in post-infected ferret sera against homologous and

heterologous viruses were determined by HAI and microneutralization assays. Sera

collected day 14 post-intranasal infection were treated with receptor-destroying enzyme

(RDE, Denka-Seiken, Tokyo, Japan) at 37oC overnight, and heat inactivated at 56°C for

45 min. Treated serum samples (25 µl) were serially diluted 2-fold and incubated with

25 µl virus containing 8 hemagglutination units in V bottom microtiter plates for 1 hr

followed by incubation with 50 µl of 0.5% turkey RBCs for 45min. The HAI titer was

defined as the reciprocal of the highest serum dilution that inhibited hemagglutination.

The microneutralization assay was performed with the RDE-treated serum. Two-fold

serially diluted serum in EMEM medium containing 1 µg/ml of TPCK-trypsin was

incubated with 100 µl of virus containing 100 TCID50 in 96-well U-bottom microplates

for 1 h at 33°C. The antiserum-virus mixtures were transferred to the MDCK cells in 96-

well plates and incubated at 33°C for 4 days. The cytopathic effect (CPE) was observed

under a microscope and the microneutralization titer was defined as the reciprocal of the

highest serum dilution that inhibits 50% CPE caused by virus infection.

Ferret tissue processing


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For histology studies, respiratory tract tissues of ferrets including nasal turbinate

(NT), trachea, and lungs (inflated with 10% formalin) were collected and fixed in 10%

formalin for a minimum of 24 hr. Paraffin-embedded tissue sections were prepared by

Cureline Inc (Burlingame, CA). The tissue sections were deparaffinized with xylene and

dehydrated by using graded alcohols. The sections were then blocked with 3% H2O2 for

15 minutes followed by incubation with a Carbo-Free blocking solution (Vector Labs,

Burlingame, CA) at room temperature for 1 hr.

Immunohistochemistry staining for influenza A virus antigen

Ferret tissue sections were incubated with goat anti-influenza A polyclonal

antibody (Millipore, Beford, MA) diluted 1:40 in blocking solution at room temperature

for 1 hr followed by incubation with horse radish peroxidase (HRP)-conjugated rabbit

anti-goat Ig (Dako, Carpenteria, CA) diluted 1:100 in blocking solution at room

temperature for 30 min. The sections were washed 3 times with PBST (0.05% Tween-20

in PBS, pH7.2) and developed with 3-amino-9-ethylcarbazole (AEC) substrate (Dako) for

influenza virus antigen (red) and hematoxylin for nuclear staining (blue-violet).

Lectin staining of tissue sections

To block all endogenous avidin/biotin binding sites, ferret tissue sections were

treated with an avidin/biotin blocking kit (Vector Labs). The sections were then

incubated with 10 µg/ml biotinylated agglutinins from Maackia amurensis (MAA1 and

MAA2) or biotinylated Sambucus nigra lectin (SNA) (Vector Labs) in blocking solution

at room temperature for 1 hr followed by incubation with HRP-streptavidin (Vector Labs)

diluted 1:300 in blocking solution for 30 minutes. The sections were subsequently


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developed with AEC substrate for lectin distribution (red) and hematoxylin (Vector Labs)

for nuclear staining (blue-violet).

Tissue binding of labeled virus

Virus labeling was done as previously described (van Riel et al., 2007). Briefly,

influenza viruses amplified in embryonated chicken eggs were harvested and clarified by

filtration through a 0.22 µm filter (Millipore, Beford, MA). The viruses were then

purified through a 20-60% sucrose gradient and suspended in 2 ml PBS. The purified

viruses were inactivated by incubation with an equal volume of 10% formalin at room

temperature for 1 hr followed by dialysis in PBS overnight. The viruses were mixed with

equal volume of 0.1 mg/ml fluorescein isothiocyanate (FITC) (Fisher Scientific,

Pittsburgh, PA) in 0.5 M bicarbonate buffer (pH 9.5) for 1 hr with constant stirring, and

unbound FITC was subsequently removed by overnight dialysis in PBS.

Formalin-inactivated FITC-labeled viruses were diluted with PBS to a titer of 50-

100 hemagglutination units per 50 microliters. The viruses were then incubated with

ferret tissue sections overnight at 4°C followed by incubation with HRP-conjugated rabbit

anti-FITC antibody (Dako) diluted 1:50 in blocking solution at room temperature for 1 hr.

The tissue sections were subsequently developed with AEC for influenza virus antigen

(red) and hematoxylin for nuclear staining (blue-violet).


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Results

The HA gene determined viral replication in the lower respiratory tract of ferrets

A/Solomon Island/3/06 (H1N1) received from the CDC had been amplified 3

times in embryonated chicken eggs and its genomic sequence was determined by

sequence analysis. Recombinant Wt A/SI/3/06 (rWt SI06) virus was produced from the

transfected 8 cDNA plasmids cloned from biologically derived A/Solomon Island/3/06

(bWt SI06) and represented the consensus sequence of bWt SI06. The genomic sequence

of rWt SI06 was confirmed to be identical to bWt SI06. Both rWt and bWt SI06 viruses

replicated efficiently in embryonated chicken eggs with titers of > 8.0 log10PFU/ml.

Replication of bWt and rWt SI06 in the respiratory tract of ferrets was examined.

Ferrets were administered intranasally 107 PFU of either bWt or rWt SI06, at three days

postinfection virus titers in the nasal turbinates (NT) and lungs were determined by

(50%) egg infectious dose (EID50) assay. Both bWt and rWt SI06 replicated efficiently in

the upper respiratory tract of ferrets at levels of 5.8 and 5.4 log10EID50/g NT tissues,

respectively. However, in contrast to bWt SI06 that replicated at a titer of 5.5

log10EID50/g tissue in the lungs, rWt SI06 was not detected in the lungs of infected

ferrets. To determine if bWt SI06 recovered from lungs of ferrets were identical to virus

inoculum, bWt SI06 isolated from NT and lungs were sequenced. The virus recovered

from the lung tissue was found to contain two amino acid changes in the HA protein: at

residue 186 (190 in the H3 numbering), amino acid change from aspartic acid to alanine

(D190A), and at residue 222 (226 in the H3 numbering), amino acid change from

arginine to glutamine (R226Q). The virus recovered from the NT tissue had mixed

residues at these two positions (Table 1).


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To determine whether the residues 190 and 226 (in H3 numbering) of the HA

protein of bWT SI06 isolated from lung or NT tissues were mutated in vivo or were

selected from the existing heterologous viral pool, bWT SI06 was subjected to plaque

assay and the HA gene from each individual plaque was sequenced. As shown in Table 1,

from 48 plaques sequenced, the majority (90%) plaques (plaque A) had D190 and R226

(DR) residues in the HA protein; 10% of the plaques (a total of 5 plaques, plaques B, C

and D) had Q226 in the HA protein, but their residue at 190 was V, N or A. Genetic

stability of these bWT SI06 variants was examined by 3-4 rounds of passages in eggs.

Except for plaque B that has V190 and Q226 changed to D190 and R226 after its

replication in eggs, the rest of the variants maintained their HA protein sequences. Since

virus with A190 and Q226 (AQ) was present in the inoculated viral population of bWt

SI06 and rWt SI06-DR could not replicate in ferret lungs, it is concluded that bWt SI06-

AQ isolated from infected ferret lungs was selected from virus inoculum, not derived

from in vivo mutations. This speculation was further supported by the HA sequence

analysis of the H1N1 viruses that circulated before, at the same time, and after

A/Solomon Island/3/06 virus. A/Beijing/262/95, A/New Caledonia/20/99 (A/NC/20/99),

A/HongKong/2652/06 (A/HK2652/06), A/St Petersburg/8/06 (A/S.P/8/06) and A/South

Dakota/6/07 (A/SD/6/07) are egg isolates, A/Singapore/23/04 and A/Canada/591/04 are

MDCK isolates (Fig.1). All of these strains have Q226 but with D or N at 190. A/New

Caledonia/20/99 vaccine strain (ca) has the N190D change and A/SI/3/06 ca has the

same HA sequence as the HA of the bWt SI06 virus.

The HA protein residue 226 played a critical role in viral replication in ferret lungs


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To further examine which amino acid was critical for virus to replicate in the

lower respiratory tract of ferrets, replication of bWt SI06 and the three bWt SI06 HA

variants in ferrets was evaluated (Table 2). All viruses replicated efficiently in the upper

respiratory tract of ferrets. The titer of bWt SI06-DR (4.4 log10EID50/g) in the NT was

approximately 10-fold lower than those of SI06-AQ (5.5 log10EID50/g) and bWt SI06-NQ

(5.6 log10EID50/g) and its replication was not detected in the lungs. In contrast, both bWt

SI06-NQ and -AQ replicated in the lungs efficiently (5.4 and 5.1 log10EID50/g).

To confirm that the R226 residue indeed rendered the virus unable to replicate in

ferret lungs, rWt SI06 variants that had DR, AR, DQ or AQ at residues 190 and 226 were

produced and evaluated for their ability to replicate in the upper and lower respiratory

tract of ferrets (Table 2). These four variants replicated efficiently in the upper respiratory

tract of ferrets at levels of 5.8, 6.1, 7.2 and 6.9 log10EID50/g NT tissue although the titers

of the viruses with DR or AR were more than 10-fold lower than those with DQ and AQ.

rWt SI06-DR and -AR were not detected in the lungs, rWt SI06-DQ and -AQ replicated

at titers of 6.3 log10EID50/g tissue. Thus, these data demonstrated that residue Q226 is

critical for replication of Wt SI06 virus in the ferret lungs.

The upper and lower respiratory tracts of ferrets infected with rWt SI06-AQ and

rWt SI06-DR were examined by immunohistopathology (Fig. 2). Viral NP antigens were

detected in the nasal cavity and nasopharynx of the ferrets infected with the AQ and DR

variants. Viral antigens were barely detected in the trachea, bronchus and alveoli of

ferrets infected with rWt SI06-DR. In contrast, viral antigens were present in the

epithelial cells and the glands of the trachea and bronchus, and pneumocytes of alveoli in

the animals infected with rWt SI06-AQ. Thus, consistently with the results from viral


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replication, the immuno-staining results confirmed that rWt SI06-DR virus did not

replicate in the lower respiratory tract of ferrets.

Viral replication in the lungs of ferrets correlated with disease symptoms

rWt SI06 HA variants were assessed for their ability to cause influenza illness in

ferrets. Virus infected ferrets were monitored for body weight gain/loss, body

temperatures, nasal symptoms, stool change and activities (Fig. 3). Ferret body weight

gain was the greatest for the PBS control group with the mean body weight gain of 8.5%

on day 3. Ferrets infected with rWt SI06-DR did not loss body weight with the maximal

body weight gain of 7.5% on day 3. rWt SI-AQ and -DQ infected ferrets lost weight on

day 1, but gradually gained weight and had maximal body weight gain of 2.2-2.7%.

These ferrets also had fever of greater than 39.8oC at 30 h postinfection. rWt SI06-DR

had temperature of higher than PBS control group at 48-54 h but did not reach 39.8oC.

Except for the PBS group, some of the virus infected ferrets had low scores (1-2) of nasal

symptoms, stool change and activity changes, however, these scores did not correlate

with temperature and body weight changes. The data obtained with the body weight

change and fever demonstrated that viral replication in the lower respiratory tract

correlated with disease symptoms.

The effect of the HA residue 226 on viral antigenicity and immunogenicity

To examine whether the residues 190 and 226 of the SI06 HA protein could affect

virus antigenicity and immunogenicity, rWt SI06 variants DR, AQ, AR or DQ were

evaluated for their immunogenicity and cross reactivity (Table 3). Serum antibody titers


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were measured by the HAI and microneutralization assays and both assays provided

comparable results. Overall, rWt SI06-DR infected ferrets had a lower serum antibody

titers than the animals infected with other variants but its serum cross reacted well with

the other three variants. The HAI and neutralizing (Nt) antibody titers in ferrets infected

with rWt SI06-DQ was about 2-fold higher than that of DR and reacted with the other

three variants well. While rWt SI06-AQ serum had the highest homologous antibody

titer, it had 7 to 8- or 14 to 16-fold lower antibody titers against viruses with DR and DQ

respectively, yet it had the similar titer against the virus with AR. rWT SI06-AR serum

had antibody titer of 4096 (HAI) or 6400 (Nt) and reacted well with the virus with AQ,

but reacted less well with the viruses with DR or DQ.

The impact of the 190 and 226 residues on immunogenicity of ca SI06 vaccine

virus was also investigated. Ferrets in groups of three were immunized with ca SI06-DR

and ca SI06-AQ and levels of serum HAI and Nt antibodies collected 14 days post

vaccination were determined (Table 3). Consistent to the data obtained with Wt SI06

variants, ca SI06-AQ induced a much higher level of HAI and Nt antibody titers than ca

SI06-DR (7 to 8-fold difference). ca SI06-DR antiserum cross-reacted well with the AQ,

AR, but had 4-fold reduction against DQ. ca SI06-AQ antiserum had very high

homologous antibody titers (HAI titer of 6502, Nt titer of 15769) and reacted with the

virus with AR, but its reactivity to DR and DQ were reduced by up to 20- and 5-fold,

respectively. Thus, the 190 and 226 residues had a significant impact on vaccine virus

antigenicity and immunogenicity.

The HA residue 226 determined viral receptor binding preference


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The HA protein residue 226 in H3N2 viruses has been previously reported to

affect viral preference for receptor binding (33) and influences severity of illness in

ferrets (19). To determine if the HA protein residue 226 in the H1N1 virus also affects its

receptor binding preference, the H1N1 variants were analyzed by hemagglutination assay

using RBC that contained either α2-3SAL or α2-6SAL. As shown in Table 4, all the

variants hemagglutinated untreated cRBC that contained both α2-6SAL and α2-3SAL.

Desialylation removed both types of receptors from the cRBC and none of the viruses

bound to desialylated RBC. Interestingly, rWt SI06-DR and AR were only able to bind

α2-3SAL resialylated RBC. Although rWt SI06-AQ and DQ both bound α2-6SAL, AQ

lost the ability to bind α2-3SAL. rWt SI06-DR and AQ had distinctive receptor binding

preference, exclusively to α2-3SAL or α2-6SAL, respectively. Thus, amino acid change

Q226R abolished virus viral receptor binding specificity to α2-6SAL.

Receptor distribution in the respiratory tract of ferrets

The distributions of α2-6SAL and α2-3SAL in the respiratory tract of ferrets is

similar to humans as reported previously (41). To determine if receptor distribution in the

lower respiratory tract of ferrets correlated with viral replication, tracheal, bronchial, and

alveolar tissues were stained with MAA1/MAA2 lectins that recognize α2-3SAL and

SNA lectin that recognizes α2-6SAL, respectively. To ensure that the three lectins could

bind to the respective sialic acid, MDCK cells that contain both α2-6SAL and α2-3SAL

were used as lectin binding assay control (Fig. 4). MAA1 stained trachea and bronchus

poorly, but MAA2 stained lamina propria or connecting tissue and submucosal area of

the trachea and bronchus well. Both MAA1 and MAA2 stained pneumocytes in alveoli


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modestly. In contrast, SNA bound strongly to the ciliated cells and submucosal glands of

trachea and bronchus, and pneumocytes and walls of alveoli. Thus, α2-6SAL is more

abundant in the lower respiratory tract of ferrets than α2-3SAL.

Virus with αααα2-6SAL receptor binding preference had increased binding to the lower

respiratory tract of ferrets

rWt SI06-AQ and rWt SI06-DR were further examined for their binding

capability to the respiratory tissues using FITC-labeled viruses (Fig. 5). Both viruses

were prepared at the same time and at the same condition, we reproducibly showed that

FITC labeled rWt SI06-AQ bound to the MDCK cells more intensely than rWt SI06-DR,

which correlated with the receptor distribution in the MDCK cells. rWt SI06-AQ had

strong binding avidity for the submucosal glands of bronchus, which contain α2-6SAL as

shown earlier, and pneumocytes of alveoli. rWt SI06-DR bound weakly to the connecting

tissue in bronchus and to the pneumocytes in alveoli. We can not exclude the possibility

that the rWt SI06-DR had poor binding in this assay. Nevertheless, these data confirmed

that viral binding correlated well with distribution of α2-6SAL and α2-3SAL receptors in

the respiratory tract. Moreover, the preference of receptor binding played a critical role in

viral replication in the lungs.


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Discussion

In this study, we showed that the α2-6SAL receptor is much more abundant in

the lower respiratory tract of ferrets than α2-3SAL. Thus, Wt SI06 with binding

preference to α2-6SAL replicated efficiently in the lungs. In contrast, the virus with

amino acid mutation in the receptor binding regions with binding specificity to α2-3SAL

did not replicate in the lungs. Replication of virus in ferret lungs correlated with disease

symptoms. Hence, receptor binding preference of an influenza virus is a critical factor

that determines viral replication in the lower respiratory tract and viral pathogenicity.

Single amino acid changes of residues 190 or 226 in the HA protein receptor-

binding region have been previously identified to be responsible for receptor binding

preference to either human-like or avian-like receptor (12, 21, 29, 30, 32, 33). By

introduction of different amino acid variations into the HA protein of rWt SI06 viruses,

we demonstrated that only the virus with Q226 in the HA protein could replicate in the

lungs and that Q226 is a critical residue to enable the virus to replicate in ferret lungs. In

contrast to the H1N1 viruses where Q226 confers binding to α2-6SAL, Q226 confers

binding to α2-3SAL for the H3N2 viruses (24, 33). The Q226L change switched the

binding specificity of the H3N2 viruses from α2-6SAL to α2-3SAL and influenced

severity of illness of the infected ferrets (19). Our recent study on the swine-origin 2009

pandemic H1N1 virus also demonstrated that the Q226R change resulted in the loss of

α2-6SAL binding (6). Thus, H1N1 viruses with the Q226R change have different

receptor binding preference than the H2, H3, H4 and H9 subtypes of influenza viruses (2,

9, 21, 22, 24). The residue 190 was reported to be important for receptor binding

preference of the 1918 H1N1 pandemic virus and avian H1N1 viruses (12). The loss of


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α2-6SAL in the 1918 pandemic virus correlated with the loss of ferret-to-ferret

transmission (39). We also found that the 190 residue influences binding to α2-3SAL.

SI06-AQ could not bind to α2-3SAL; in contrast, SI06-DQ could bind to SAα2-3,

indicating that 190 residue also affect virus receptor binding preference.

The receptor distribution in the respiratory tract of ferrets has not been well

studied. MAA and SNA lectins are normally used to determine the cellular localization of

ligands by their differential binding of Sia-terminated oligosaccharides on the cell surface

(24). In humans, conflicting results on receptor distribution in respiratory tract have been

reported; however, these could be a result of using different lectins in the studies (24).

MAA1 and MAA2 both bind to α2-3SAL, and MAA1 also binds to non-Sia residues.

Therefore, both MAA1 and MAA2 were used in our study. In contrast to SNA lectin that

binds avidly to cilial cells in the luminal surface of airway epithelium, MAA1 and MAA2

bind to epithelial surface of the trachea less well but bind avidly to the submucosal

connective tissue. These results are in agreement with those reported previously for the

distribution of receptors in respiratory tract of ferrets (18, 19). In addition, we also

showed that submucosal glands and alveoli are rich in α2-6SAL, although α2-3SAL was

also detected in alveoli. Kirkeby et al (18) pointed out that mucinous cells in the airways

of ferrets may become metaplastic and change their expression of sialoglycans as a

consequence of an influenza infection. However, Kerkeby et al. were unable to detect α2-

6SAL and α2-3SAL in alveoli. Shinya et al (34) reported that human lung contained α2-

3SAL; they hypothesized that infection with the H5N1 virus with binding preference for

avian receptor is thus restricted to lungs. We discovered that human alveoli also contain

abundant levels of α2-6SAL (data not shown), which would allow human influenza virus


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to replicate efficiently in the lung tissue and cause pneumonia. Our study shows that Wt

SI06-DR could only bind α2-3SAL and that the virus replicated efficiently in the upper

respiratory tract, but was unable to replicate in the lungs. This result is different from the

highly pathogenic H5N1 A/Vietnam/1203/2004 (VN04) virus. Despite its preference for

the α2-3SAL receptor, VN04 replicates very efficiently in the lungs of the infected

ferrets (37). The H5N1 VN04 virus has multi-basic amino acids in the HA1-HA2

cleavage site enabling viral replication independent of exogenous trypsin and spreading

systematically. In addition, other viral proteins such as the PB2, PB1-F2 and NS1

proteins of the VN04 virus also contribute to virus virulence (4, 8, 17, 36). The H1N1 Wt

SI06 virus is far less virulent than the H5N1 VN05 virus. We speculate that the inability

of the virus with the R226 residue to replicate in the lower respiratory tract is due to the

paucity of α2-3SAL in the trachea and lungs. rWt SI06-AQ with an α2-6SAL specificity

thus has the advantage of replicating in the cells of trachea and lungs that contain α2-

6SAL receptor. Lack of viral replication in the respiratory tract is not necessarily due to

lack of viral binding to the cells, other factors may also contribute to virus pathogenicity.

rWt SI06 variants, which have the identical genomic sequence with the exception of only

one or two amino acid difference in the HA protein, replicated drastically differently in

ferret lungs, suggesting that the receptor binding preference of a virus is an important

factor in determining viral replication in the host.

Human H1N1 influenza virus binds to both α2-6SAL and α2-3SALwith

preference to α2-6SAL (11, 14). Human H3N2 influenza virus normally binds to α2-

6SAL, whereas avian virus preferentially binds to α2-3SAL. Receptor binding

preference has been proposed to be an important species barrier; it prevents avian viruses


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from transmitting to humans (1, 35). Vaccines against influenza are mainly produced in

embryonated chicken eggs. However, some Wt influenza viruses and reassortant vaccine

strains do not grow well in eggs. To select variants that grow well in eggs, egg passage is

routinely used. The egg adaptation process frequently introduces amino acid changes into

the HA protein that affect viral receptor binding and sometimes even viral antigenicity

(21, 29, 30, 32, 33). Egg adaptation of human influenza virus results in increased binding

of α2-3SAL (avian-like receptor), and impaired ability to bind α2-6SAL (human-like

receptor) (16, 21). Recently, Hensley et al. (13) reported that passage of an influenza

virus in mice with preexisting immunity frequently selected single amino acid change at

residue 158, 246 or 156 in the HA globular domain, which decreased cellular receptor

binding avidity and affected viral antigenicity. Thus, influenza viruses evolve to be able

to escape from the pressures of existing polyclonal antibodies or other growth pressure

through single amino acid change in the receptor binding region.

Sequence analysis of bWt SI06 virus revealed heterogeneity of the HA protein at

residues 190 and 226; it represents sequence changes from viral passages in eggs.

Although the virus with residues A190 and Q226 was present as a minor population by

sequencing analysis of single plaques, it was the only variant recovered from lung tissue

of ferrets infected with bWt SI06. The virus with residues D190 and Q226 in the HA

protein was also present in Wt virus pool and replicated well in ferret lungs, but it was

not isolated from Wt virus infected lungs. The original clinical isolate of bWt SI06 most

likely contained residue Q226 with affinity to α2-6SAL. Q226 was conserved in H1N1

viruses that circulated in human population from 1995 to 2007 as shown in Fig. 1;

however, the residue 190 varied among the H1N1 natural isolates (see Fig. 1). More


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recent H1N1 strains have N190 in the HA protein. A/NC/20/90, A/SD/6/07, and

A/HK/2652/06 with Q226 replicate well in the lungs of ferrets (data not shown).

Residues D, V, N, or A were found in the HA protein of bWt SI06 variants, indicating

that this 190 residue had a strong pressure to mutate. Some of these residues were the

intermediate changes accruing in the egg adaptation process. Wt SI06-DR, with a

preference for avian-like receptor, must have been selected in egg amplification process.

The Q226R change in the earlier H1N1 influenza viruses that resulted in the loss of

binding specificity for α2-6SAL has been reported by Mochalova et al (23).

The virus with residues DR is less immunogenic than that with residues AQ, DQ

or AR as examined for the rWt SI06 variants although its antiserum cross-reacts with

other HA protein variants well. However, ferret sera generated against viruses with AQ

or AR reacted less well with SI06-DR and -DQ. Live attenuated ca SI06-DR vaccine

virus replicated slightly less well than ca SI06-AQ in the upper respiratory tract of ferrets

and was not as immunogenic as ca SI06-AQ. A/HK/2562/06 (A/Solomon Island/3/06-

like strain) that contained N190 and Q226 was also very immunogenic and had 4-fold

reduction in reactivity with rWt SI06-DR (data not shown). Because Wt SI06 egg isolate

with DR residues in the HA protein was used as the reference strain, A/HK/2562/06 was

found to be antigenically different from the SI06 strain and was not considered as the

vaccine strain. Instead, SI06 with D190 and R226 was chosen as the H1N1 vaccine

component for the live attenuated influenza vaccine used in the 2007-2008 influenza

season. Our data indicate that A/HK/2562/06 with residues that confer to α2-6SAL

binding preference would be a better choice for the H1N1 vaccine in comparison to the

A/Solomon Island/3/06 strain that has the binding preference to α2-3SAL. In the


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following season, a later isolate, A/South Dakoda/6/07 with residues N190 and Q226,

was selected as the vaccine strain. In summary, our studies on the seasonal Wt H1N1

viruses further emphasize the need of carefully choosing appropriate Wt viruses as

reference strains and the need of thoroughly evaluating influenza vaccine variants for

their receptor binding preference, their antigenicity and immunogenicity in order to select

suitable vaccine strains for immunization.


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Acknowledgements

We are grateful to Joseph Show and Gorazd Drozina for editing this manuscript.

We thank Dan Ye and Chin-Fen Yang for their sequencing support, Scott Jacobson,

Stephanie Gee, Armando Navarro, Paulyn Cha and Brett Pickell for ferret studies, Bin Lu

and Zhongying Chen for discussions, Jennifer Woo and Chengjun Mo for help with the

microscope imaging, and Gary Van Nest for review of the manuscript. We also thank the

CDC for providing the Wt influenza viruses.


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Figure Legends

Figure 1. Hemagglutinin sequence alignment to compare the HA sequences of the H1N1

viruses circulated from 1995 to 2007. Cold-adapted (ca) vaccine strains of A/New

Caledonia/20/99 and A/Solomon Island/3/06 are also included. Residues 190 and 226 are

highlighted in green or yellow, three A/HK/2652/06 specific residues are highlighted in

grey.

Figure 2. Detection of virus replication in the respiratory tract of ferrets. Ferrets were

infected with rWt SI06-AQ or rWt SI06-DR intranasally, and three days later the tissues

were processed for immunohistochemistry staining using goat anti-influenza A

polyclonal antibody followed by incubation with horse radish peroxidase-conjugated

rabbit anti-goat antibody. Viral antigens in the infected cells are indicated in red.

Figure 3. Monitory of the infected ferrets for their body weight and temperature changes.

Ferrets in groups of three were inoculated with 7.0 log10PFU virus intranasally and

monitored for their body weight daily (upper graph) and rectal temperature twice daily

(lower graph) at a 6 h interval during the day. Each data value is a mean of the three

ferrets. The dotted line in the temperature graph represents the fever temperature.

Figure 4. Receptor distribution in the respiratory tract of ferrets. MAA1, MAA2 or SNA

lectin was used to stain the tissues in the respiratory tract of ferrets to detect α2-3SAL

(MAA1 and MAA2) or α2-6SAL (SNA). The Madin-Darby canine kidney cells served as

controls.

Figure 5. Virus binding to the respiratory tract tissues. FITC-labeled rWt SI-AQ or rWT

SI-DR virus was incubated with ferret bronchus or lung tissues followed by incubation


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with rabbit anti-FITC antibody and developed with AEC substrate. The virus antigens

bound to the tissues are shown in red.


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Table 1. Sequence analysis of bWt SI06 virus isolated from ferrets and variants isolated

from individual plaques

HA residue Virus Virus isolate

190a 226

Stability in eggs

SI06-NT Ferret NT A/D Q/R ND

SI06-lung Ferret lung A Q yes

SI06 Original D R yes

SI06 plaque-A 90% (43/48) D R yes

SI06 plaque-B 6% (3/48) V Q Changed to DR

SI06 plaque-C 2% (1/48) N Q yes

SI06 plaque-D 2% (1/48) A Q yes

ND: not determined

a Standard 1-letter codes for amino acids


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Table 2. Replication of wild type SI06 variants in the respiratory tract of ferrets

1 The virus contained sequence variations in the HA; standard 1-letter amino acid codes

apply.

Virus Titer (Log10EID50 ± SE) Virus HA residue

190 and 226 NT Lung

Original1 5.8±0.4 5.8±0.2

DR 4.4±0.8 <1.5

NQ 5.6±0.4 5.4±0.4

bWt SI06

AQ 5.5±0.1 5.1±0.2

DR 5.8±0.2 <1.5

AR 6.1±0.9 <1.5

DQ 7.2±0.2 6.3±0.5

rWt SI06

AQ 6.9±0.2 6.3±0.3


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Table 3. The effect of the HA residues on virus antigenicity and immunogenicity

Ferrets in groups of 2 (rWt SI06) or 3 (ca SI06) were inoculated with 1x107

PFU virus. Serum samples were collected on day 14 and

examined by the hemagglutination inhibition (HAI) assay using turkey red blood cells and by the neutralization assay against

homologous (bold and underlined) and heterologous viruses. The values represent the geometric mean titers of reciprocal of the

highest serum dilutions that inhibited hemagglutination or neutralized virus infectivity.

GMT of HAI antibody of postinfected ferret serum GMT of Nt antibody of postinfected ferret serum

rWt SI06 ca SI06 rWt SI06 ca SI06

Virus

DR DQ AQ AR

DR AQ

DR DQ AQ AR

DR AQ

rWt SI06-DR 724 724 724 362 813 323 1600 1386 1386 980 2032 923

rWt SI06-DQ 512 1448 1448 1024 203 1625 693 3200 2771 1200 508 2930

rWt SI06-AQ 1024 5793 11585 5793 512 6502 2771 12800 20239 9600 2113 14769

rWt SI06-AR 724 4096 8192 4096 813 4096 1131 4525 11085 6400 1677 6451


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Table 4. Receptor binding preferences of hemagglutinin variants by hemagglutination

assay

Hemagglutination titer in cRBC with indicated treatment

Virus untreated desialylated α2-3SAL

resialylated

α2-6SAL

resialylated

rWt SI06-DR 64 < 2 16 < 2

rWt SI06-AQ 64 < 2 < 2 64

rWt SI06-AR 128 < 2 64 < 2

rWt SI06-DQ 64 < 2 16 64

The values represent the reciprocal of the highest virus dilutions that hemagglutinated red

blood cells.


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Fig. 1

A/Beijing/262/95 PPNIGDQRAI YHTENAYVSV VSSHYSRRFT PEIAKRPKVR GQEGRINYYW

A/NC/20/99 PPNIGNQRAL YHTENAYVSV VSSHYSRRFT PEIAKRPKVR DQEGRINYYW

A/NC/20/99 ca PPNIGDQRAL YHTENAYVSV VSSHYSRRFT PEIAKRPKVR DQEGRINYYW

A/Singapore/23/04 PPNIGDQRAL YHTENAYVSV VSSHYSRKFT PEIAKRPKVR DQEGRINYYW

A/Canada/591/04 PPNIGDQRAL YHTENAYVSV VSSHYSRKFT PEIAKRPKVR DQEGRINYYW

A/SI/3/06 PPNIGDQRAL YHTENAYVSV VSSHYSRKFT PEIAKRPKVR DREGRINYYW

A/SI/3/06 ca PPNIGDQRAL YHTENAYVSV VSSHYSRKFT PEIAKRPKVR DREGRINYYW

A/HK/2652/06 PPNIGNQMTL YHKENAYVSV VSSHYSRKFT PEIAKRPKVR DQEGRINYYW

A/S.P/8/06 PPNIGNQRAL YHTENAYVSV VSSHYSRKFT PEIAKRPKVR DQEGRINYYW

A/SD/6/07 PPNIGNQKAL YHTENAYVSV VSSHYSRKFT PEIAKRPKVR DQEGRINYYW

190

186

226222

H3#H1#


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rWt SI-DR rWt SI-AQ

Fig. 2

trachea

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Nasal pharynx


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2

3

4

5

6

7

8

9 PBS

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AQx

Fig. 3

38.6

38.8

39.0

39.2

39.4

39.6

39.8

40.0

40.2

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0 24 48 72

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MAA1 MAA2 SNA

MDCK

Fig.4


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alveoli

rWt SI-AQ rWt SI-DR

bronchus

Fig. 5

MDCK on February 1, 2019 by guest

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JVI Accepts, published online ahead of print on 3 March...

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