Monoclonal antibodies isolated from human B cells neutralize a broad range of H1subtype influenza A viruses including swine-origin Influenza virus (S-OIV)

Roberto Burioni ⁎, Filippo Canducci 1, Nicasio Mancini 1, Nicola Clementi, Monica Sassi,Donata De Marco, Roberta Antonia Diotti, Diego Saita, Michela Sampaolo,Giuseppe Sautto, Matteo Pianezze, Massimo Clementi

Laboratorio di Microbiologia e Virologia, Università Vita-Salute San Raffaele, via Olgettina 60, 20132 Milan, Italy

a b s t r a c ta r t i c l e i n f o

Article history:

Received 10 November 2009

Returned to author for revision

4 December 2009

Accepted 14 December 2009

Available online 22 January 2010

Keywords:

Monoclonal antibodies

Immunotherapy

Pandemics

Influenza

Oseltamivir

Antiviral therapy

The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the

virus–host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab

fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned

before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The

two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC50 ranging from 2

to 7 μg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies

neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating

full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and

the treatment of this human infection.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Recently, a new strain of human H1N1 influenza A virus (swine-

origin influenza virus; S-OIV) was identified (Fraser et al., 2009a). As

of September 11th, 2009, the virus has spread to many countries with

more than 277,607 cases and 3205 deaths. These figures, together

with the fact that the new virus has been assessed as having pandemic

potential, make this new infection a serious concern for the global

health(Fraser et al., 2009b; Trifonov et al., 2009b). The elucidation of

the interplay between the new influenza virus and the human host,

mainly the identification of S-OIV antigenic regions that are able to

elicit broad-range and neutralizing antibodies, is crucial for the

understanding of the epidemiology of this disease.

Indeed, human monoclonal antibodies with broad-range neutral-

izing activity against S-OIV can be key reagents in this scenario, as

they can be used to probe in vitro the vaccine candidates and

providing useful information for understanding data generated by

preliminary in vivo studies(Parren et al., 1996). Furthermore, human

monoclonal antibodies neutralizing influenza viruses are molecules

potentially useful in prophylaxis and therapy (Simmons et al., 2007;

Trifonov et al., 2009b). Even if antibody-based prophylaxis and

therapy of influenza is at present an unexplored option, polyclonal

and monoclonal human antibodies are effectively used in a number of

different viral infections (Sawyer, 2000). It has been demonstrated

that anti-influenza mouse and human monoclonal antibodies are

effective in prophylaxis and therapy in mice (Palladino et al., 1995;

Renegar et al., 2004; Smirnov et al., 2000), and passive immunization

by vertical transmission of anti-influenza antibodies is protective

against infection both in animal models and in human subjects (Luke

et al., 2006; Martinez, Tsibane, and Basler, 2009; Prabakaran et al.,

2009; Prabhu et al., 2009; Puck et al., 1980; Reuman et al., 1983;

Simmons et al., 2007; Sui et al., 2009; Sun et al., 2009; Sweet et al.,

1987a, 1987b; Throsby et al., 2008; Yoshida et al., 2009; Yu et al.,

2008). All these observations indicate that passive antibody adminis-

tration could be a potentially useful adjunctive treatment and

preventive option in the S-OIV infection.

In this paper, we describe the molecular cloning, the binding

characteristics, and the broad-range neutralizing activity of two

human monoclonal antibody Fab fragments (HMab) directed against

S-OIV hemagglutinin (S-HA). These antibodies, cloned from a patient

before the emergence of the S-OIV strain, demonstrate that exposure

to previous H1N1 strains elicits the production of S-OIV-neutralizing

antibodies. The data presented here document not only that previous

influenza H1N1 infections can provide a certain degree of protection,

but also that some epidemiological features of the S-OIV infection as

well as some unexplained results of the preliminary in vivo vaccine

trials can be clarified at the molecular level.

Virology 399 (2010) 144–152

⁎ Corresponding author. Fax: +39 02 26434288.

E-mail address: Burioni.roberto@hsr.it (R. Burioni).1 Both authors contributed equally to the presented work.

0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2009.12.014

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Results

Two distinct HMabs, named PN-SIA28 and PN-SIA49, were cloned

from a patient exposed to previously circulating H1N1 influenza A

strains, with a negative clinical history for influenza infection in the

last years and with a detectable serum neutralizing activity against a

1934 influenza A isolate.

When tested in immunofluorescence assay, both Mabs recognized

cells infected with the S-OIV strain (A/Milan/UHSR1/2009), with the

human strains A/Puerto Rico/8/34 (A/PR/8/34); A/Wilson-Smith/33

(A/WS/33); A/Malaya/302/54 (A/Mal/302/54); A/New Caledonia/

20/99 (A/NC/20/99), and with a swine strain (A/swine/Parma/1/

97). No reactivity was demonstrated against the A/Victoria/3/75

H3N2 nor against the B/Lee/40 influenza B strain. In

Fig. 1. Immunofluorescence on A/Milan/UHSR1/2009-infected MDCK cells or HA-transfected cells. MDCK cells stained after 8 h of infection with HMabs PN-SIA28 (a) and PN-SIA49

(b, c [100 magnification]), mouse anti-NP1 (d), the negative control Fab509 (e) and a double stain with HMabs PN-SIA28 and PN-SIA49 of mock infected cells (f). HEK293T cells

transfected with recombinant A/Puerto Rico/8/1934 HA expression vector and stained with positive control M145 Monoclonal antibody (g) HMabs PN-SIA28 (h) and PN-SIA49 (i).

HEK293T cells transfected with recombinant A/Milan/UHSR1/2009 (S-OIV) HA expression vector and stained with positive control M145Monoclonal antibody (l), HMabs PN-SIA28

(m) and PN-SIA49 (n). HEK293T cells transfected with recombinant A/South Carolina/1/18 HA expression vector and stained with positive control M145 Monoclonal antibody (o),

HMabs PN-SIA28 (p) and PN-SIA49 (q). Mock-transfected HEK293T cells stained with control M145 monoclonal antibody (r). HMabs PN-SIA28 (s) and PN-SIA49 (t).

145R. Burioni et al. / Virology 399 (2010) 144–152

immunofluorescence staining on infected cells, both HMabs featured a

clear cytoplasmic pattern with plasma membrane reinforcement (Fig.

1) against S-OIV and all tested influenza A strains. No differences were

observed among cells infectedwith the different isolates. Notably, cells

transfected with the recombinant hemagglutinin (HA) genes of A/

Puerto Rico/8/1934, A/Milan/UHSR1/2009 and A/South Carolina/1/

18 were recognized by both HMab (Fig. 1), thus suggesting that both

PN-SIA28 and PN-SIA49 bind to extremely conserved antigenic

epitopes that are present in all H1 HAs we analyzed.

The epitope recognized by both antibodies is conformational as

evaluated by Western blot analysis and present only in the HA0 form

of hemagglutinin. In fact, no binding was observed under denaturing

conditions and nor HA1 or HA2 subunits were separately recognized

by PN-SIA28 and PN-SIA49 (Fig. 2).

PN-SIA28 and PN-SIA49 showed a strong neutralization activity

against S-OIV and all other H1N1 strains used in the study. When

neutralization activity was evaluated with immunofluorescence

reduction assay PN-SIA49 featured IC50 (Fab concentration giving

50% of neutralization) ranging from 2.1 μg/mL against A/H1N1/PR/8/

34 to 6.9 μg/mL against A/NC/20/99. PN-SIA49 neutralized S-OIV

strain (A/Milan/UHSR1/2009) with an IC50 of 2.8 μg/mL. PN-SIA28

was demonstrated to have an IC50 ranging from 3.0 μg/mL against A/

H1N1/WS/33 to 7.0 μg/mL against A/H1N1/NC/20/99. PN-SIA28

neutralized S-OIV strain with an IC50 of 4.0 μg/mL. No neutralizing

activity was demonstrated for both Mabs against the B/Lee/40

influenza B strain nor against A/Victoria/3/75 H3N2 strain. (Table 1,

Fig. 3). Similar results were obtained with plaque reduction assay

(Fig. 4). When both Mabs were tested against H5N1 influenza viruses,

no reductions of EID50 values were observed comparedwith untreated

viral inoculums (A/turkey/Turkey/1/2005: negative control: 107.5

EID50/100μl, PN-SIA28: 106.83EID50/100μl, PN-SIA49: 10

7EID50/100μl;

A/chicken/Egypt/A6/2008: negative control: 106.24 EID50/100μl,

PN-SIA28: 106.7 EID50/100μl, PN-SIA49: 107.2 EID50/100μl). DNA

sequence of the light and heavy variable regions demonstrated that

the two Hmabs are different. Genbank accession numbers of the

heavy and light sequences of PN-SIA28 and PN-SIA49 are

GQ867592 and GQ867595; GQ867593 and GQ867594, respectively.

PN-SIA28 and PN-SIA49 VH genes were V3-30 and V3-23,

respectively. Both antibodies were mutated with respect to the

germline encoded VH gene sequences (94.62% and 92.36% identity,

respectively) (Fig. 5).

Discussion

Pandemic influenza viruses have emerged at least three times in

the last 100 years with the most severe being the 1918 H1N1 Spanish

influenza. The risk of an influenza pandemic caused by the newly

emerged S-OIV strain is real, and the consequences of this event are

difficult to estimate but could be catastrophic. Even if the pathogenic

potential of the novel strains appears lower than that of the 1918H1N1

Influenza virus, the possibility of a further evolution of the pathogen

cannot be excluded (Lipsitch et al., 2009). The understanding of the

role played by immunity elicited by previously circulating viruses, a

better knowledge of the virus–host interplay, and more importantly,

the availability of adjunctive novel tools for preventing and treating

this disease may be crucial in contrasting a possible pandemic.

The molecular cloning of from an unexposed patient of human

monoclonal antibodies, endowed with strong neutralizing activity

against S-OIV and against a broad range of human and swine H1N1

isolates, is the clear demonstration that some conserved antigenic

determinants present in a wide range of H1N1 viruses are also present

in the S-OIV strain. The two Mabs are displaced in competition

experiments by the mouse Mab C179 (Takara), and they compete the

one with the other (data not shown). This indicates that the two

epitopes recognized by the two HMabs described in this paper are at

least overlapping and situated in the stem region of the HA molecule.

The presence of antibodies neutralizing a wide range of H1N1

influenza isolates (including S-OIV) in the antibody repertoire elicited

by infection with previously circulating strains can explain the milder

clinical syndrome and the lower rate of this infection observed in

older subjects (Trifonov et al., 2009a, 2009b). Itoh et al. (2009) have

shown that only sera of those born before 1918 have some

neutralizing activity against S-OIV, suggesting a close serological

relation between the novel strain with the 1918 pandemic strain but

not with the antigenically divergent human H1N1 viruses circulating

in the 1920s to 1950s and since 1977. This is apparently in contrast

with our observation, as our antibodies are derived from the

repertoire of a patient born well after 1918. However, neutralization

assays performed with polyclonal sera cannot fully dissect the

humoral immune response and it is likely that only the minority of

antibodies elicited after the infection is endowed with cross-

neutralizing activity, being the majority directed against more

antigenic and variable viral antigens.

Furthermore, recently published data on the preliminary vaccine

trials (Galli et al., 2009; Hancock et al., 2009) noted an unanticipated

robust immune response to the H1N1 vaccine after a single dose

administration even in patients with no measurable antibodies

against S-OIV at the baseline. This unexpected brisk response to the

vaccination can be explained by an immunotypic similarity not yet

recognized between the 2009 H1N1 virus and recent seasonal strains.

The data presented in this paper are providing a molecular evidence

supporting this hypothesis, being important in the current time-

limited run for the quest of an effective vaccination strategy and

schedule.

Finally, these HMabs have the potential of being useful as

adjunctive tools in treatment and prophylaxis in the unfortunate

case of a S-OIV pandemic. In a mouse model of H5N1 influenza

infection, neutralizing human monoclonal antibodies were proven to

Fig. 2.Western blot on transfected cell lysates. Western blot on lysates of HEK293T cells

transfectedwith A/Milan/UHSR1/2009 (S-OIV) HA expression vector (lanes a–d and h)

or onmock-transfected cells (lanes e and g). Lanes: (a) positive serumdiluted 1:500; (b)

positive serum diluted 1:1000; (c) PN-SIA28; (d) PN-SIA49; (e) PN-SIA28 (on mock

lysate); (f) MagicMark XP (Invitrogen); (g) PN-SIA49 (on mock lysate); (h) anti-H1

positive control Anti-H1(GeneTex).

Table 1

IC50 values against H1N1 strains tested in this study (rounded to the first decimal).

PN-SIA28 IC50 PN-SIA49 IC50

A/PR/8/34 4.1 μg/ml 2.1 μg/ml

A/WS/33 2.9 μg/ml 4.5 μg/ml

A/Mal/302/54 3.4 μg/ml 2.7 μg/ml

A/NC/20/99 7.0 μg/ml 6.9 μg/ml

A/swine/Parma/1/97 3.9 μg/ml 2.3 μg/ml

A/Milan/UHSR1/2009 (S-OIV) 4.0 μg/ml 2.8 μg/ml

Fig. 3. Fluorescence inhibition assay. Sigmoidal dose–response curve fit nonlinear regression is reported for PN-SIA28 and PN-SIA49 (red line) against all viral strains studied in this

paper. IC50 values for H1N1 strains are reported for in Tab I. A control unrelated Fab e509 was also used (black line).

146 R. Burioni et al. / Virology 399 (2010) 144–152

147R. Burioni et al. / Virology 399 (2010) 144–152

Fig. 4. Plaque inhibition assay. Sigmoidal dose–response curve fit nonlinear regression is reported for PN-SIA28 and PN-SIA49 against all viral strains studied in this paper.

148 R. Burioni et al. / Virology 399 (2010) 144–152

be extremely efficient in conferring a significant immunity to mice,

causing a milder disease and strongly reducing virus dissemination

(Simmons et al., 2007). Therapy and prophylaxis of S-OIV infection by

administration of human monoclonal antibodies is a plausible

strategy. Several reports related to the 1918 “Spanish” pandemic

indicated as early treatment with blood product from recovered

patients was associated with a strong reduction in mortality, which

was more evident in the case of a early treatment (Luke et al., 2006),

with the speculation that the administration of neutralizing anti-

bodies present in the blood-derived products favorably modified the

virus–host balance causing a milder disease and preventing the

development of respiratory complications (Luke et al., 2006).

Administration of human monoclonal antibodies neutralizing S-OIV

has the potential of having the same effect. The IC50 values of PN-

SIA28 and PN-SIA49 Fabs are in the micromolar range (2.1–7.0 μg/

ml), comparable to those described previously by Throsby et al. (IC50

ranging from 0.55 to 15 μg/ml) (Throsby et al., 2008) or remarkably

lower than those described by Sui (IC50 in the order of 10 μg/ml) (Sui

et al., 2009). It should be noted that both works described the

neutralizing activities of recombinant full IgGs while our HMabs are in

the Fab form, and usually the neutralization capacity of Fabs strongly

increases once expressed in IgG form (Lamarre and Talbot, 1995).

Finally, even if most of the currently circulating S-OIV strains are

sensitive to mono-therapy with Oseltamivir and Zanamivir, in the

absence of additional drugs they will probably give rise to resistant

strains, as recently observed in these pandemics in Denmark, Japan,

and Hong Kong (http://www.who.int/csr/disease/swineflu/notes/

h1n1_antiviral_resistance_20090708/en/index.html) and as already

happened with the vast majority of seasonal H1N1 that have now

become resistant (Shetty, 2009). In the current situation, not knowing

in advance the efficacy and of an anti-S-OIV vaccine, having an

alternative path open to the scientific community could be important.

Humanmonoclonal antibodymass production is feasible, scalable, the

final product is free of adventitious agents and it is utilizing well-

known procedures that are used formany drugs. Finally, given the fact

that these antibodies are of human origin, the risk of unwanted effects

is minimal.

In summary, the human monoclonal antibodies neutralizing the

pandemic H1N1 S-OIV strain can be useful tools for the understanding

of the disease, for the understanding of vaccine trial data in the

shortest possible time, and can constitute the basis, alone or in a

combination with other monoclonal antibodies, for a new class of

drugs to be used in the treatment and in the prophylaxis of this disease.

Materials and methods

This work was approved by the San Raffaele ethical committee,

and all human samples were collected after informed consent was

obtained.

Human monoclonal antibodies

A patient, of the age of 55, with a negative clinical history of

infection with influenza virus in the past 10 years, and with a serum

neutralizing titer (IC50N1:20) against a reference H1N1 1934 strain

(A/PR/8/34), was used as a source of lymphocytes 3 weeks after a

routine influenza seasonal vaccination and after written informed

consent was given. This procedure was performed well before the

emergence of the novel S-OIV strain. Fifteen B-cell lines producing

antibodies reacting in immunofluorescence with influenza A-infected

cells were obtained by Epstein-Barr Virus (EBV) transformation (Cole

et al., 1984), and subsequently cDNA coding for Fab fragments were

PCR amplified and cloned in appropriate bacterial expression vector

(Burioni et al., 1997, 1998a) for avoiding instability of antibody

Fig. 5. Sequence analysis of VH genes of PN-SIA28 and FN-SIA HMabs. Alignment of VH sequences (nucleotides and amino acids) with each germline IGHV gene was performed using

the International ImMunoGeneTics database. The CDR3 gene segments are underlined.

149R. Burioni et al. / Virology 399 (2010) 144–152

production of EBV-transformed cell lines and for DNA sequencing.

Transformed bacteria were used for production of the recombinant

Fabs (Burioni et al., 1994; Perotti et al., 2008), which were purified as

previously described (Burioni et al., 1998b). Only two of the Fabswere

demonstrated to be endowed with neutralizing activity. An anti-

hepatitis C virus Fab, named e509 (Burioni et al., 1998b), produced

and purified with an identical procedure, was used as a negative

control in all experiments. Mutations identified by comparing each

sequence with germline sequences (International ImMunoGeneTics

database at http://imgt cines.fr/) were defined on the basis of

nucleotide changes in the VH segment.

Isolation and identification of influenza A/Milan/UHSR1/2009 strain

All experiments were conducted in the BLS3 laboratory of the

Università Vita-Salute San Raffaele. The swine origin influenza virus

(S-OIV) used in this study was isolated from the pharyngeal swab of a

26-year-old Italian patient of our hospital referring fever and malaise

after a recent travel to the United States. The swab was seeded on 80%

confluent MDCK (Madin-Darby canine kidney) cells (ATCC no. CCL-

34TM). The cells were infected in modified Eagle medium (MEM,

GIBCO) with the addition of 2 μg/ml of trypsin. After 1 h, 10% fetal

bovine serum (GIBCO), 50 μg/ml of penicillin (GIBCO), 100 μg/ml of

streptomycin (GIBCO) and of L-glutamine (2 mM) (EuroClone) were

added and cells were incubated at 35 °C, in 5% CO2 atmosphere for 5

days. Identification was performed directly on patient's swab sample

and on culture supernatant by whole length amplification and

sequencing of the S-HA gene by using a previously described RT-

PCR protocol (Puthavathana et al., 2005) with minor modifications

(Baek et al., 2009). Primer forward was Bm-HA-1-Fw 5′-TAT TCG TCT

CAG GGA GCA AAA GCA GGG G-3′, primer reverse was Bm-NS-890-

Rev 5′-ATA TCG TCT CGT ATT AGT AGA AAC AAG GGT GTT T-3′. The

reaction was performed using the SuperScript III One-Step RT-PCR

System with Platinum® Taq High Fidelity (Invitrogen) and the

following thermal profile: 30 min 50 °C; 10 min 94 °C; 30 s 94 °C,

1 min 53 °C, 1 min 72 °C (45 cycles). Sequencing was performed by

using BigDye Terminators 3.1 with the automatic sequencer AbiPr-

ism3130 (Applied Biosystems); the primers used in the amplification

are: F1 5′-TAG GAA ACC CAG AAT GCG-3′, F2 5′-TAC TGG ACC TTG CTA

GAA-3′, F3 5′-TCT ATT TGG AGC CAT TGC-3′.

Other influenza strains of human, swine or animal origin used in

this study

The following reference strains were used: A/Puerto Rico/8/34

(A/PR/8/34); A/Wilson-Smith/33 (A/WS/33); A/Malaya/302/54

(A/Mal/302/54); A/New Caledonia/20/99 (A/NC/20/99). A swine

strain (A/swine/Parma/1/97) was kindly provided by the Istituto

Zooprofilattico di Brescia, Brescia, Italy. The influenza B strain B/Lee/

40 and the influenza A/Victoria/3/75 were also tested in all studies.

The avian A/chicken/Egypt/A6/2008 and A/turkey/Turkey/1/2005

influenza A (H5N1) strains were also tested. All viruses, but A/swine/

Parma/1/97, were cultured on MDCK cells propagated in MEM

(GIBCO), supplemented with 10% fetal bovine serum (GIBCO),

50 μg/ml of penicillin (Gibco), 100 μg/ml of streptomycin (Gibco)

and of L-glutamine (2 mM) (EuroClone). The A/swine/PR/1/97

isolate was analogously grown on a different, more permissive cell line

(NSK-newborn swine kidney), kindly provided by the Istituto Zoopro-

filattico di Brescia. The cells were infected with each strain at 80%

confluence, after addition to themediumof 1 μg/ml of trypsin. After the

infection, the cells were incubated at 35 °C, in 5% CO2 atmosphere.

Immunofluorescence assays

Seven hours after infection, the MDCK cells were trypsinized,

washed twice in PBS and spotted on a slide by cytocentrifugation

(2×105 cells in each spot). The cells were then fixed and permeabi-

lized by cold methanol/acetone (−20 °C) for 10 min at room

temperature. After three washes in PBS, the cells were incubated for

30 min at 37 °C with each one of the HMabs (10 μg/mL). The cells

were then washed again in PBS and incubated for 30 min at 37 °C

with a fluoresceinated anti-human Fab monoclonal diluted in Evans

Blue. Uninfected cells were used as control. Commercially available

anti-NP protein mouse monoclonal (Anti-influenza A group, Argene,

cod 11-030) was used as an infection control and as negative control

e509Fab was used on infected cells. The same protocol was used to

stain HEK 293T cells 48 h after transfection, but Hoechst (SIGMA)

was used for counterstaining. M145 Monoclonal Anti-Human

Influenza A (clone C179, Takara) directed against a conformational

epitope of the viral hemagglutinin was used as positive control in

transfection experiments.

Transfection of 293T cells with A/Puerto Rico/8/1934, A/South

Carolina/1/18 and A/Milan/UHSR1/2009 strain hemagglutinin genes

The full-length HA gene of A/Puerto Rico/8/1934 was amplified

with the above-described protocol and directly cloned into the

pcDNA3.1 expression vector (Invitrogen). In particular, the HA gene

was amplified by using the following pair of primers APR834 CAC CAT

GAA GGC AAA CCT ACT GGT CCT GTT ATG TG and APR834_as TCA GAT

GCA TAT TCT GCA CTG CAA AGA TCC ATT AGA and directly cloned

followingmanufacturer's instructions. Both the A/Milan/UHSR1/2009

and the A/South Carolina/1/18 HA genes were artificially synthesized

(Geneart AG, Germany) flanking the full-length HA sequence with the

recognition sites of Hind III and AgeI restriction enzymes at 5′ and 3′

extremities, respectively. Each artificially synthesized HA gene was

subcloned from the shuttle vector supplied by the manufacturer

(Geneart, AG, Germany) into the pcDNA3.1 expression vector after

digestion with Hind III and AgeI enzymes. HEK 293T cells were

transfected with lipofectamine 2000 reagent (Invitrogen) with the

recombinant HA expression vectors following the manufacturer's

instructions. Forty-eight hours after transfection, cells were collected,

washed twice with PBS and spotted on a slide by cytocentrifugation.

Immunofluorescence was performed as described above.

Western blot

A Western blot assay was performed on lysates obtained from

HEK293T cells transfected or mock transfected with A/Milan/UHSR1/

2009 (S-OIV) HA expression vector. Ten micrograms of lysates

obtained by mechanical homogenization in NativePAGE™ Sample

Buffer (Invitrogen), were loaded on a 10% polyacrilamide gel. After

running, samples were transferred on a nitrocellulose membrane

(Hybond-ECL; Amersham Bioscience) overnight at 30 V and 90 mA.

The membrane was then blocked with PBS–milk (5%) for 1 h and then

washed three times with PBS–Tween20 (0.1%). Each HMab (5 μg/ml)

was then added together with the serum from a convalescent patient

(obtained 30 days after infection) and a commercially available

monoclonal antibody as positive controls (Anti-H1, GeneTex,

GTX28262). All antibodies were incubated for 1 h at room tempera-

ture. After washing with PBS–Tween 20 (0.1%), secondary antibodies

were added for 1 h and after an additional washing step, the substrate

solution was added (SuperSignal® West Pico Chemiluminescent

Substrate, PIERCE) and incubated for 2 min.

Virus neutralization assays

Fluorescence inhibition assay

Each viral isolate was titrated by the limiting dilution method, and

the viral titer calculated by the Reed-Muench formula. 100 TCID50 of

each isolate were preincubated 1 h at 37 °C with scalar concentrations

of each human Fab (from 0.125 to 20 μg/mL). 250 μL of each pre-

150 R. Burioni et al. / Virology 399 (2010) 144–152

incubated solutionswere then added onMDCK cells and incubated 1 h

at 37 °C (5% CO2). The cells were then washed twice in PBS; 1.5 mL of

serum-free medium with trypsin (1 μg/mL) was added, and the cells

were incubated 6 h at 37 °C. The cells were then washed again with

PBS, fixed and permeabilized with cold methanol/acetone. The

number of infected cells on each slide was then revealed using the

anti-NPmonoclonal antibodies, as above. An infection control without

antibody was added, as well as a negative control with e509 anti-

HCV/E2 monoclonal Fab. Each neutralization assay was performed in

triplicate and repeated in three different sessions. The neutralization

activity for each Fab concentration was expressed as the percentage

reduction of fluorescent nuclei compared with the nuclei counted in

the infection control. The neutralization curves were then fitted by

nonlinear regression with the GraphPad Prism software, allowing IC50

calculation (HMab concentration giving 50% neutralization).

Plaque inhibition assay

Each viral isolate was titrated by the limiting dilution method, and

the viral titer was calculated either as plaque forming units (PFU) on

six-well flat-bottomed plates either as TCID50. 100 TCID50 of each

isolate were preincubated 1 h at 37 °C with different concentrations of

the human Fabs. The same controls used in IF assay were also used in

the plaque reduction assay. One milliliter of each neutralization mix

was added on MDCK monolayer and the infection was performed 1 h

at 35 °C in presence of 5% CO2. After the adsorption, the medium was

removed and the monolayer washed twice with sterile PBS. Two

milliliters of MEM-agarose 0.8% supplemented with antibiotics

(penicillin (50 μg/ml) (GIBCO), streptomycin (100 μg/ml) (GIBCO)

and of L-glutamine (2 mM)) and trypsin (1 μg/ml) were gently added

on each well, and the plates were incubated 48 h at 34 °C in presence

of 5% CO2. After 48 h the agarose medium was removed from each

well and 1 ml of 70% methanol-crystal violet 1% was added to each

well at room temperature. Finally, the wells were washed with tap

water and dried to allow the count of PFU. The neutralization was

determined counting the PFU reduction in presence of antibodies, in

comparison with PFU observed in the infection control.

EID50 (embryonated infectious dose) reduction assay for H5N1

influenza strains

Serial 10-fold dilutions (10−3 to 10−8) of Highly Pathogenic Avian

Influenza (HPAI) H5N1 viruses (A/chicken/Egypt/A6/2008 and A/

turkey/Turkey/1/2005) were prepared in PBS and each dilution was

mixedwith equal volume of HMAbs PN-SIA28 or PN-SIA49 (50 μg/ml)

or negative control (PBS). After incubation for 1 h at 37 °C, themixture

was inoculated into the allantoic cavities of 10-day-old embryonated

chicken eggs (five eggs for each viral dilution).

After 72 h of incubation at 37 °C, the eggs were checked for vitality,

chilled and allantoicfluidswere tested individually for HA activity. The

EID50 per 100 μl was calculated using the method of Reed-Muench.

Acknowledgments

We would like to express our gratitude to Ilaria Capua and

Calogero Terragino for assessing neutralizing potential of our HuMAbs

against H5N1 virus in emrbyonated eggs experiment. We would like

to thank Elena Comoglio for helpful discussions and for support.

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