Published Ahead of Print 8 January 2014. 2014, 88(6):3432. DOI: 10.1128/JVI.03004-13. J. Virol. 

Randy A. AlbrechtJohn K. Rose, Peter Palese, Adolfo García-Sastre andVictor H. Leyva-Grado, Alex B. Ryder, Matthew S. Miller, Florian Krammer, Rong Hai, Mark Yondola, Gene S. Tan, FerretsHemagglutinin Stalk-Based Immunity in Assessment of Influenza Virus

http://jvi.asm.org/content/88/6/3432Updated information and services can be found at:

These include:

REFERENCEShttp://jvi.asm.org/content/88/6/3432#ref-list-1at:

This article cites 73 articles, 37 of which can be accessed free

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

on June 28, 2014 by E

AS

T C

AR

OLIN

A U

NIV

http://jvi.asm.org/

Dow

nloaded from

Assessment of Influenza Virus Hemagglutinin Stalk-Based Immunityin Ferrets

Florian Krammer,a Rong Hai,a Mark Yondola,a* Gene S. Tan,a Victor H. Leyva-Grado,a Alex B. Ryder,b Matthew S. Miller,a

John K. Rose,b Peter Palese,a,c Adolfo García-Sastre,a,c,d Randy A. Albrechta,d

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USAa; Department of Pathology, Yale University School of Medicine, NewHaven, Connecticut, USAb; Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USAc; Global Health & Emerging Pathogens Instituteat Icahn School of Medicine at Mount Sinai, New York, New York, USAd

ABSTRACT

Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus hemagglutinin and stalk-baseduniversal influenza virus vaccine strategies are being developed as promising countermeasures for influenza virus infections.The pan-H1-reactive monoclonal antibody 6F12 has been extensively characterized and shows broad efficacy against divergentH1N1 strains in the mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret modelof influenza disease. Furthermore, we recently developed a universal influenza virus vaccine strategy based on chimeric hemag-glutinin constructs that focuses the immune response on the conserved stalk domain of the hemagglutinin. Here we set out totest this vaccination strategy in the ferret model. Both strategies, pretreatment of animals with a stalk-reactive monoclonal anti-body and vaccination with chimeric hemagglutinin-based constructs, were able to significantly reduce viral titers in nasal turbi-nates, lungs, and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal wash viral titers. In summary, bothstrategies showed efficacy in reducing viral loads after an influenza virus challenge in the ferret model.

IMPORTANCE

Influenza virus hemagglutinin stalk-reactive antibodies tend to be less potent yet are more broadly reactive and can neutralizeseasonal and pandemic influenza virus strains. The ferret model was used to assess the potential of hemagglutinin stalk-basedimmunity to provide protection against influenza virus infection. The novelty and significance of the findings described in thisreport support the development of vaccines stimulating stalk-specific antibody responses.

In the United States, epidemics of seasonal influenza cause sub-stantial morbidity (1) and significant mortality (2). Despite the

proven ability of inactivated and live attenuated influenza virusvaccines to reduce the impact of influenza, the potential of cur-rently licensed influenza vaccines is not fully manifested becauseof several factors. First, influenza vaccination coverage rates re-main low (3). In particular, a recent survey of 11,963 adults (18 to64 years of age) revealed that only 28.2% reported receiving the2008-2009 influenza vaccine (4). Second, influenza vaccines in-duce immune responses that specifically neutralize influenza vi-ruses that are closely related to the vaccine strain, yet the potencyof these neutralizing responses diminishes with antigenic drift.Thus, annual influenza vaccination is required to maintain pro-tective immune responses against a “moving target” (5). Third,the emergence of pandemic influenza virus strains is difficult topredict, and once an influenza pandemic emerges, it is even moredifficult to redirect vaccine production in a timely fashion to re-spond to a pandemic, as happened during the 2009 H1N1 influ-enza pandemic (6, 7). Predictions of influenza pandemics is fur-ther complicated by the realization that several influenza virussubtypes possess pandemic potential, as evidenced by the emer-gence of avian influenza A (H7N9) virus in March 2013 (8) andsporadic human infections with H4, H5, H6, H7, H9, and H10avian influenza viruses (9–14).

Hemagglutinin (HA)-specific universal influenza vaccineshave the potential to mitigate these limitations by focusing hu-moral immune responses on its antigenically conserved stalk re-gion. Approaches to developing stalk-focused universal vaccineshave included headless HA (15–17), recombinant soluble HA

(18–22), synthetic polypeptides (23), prime-boost regimens (24,25), nanoparticles (26), and recombinant influenza viruses ex-pressing chimeric HA (cHA) (19, 21). Stalk-specific vaccineswould shift the humoral immune responses away from the immu-nodominant globular-head domain to the more conserved stalkdomain. Universal vaccines stimulating stalk-specific antibody re-sponses would have several desirable aspects, including (i) confer-ring protection against homologous and drifted influenza virusstrains, (ii) obviating the need for annual influenza vaccinationswith reformulated H1, H3, and B virus strains that antigenicallymatch prevalent circulating strains, and (iii) conferring increasedprotection against newly emerging influenza viruses with pan-demic potential (27, 28). Importantly, stalk-reactive antibodiesoccur naturally in humans, albeit in general at low frequencies,and have been detected in experimentally vaccinated mice (21,29–37). On the basis of sequence conservation, a universal influ-enza vaccine targeting the HA stalk would likely require threecomponents to cover group 1 (H1, H2, H5, H6, H8, H9, H11,

Received 12 October 2013 Accepted 23 December 2013

Published ahead of print 8 January 2014

Editor: T. S. Dermody

Address correspondence to Randy A. Albrecht, randy.albrecht@mssm.edu.

* Present address: Mark Yondola, Avatar Biotechnologies, Brooklyn, NY.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.03004-13

3432 jvi.asm.org Journal of Virology p. 3432–3442 March 2014 Volume 88 Number 6

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

H12, H13, H16, H17) and group 2 (H3, H4, H7, H10, H14, H15)influenza A and B virus HAs.

In this study, we have examined in ferrets the level of protec-tion conferred by group 1 HA stalk-specific antibodies against achallenge infection with pandemic H1N1 virus. Ferrets were pas-sively immunized with stalk-reactive monoclonal antibodies(MAbs) or vaccinated with recombinant viral vectors expressingcHAs known to induce stalk-reactive antibodies in mice. Thesestudies revealed that group 1 stalk-specific antibodies could re-duce titers of infectious virus within the nasal cavity and also re-duced pulmonary virus titers in immunized ferrets challengedwith a pandemic H1N1 influenza virus that contains an HA headnot present in the cHA vaccination regimen. These findings sug-gest that ferrets produce HA stalk-reactive antibodies followingvaccination with cHAs and that stalk-reactive antibodies provideprotection from heavy viral loads after a challenge infection in thisinfluenza animal model.

MATERIALS AND METHODSCells and viruses. Madin-Darby canine kidney (MDCK), 293T, 293,A549, and baby hamster kidney 21 (BHK-21) cells were propagated inDulbecco’s modified Eagle’s medium (DMEM) or minimum essentialmedium (both from Gibco). A/Netherlands/602/09 pandemic H1N1 vi-rus and the recombinant B-cH9/1 virus (a B/Yamagata/16/88 virus thatexpresses a cH9/1 HA as described in reference 35) were grown in embry-onated chicken eggs, and titers were determined on MDCK cells in me-dium containing tosyl phenylalanyl chloromethyl ketone (TPCK)-treatedtrypsin as described before.

Generation of a VSV vector expressing cH5/1 protein. The cH5/1gene (an A/Viet Nam/1203/04 H5 head on top of an A/PR/8/34 H1 stalkdomain [19, 21]) was amplified by PCR, and the SalI-NheI restrictionenzyme-digested PCR product was then cloned into the XhoI and NheIsites of the pVSV-XN2 (38) vector to generate pVSV-cH5/1. Recombinantvesicular stomatitis virus (VSV) expressing cH5/1 HA (VSV-cH5/1) wasrecovered with the above plasmid with minor modifications to the previ-ously described method (39). Briefly, BHK-21 cells were infected with theT7 polymerase-expressing vaccinia virus vTF7-3 (40) at a multiplicity ofinfection (MOI) of 20. At 1 h postinfection, the cells were transfected withthe pVSV-cH5/1 plasmid and support plasmids pBS-N, pBS-P, pBS-G,and pBS-L. At 48 h posttransfection, the cell culture medium was col-lected, filtered through a 0.1-�m filter, and passaged onto BHK-21 cells.After a cytopathic effect (CPE) became evident, the culture medium wascollected and virus was plaque purified and used to grow stocks. A VSVvector expressing green fluorescent protein (GFP) was used as a control.

Generation of an adenovirus 5 vector expressing cH6/1 protein.Prior to virus generation, cH6/1 (an A/mallard/Sweden/81/02 H6 globu-lar-head domain on top of an H1/PR8 stalk domain [21, 35]) was clonedinto a previously described transfer plasmid (pE1A-CMV, lacking the HAepitope tag) (41). For virus generation, 2.0 � 106 human embryonic kid-ney 293 (HEK-293) cells (generously supplied by Patrick Hearing) wereplated per well of a six-well dish and transfected the following day with a3:1 ratio of X-tremeGENE 9 (Roche) to DNA according to the manufac-turer’s instructions. Cells were transfected with a total of 5.5 �g of DNAconsisting of 5 �g of PvuI-linearized cH6/1 pE1A-CMX plasmid and 500ng of dl309 viral DNA that had been digested with ClaI/XbaI to remove theleft end of the adenoviral genome (bp 1 to 920). X-tremeGENE 9-trans-fected, ClaI/XbaI-digested viral DNA was used as a negative control. After24 h of incubation, cells were overlaid with 2� DMEM-supplemented 1%agarose for plaque selection. Overlays were reapplied approximately every3 days for 1 week, and then plaques were isolated for screening and usedfor 10 lysate generation. Once a CPE was evident (2 to 3 days), cells wereharvested and frozen at �80°C. Cells underwent four freeze-thaw cycles,and then viral DNA was prepared by an established method for sequenc-ing (42). Once the cH6/1 sequence was confirmed, virus stocks were am-

plified on HEK-293 cells and purified by consecutive banding on step andequilibrium cesium chloride gradients. Expression of the cH6/1 proteinwas confirmed by immunofluorescence staining on A549-infected cellswith anti-stalk MAb 6F12 (43), and virus titers were determined by stan-dard plaque assay on HEK-293 cells. The empty control adenovirus vector(in the same genomic background) was kindly provided by Patrick Hear-ing.

Immunostaining. MDCK cells were infected at a MOI of 1 withB-cH9/1 or wild-type B/Yamagata/16/88 and fixed (0.5% paraformalde-hyde) at 24 h postinfection. A subset of cells was permeabilized with 0.1%Triton X-100 and stained with an anti-influenza B virus nucleoproteinantibody (Abcam; 1:1,000). The rest of the cells were stained with anti-H1stalk antibody 6F12 (10 �g/ml) or anti-H9 head antibody G1-26 (BEIResources NR-9485; 1:1,000). 293T and A549 cells were infected/trans-duced with empty or cH6/1-expressing adenovirus at an MOI of about100. Cells were permeabilized with 0.5% Triton X-100 and stained with ananti-hexon antibody (Abcam; 1:1,000), an anti-H1 stalk antibody 6F12(10 �g/ml), or an anti-H6 head antibody NatalieC (10 �g/ml). An Alexa488-conjugated anti-mouse antibody (Life Technologies; 1:1,000) wasused as the secondary antibody for immunofluorescence analysis. Verocells were infected at a low MOI with VSV expressing GFP or cH5/1 HA.Cells were fixed at 24 h postinfection and stained with mouse anti-VSVserum (1:1,000), MAb 6F12 (10 �g/ml), or anti-H5 head antibodyVN4-10 (BEI Resources NR-2737; 1:1,000). A horseradish peroxidase(HRP)-linked anti-mouse antibody (Santa Cruz; 1:3,000) was used as thesecondary antibody, and stained cells were visualized with aminoethylcarbazole substrate solution (Millipore).

Antibodies and recombinant proteins. Mouse MAbs 6F12 (H1 stalkreactive, IgG2b) (43) and XY102 (A/Hong Kong/1/68 HA head reactive,hemagglutination inhibition [HI] active, IgG2b) (44) were purified fromsupernatants of hybridoma cultures as described before. Briefly, the su-pernatants were passed over a column loaded with protein G-Sepharose(GE Healthcare), washed, eluted, and concentrated, and the buffer wasexchanged for phosphate-buffered saline (PBS; pH 7.4) with Amicon Ul-tra centrifugation units (Millipore). Protein concentrations were deter-mined by the A280 method with a NanoDrop device. Recombinant HAswere expressed as ectodomains with a C-terminal trimerization domainand a hexahistidine tag with the baculovirus system as described before(20, 45). Protein concentrations were measured by the Bradford method.

Animals, passive transfer, immunization, and challenge. Five-month-old male Fitch ferrets were confirmed to be seronegative for cir-culating H1N1, H3N2, and B influenza viruses prior to purchase fromTriple F Farms (Sayre, PA). Ferrets were housed in PlasLabs poultry in-cubators with free access to food and water (46–48). All of the animalexperiments described here were conducted by using protocols approvedby the Icahn School of Medicine at Mount Sinai Institutional Animal Careand Use Committee. Animals were anesthetized by intramuscular admin-istration of ketamine/xylazine for all of the procedures described here,including bleeding, nasal washes, vaccination, infection, and passivetransfer.

For passive-transfer experiments, animals were bled to obtain baselineserum samples 2 weeks before the transfer. On day �1, 30 mg/kg of mouseMAb 6F12 or XY102 (n � 2 per group) was transferred intravenously viathe vena cava (Fig. 1A). At 24 h postinoculation, animals were bled andinfected with 104 PFU of A/Netherlands/602/09 (pandemic H1N1) virus.Nasal washes were then taken on days 1 and 3 postinfection, and bodyweights were monitored daily. Animals were observed for approximately30 min daily for signs of morbidity (e.g., sneezing). On day 4 postinfec-tion, animals were sacrificed and exsanguinated and tissue samples weretaken from the upper left and right lobes of the lungs, olfactory bulb, andnasal turbinates.

For vaccination experiments, animals (n � 5) were intranasally in-fected with 2 � 107 PFU (in 1 ml of PBS) of influenza B virus vectorB-cH9/1 HA (an H9 head on top of an H1 stalk domain [21, 35]) (see Fig.4A). At 3 weeks postinfection, animals were boosted by the intramuscular

Hemagglutinin Stalk Antibodies in the Ferret Model

March 2014 Volume 88 Number 6 jvi.asm.org 3433

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

FIG 1 Persistence and distribution of MAb 6F12 in two passively immunized ferrets. (A) Schematic representation of the passive immunization and challengestudy. A baseline serum sample was collected prior to the passive immunization of ferrets with 30 mg/kg of MAb 6F12 on day �1. On day 0 postimmunization,a serum sample was taken and ferrets were challenged by infection with 104 PFU of A/Netherlands/602/09. (B) Titers of MAb 6F12 in serum samples collectedon days �1, 0, and 4 of passive immunization were measured by ELISA reactivity against baculovirus-produced H1 from A/California/04/09. (C) Titers of MAb6F12 in nasal wash samples collected on days 1 and 3 of passive immunization were measured by ELISA reactivity against baculovirus-produced H1 fromA/California/04/09. (D) Titers of MAb 6F12 in lung homogenate samples collected on day 4 of passive immunization were measured by ELISA reactivity againstbaculovirus-produced H1 from A/California/04/09. In the experiments whose results are shown in panels C and D, nasal wash or lung samples from ferretspassively immunized with MAb XY102, which specifically recognizes H3 of A/Hong Kong/1/1968, served as negative controls (n � 2 ferrets). OD, optical density.

Krammer et al.

3434 jvi.asm.org Journal of Virology

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

administration of 2 � 105 PFU (in 0.5 ml) of recombinant VSV-cH5/1 HA(an H5 globular-head domain on top of an H1 stalk domain [19, 21]). Asecond boost consisting of a replication-deficient recombinant adenovi-rus 5 vector expressing the cH6/1 protein (an H6 globular-head domainon top of an H1 stalk domain) was given intranasally and intramuscularly(1.2 � 108 PFU in 0.5 ml per site) 3 weeks after the first boost. Controlgroup animals received the same empty or GFP-expressing virus (VSV)vectors in the same sequence (n � 4). Four weeks after the last priming,animals were challenged with 104 PFU of A/Netherlands/602/09 (pandemicH1N1) virus. Nasal washes were then taken on days 1 and 3 postinfection, andweight was monitored daily. Animals were observed for approximately 30min daily, and signs of morbidity (e.g., sneezing) were recorded. On day 4postinfection, animals were sacrificed and tissue samples were taken from thelung (upper right lobe), olfactory bulb, and nasal turbinates.

HI assays. HI assays were performed as described elsewhere (46, 49).Working stocks of each influenza virus strain were prepared by dilutingthe virus stock to a final HA titer of 8 HA units/50 �l. Each serum samplewas serially diluted 2-fold in PBS (25 �l per well) in 96-V-well microtiterplates. Then, 25 �l of working stock of the influenza virus strain was addedto each well so that all of the wells contained a final volume of 50 �l. Theserum-virus samples were then incubated at room temperature for 45 minto allow HA head-specific antibodies to neutralize the influenza virus. Toeach well, 50 �l of a 0.5% suspension of turkey or chicken red blood cellswas added. The assay plates were then incubated at 4°C until red bloodcells in the PBS control sample formed a button and red blood cells hem-agglutinated in control wells containing virus and no antibody. The HItiter was defined as the reciprocal of the highest dilution of antibody thatinhibited red blood cell hemagglutination by influenza virus.

ELISAs. Enzyme-linked immunosorbent assays (ELISAs) were per-formed as described before (20, 31). Briefly, plates were coated with 2�g/ml of recombinant, baculovirus-produced H1 (from A/California/04/09, A/New Caledonia/20/99, and A/South Carolina/1/18), H2 (from A/Ja-pan/305/57), or H17 (from A/yellow shouldered bat/Guatemala/06/10)HA protein (20). Wells were then incubated with serial (2-fold) dilutionsof ferret sera, nasal washes, or lung homogenates for 1 h at room temper-ature. After extensive washes, plates were incubated with anti-mouse an-tibody (for MAbs; Santa Cruz) or anti-ferret (Alpha Diagnostics Interna-tional) IgG HRP-labeled secondary antibody for another hour at roomtemperature. Plates were washed again and then developed with Sigma-Fast o-phenylenediamine dihydrochloride substrate and read on a Syn-ergy H1 (BioTek) plate reader.

RESULTSPersistence and tissue distribution of 6F12 in the ferret. Previ-ously, we have shown that HA head-reactive IgA but not IgG an-tibody is able to prevent transmission in the ferret and guinea pigmodels of influenza virus infection (49). We reasoned that at anespecially low concentration (3 mg/kg), IgG is not efficientlytransported to mucosal surfaces. This transport might be addi-tionally inhibited by the lower Fc-Fc receptor interactions be-tween mouse MAbs and the ferret host. In addition, the half-life ofmouse IgG in ferrets has not been well characterized; however, aprevious study that examined the therapeutic potential of a hu-manized MAb, m102, in the ferret model of Nipah virus infectionreported an elimination half-life of 3.5 days following the intrave-nous administration of 25 mg of MAb (50). We were thereforecurious if treatment with a large dose of MAb (30 mg/kg) wouldincrease the Ab concentration on mucosal surfaces and protectfrom upper respiratory tract infection. Ferrets were passively im-munized by the intravenous administration of 30 mg/kg of eitherH1 stalk-specific MAb 6F12 or H3-specific MAb XY102 (isotypecontrol) (Fig. 1A). The persistence and tissue distribution of MAb6F12 were examined by ELISA with baculovirus-produced H1from A/California/04/09. MAb 6F12 could be easily detected by

ELISA within serum samples on day 4 after passive immunization(Fig. 1B). In addition, MAb 6F12 was detected by ELISA in nasal washsamples collected on day 1, but the level of MAb declined by day 3after a challenge infection (Fig. 1C). MAb 6F12 was also detected byELISA in lung homogenates on day 4 postchallenge (Fig. 1D). Theseresults suggest that passive immunization by intravenous administra-tion of MAb 6F12 would confer a window of protection against achallenge infection within the ferret respiratory tract.

Prophylactic administration of 6F12 reduces viral loads inlungs, olfactory bulbs, and nasal turbinates. Mouse MAb 6F12 isan H1 stalk domain-specific antibody that potently inhibits viralreplication of H1N1 virus isolates spanning from 1930 to 2009 andefficiently protects mice prophylactically and therapeutically froma viral challenge (43). In order to investigate whether 6F12 wouldalso be efficacious prophylactically in the ferret model of influenzadisease, a 30-mg/kg dose of this MAb was administered to ferretsintravenously 24 h prechallenge and the animals were then chal-lenged with pandemic H1N1 strain A/Netherlands/602/09. Con-trol group ferrets received the same amount of an isotype controlantibody (Fig. 1A). Viral titers from nasal wash samples taken ondays 1 and 3 were slightly lower in the 6F12-treated animals thanin the control group (Fig. 2A). The effect was more pronouncedon day 1 than on day 3, which matches the lower 6F12 titers foundin nasal washes on day 3 postchallenge. Furthermore, the day 4nasal turbinate titers of 6F12-treated ferrets were lower than thoseof control animals (Fig. 2B). A reduction of approximately 2 logsin 6F12-treated animals was also observed in the olfactory bulb(Fig. 2C), and lung titers were approximately 1 log lower thanthose of control animals (Fig. 2D). Weight loss was only minimaland similar in both groups (data not shown). In summary, pro-phylactic treatment of ferrets with MAb 6F12 reduced the viralloads in challenged animals in all of the analyzed tissues. The read-outs established for this experiment were then also used to com-pare and analyze the efficacy of a cHA vaccine regimen in ferrets.

Vaccination with cHAs induces stalk-reactive antibodies inthe ferret. We have previously shown that vaccination of inbredBALB/c mice with cHA constructs (HAs with a conserved stalkdomain but divergent head domains) induces broadly neutraliz-ing stalk-reactive antibodies (21). Here we wanted to test if vacci-nation of ferrets would also induce stalk-reactive antibodies. Tothis end, we used viral vectors expressing cHA constructs (Fig. 3).Prior to vaccination of ferrets with the viral vectors, the expressionof the cHA was demonstrated by immunostaining. The expressionof cHA by an influenza B virus vector expressing B-cH9/1 HA (21,35) was demonstrated by immunofluorescence assay with infectedMDCK cells (Fig. 3A). The expression of cHA by a VSV expressingcH5/1 HA (19, 21) was demonstrated by immunostaining of virusplaques in Vero cells (Fig. 3B). The expression of cHA by a repli-cation-deficient adenovirus 5 vector expressing cH6/1 HA (an H6head on top of an H1/PR8 stalk domain [19, 21]) was demon-strated by immunofluorescence assay with infected 293T andA549 cells (Fig. 3C).

Ferrets were first vaccinated with B-cH9/1 HA and thenboosted with VSV-cH5/1 HA (an H5 head on top of an H1 stalkdomain [19, 21]) and then with a replication-deficient adenovirus5 vector expressing cH6/1 HA (an H6 head on top of an H1/PR8stalk domain [19, 21]) (Fig. 4A). This vaccination regimen waschosen in order to avoid the generation of antibodies against anyantigen in pandemic H1N1 virus different from the HA stalk,which could also contribute to protection after a subsequent chal-

Hemagglutinin Stalk Antibodies in the Ferret Model

March 2014 Volume 88 Number 6 jvi.asm.org 3435

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

lenge. Vaccinated animals developed low seroreactivity againstpandemic H1 HA after priming. This reactivity was boosted ap-proximately 4-fold by the cH5/1 vaccination and then again 8-foldby the final cH6/1 vaccination. Sera from vector control animalsexhibited only background reactivity that was comparable to thereactivity of pooled prevaccination sera of the ferrets used. SincecHA-vaccinated animals were naive to the H1 head domain andalso tested HI negative against the pandemic H1N1 strain A/Neth-erlands/602/09, we conclude that any reactivity to H1 strains isbased on cross-reactive antibodies to the conserved stalk domain.Furthermore, our cHA vaccine constructs are based on the stalkdomain of A/PR/8/34 H1 HA. Therefore, reactivity to pandemicH1 HA already represents heterologous stalk reactivity within H1HAs. We also tested reactivity to two more H1 HAs, the HA fromprepandemic seasonal strain A/New Caledonia/20/99 and the HAfrom 1918 pandemic H1N1 strain A/South Carolina/1/18 (Fig. 4Cand D). Sera from cHA-vaccinated animals reacted strongly withboth proteins. In order to test if cHA vaccination induces cross-reactivity to other group 1 subtypes, we also tested reactivityagainst an H2 HA from A/Japan/305/57 virus (Fig. 4E) and againstan H17 HA (from recently discovered bat H17N10 influenza virusstrain A/yellow shouldered bat/Guatemala/06/10) (Fig. 4F). Sera

from cHA-vaccinated ferrets reacted strongly with both HAs,while sera from vector control animals showed only backgroundreactivity (Fig. 4E and F). Cross-reactivity against group 2 HA wasnot expected, since earlier studies with mice have shown thatgroup 1 stalk-based cHA vaccination regimens do not protectfrom a group 2 virus challenge and vice versa (21, 22). Impor-tantly, we did not detect any H1 head-specific antibody responsesagainst the challenge virus following the vaccination regimen asmeasured by HI assay (data not shown). As positive controls, con-valescent-phase reference sera from two ferrets infected withA/California/7/2009 were included in the HI assay, and each ref-erence serum yielded an HI titer of 1,280.

cHA vaccine constructs protect ferrets from a viral challenge.In order to test the protection that cHA vaccination would conferon ferrets, we challenged the animals with the pandemic H1N1strain A/Netherlands/602/09 (Fig. 5A). The readouts were thesame as for the passive-transfer experiment; we measured virustiters in day 1 and 3 nasal washes and in the lungs, olfactory bulb,and nasal turbinates on day 4 postinfection. Interestingly, nasalwash titers were lower in cHA-vaccinated ferrets than in controlanimals on day 1 (approximately 5-fold), as well on day 3 (morethan 10-fold), when the difference was highly significant (P �

FIG 2 Prophylactic administration of MAb 6F12 reduced viral titers following a challenge infection. Ferrets were passively immunized with MAb 6F12 (greenbars; n � 2 ferrets) or isotype control MAb XY102 (black bars; n � 2 ferrets). On day 0 after passive immunization, ferrets were challenge infected with 104 PFUof A/Netherlands/602/09. (A) Virus titers in nasal wash samples collected on day 1 or 3 after a challenge infection were determined by plaque assay. On day 4 afterthe challenge infection, titers of influenza virus in nasal turbinate (B), olfactory bulb (C), and lung (D) samples were assessed by plaque assay.

Krammer et al.

3436 jvi.asm.org Journal of Virology

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

0.0005) (Fig. 5A). This result is not surprising since we expectedthat the intranasally applied priming and second boost would in-duce stalk-reactive mucosal IgA antibodies. The reduction of virustiters in the nasal washes is also reflected by a significant reductionof virus titers in the nasal turbinates of about 10-fold (P � 0.0331)(Fig. 5B). Furthermore, the olfactory bulb virus titers of cHA-vaccinated animals were more than 2 logs lower than those ofvector control animals (P � 0.0062) (Fig. 5C). In fact, we wereunable to detect virus in the olfactory bulbs of four out of fivecHA-vaccinated ferrets, whereas high virus titers were found inthe olfactory bulbs of all four control ferrets. Finally, we also de-tected a reduction of approximately half a log of lung virus titers in

cHA-vaccinated ferrets compared to those of vector control fer-rets (Fig. 5D). In summary, the protective efficacy of the cHAvaccine was comparable to (nasal turbinates, olfactory bulbs, andlung titers) or better than (nasal wash titers) that of prophylacti-cally administered MAb 6F12.

DISCUSSION

In recent years, broadly neutralizing antibodies against the con-served stalk domain of the influenza virus HA have been isolated(30, 32, 36, 37, 43, 51, 52, 53–56). These antibodies can be used forprophylactic and therapeutic treatments of influenza virus infec-tions. Although the large amount of MAb needed for treatmentmight preclude the use of the antibodies in the general population,this approach might be useful for the therapy of severe influenzacases, especially when drug-resistant viruses in an immunocom-promised host are involved (57–63). We therefore wanted to eval-uate MAb 6F12 in a prophylactic setting in the ferret model. Thisantibody has pan-H1 neutralizing activity in vitro and is able toprotect mice from a challenge with H1N1 influenza viruses thatspan almost 100 years of antigenic drift (43). We show here thatMAb 6F12 is indeed efficacious against a pandemic H1N1 strain inthe ferret model as well. In particular, prophylactic administrationof MAb 6F12 resulted in a more pronounced reduction of virustiters in olfactory bulbs and lungs. Unexpectedly, we could alsodetect this mouse IgG antibody at low titers in nasal wash samplesfrom treated ferrets. These low levels of antibody found in thenasal washes correlated well with small reductions of nasal washviral titers. Several factors could contribute to the pronouncedreduction of virus titers in olfactory bulb and lung samples com-pared to the modest reduction of virus titers observed in nasalwash samples. On day 4 after intravenous injection, high levels ofMAb 6F12 could be detected in serum and lung samples, whichcontrasts with the low level of MAb 6F12 detected in nasal washsamples. In addition, MAb 6F12 liberated by the homogenizationof olfactory bulb and lung tissue samples would bind to and neu-tralize a small fraction of the virus present in the tissue samplesprior to the determination of virus titers by plaque assay. Wespeculate that 6F12-like antibodies, if transported efficiently tomucosal surfaces (e.g., locally induced by intranasally adminis-tered vaccines) would be able to efficiently reduce nasal wash virustiters and possibly have an impact on transmission as well. Werecently showed that this is the case for globular-head-reactiveMAb 30D1, which efficiently blocks replication when adminis-tered to guinea pigs as IgA (efficiently transported to mucosalsurfaces) but lacks efficacy when administered as IgG (not effi-ciently transported to mucosal surfaces) (49).

In an “antibody-guided” vaccine approach based on stalk-re-active antibodies, we have developed cHA vaccine constructs (19,21). These constructs possess a conserved, structurally integratedstalk domain in combination with divergent globular-head do-mains from “exotic” subtypes (21). By sequentially immunizingmice with these constructs, we protected them from a challengewith heterologous (H1N1) and heterosubtypic (other group 1HA-expressing viruses) influenza viruses (21). Here, we tested theefficacy of this vaccine approach in the ferret model. By immuniz-ing ferrets with combinations of divergent globular heads and aconserved stalk domain, we hoped to get an immune responsefocused on broadly neutralizing epitopes in the stalk. This strategyis based on the observation that sequential infection/vaccinationwith seasonal H1N1 and pandemic H1N1 viruses (which have

FIG 3 Expression of cHAs by viral vectors. (A) An engineered influenza Bvirus expresses cH9/1 HA (H9 head on top of an H1 stalk domain) instead ofinfluenza B HA. Shown is staining of B-cH9/1- or B-wt (wild-type influenza Bvirus)-infected cells with an anti-influenza B nucleoprotein antibody (anti-NP), anti-H1 stalk antibody 6F12 (anti-stalk), or an anti-H9 head antibody(anti-H9 head). (B) A recombinant VSV was engineered to express cH5/1 (H5head on top of an H1 stalk domain) HA as a transgene. Shown is staining ofVSV-cH5/1- or VSV-GFP-infected Vero cells with anti-VSV mouse serum(anti-VSV), anti-H1 stalk antibody 6F12 (anti-stalk), or an anti-H5 head an-tibody (anti-H5 head). (C) A replication-deficient adenovirus was engineeredto express cH6/1 HA (H6 head on top of an H1 stalk domain). Shown areinfected 293T cells stained for the presence of adenovirus (anti-hexon) andtransduced A459 cells stained with anti-H1 stalk antibody 6F12 (anti-stalk) oran anti-H6 head antibody (anti-H6 head).

Hemagglutinin Stalk Antibodies in the Ferret Model

March 2014 Volume 88 Number 6 jvi.asm.org 3437

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

highly divergent globular-head domains and highly conservedstalk domains) induces high levels of stalk-reactive antibodies inhumans (32, 35–37, 64). Similar findings were also obtained in themouse model (31). Here, in the ferret model, we show that a cHA-

based immunization strategy confers protection against a pan-demic H1N1 challenge. The observed level of protection was sim-ilar to or better than that conferred by inactivated, antigenicallymatched, unadjuvanted split vaccine administered once (65, 66)

FIG 4 Ferrets develop HA stalk-specific humoral responses after repeated immunization with viral vectors expressing cHAs. (A) Schematic representation of the HAstalk-based vaccination strategy used in this study. Ferrets (n � 5) were vaccinated with influenza B virus expressing cH9/1 HA, boosted with VSV-cH5/1 HA, andboosted a second time with an adenovirus 5 vector expressing the cH6/1 protein. Control ferrets (n � 4) were vaccinated with wild-type influenza B virus or VSV(expressing GFP) and adenovirus (empty) vectors. Ferrets were then challenged by infection with 104 PFU of A/Netherlands/602/09 virus. The development of broadlycross-reactive stalk-specific antibody responses was assessed by ELISA with baculovirus-produced H1 from A/California/04/09 (B), H1 from A/South Carolina/1/18)(C), H1 from A/New Caledonia/20/99 (D), H2 (from A/Japan/305/57) (E), or H17 (from A/yellow shouldered bat/Guatemala/06/10) (F). OD, optical density.

Krammer et al.

3438 jvi.asm.org Journal of Virology

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

or twice (67) or an antigenically matched experimental vacciniavirus-vectored construct (68). It is of note that the cHA-basedvaccine did not induce any HI-active antibodies, but vaccinatedferrets were able to produce a broadly reactive anti-stalk responseagainst divergent group 1 HA subtypes. This proof-of-principlestudy focused on protection afforded by the stalk domain of HA. Ahuman vaccine candidate based on the same principle would mostlikely consist of inactivated or attenuated cHA-expressing virusesthat also have a neuraminidase (NA). We believe that the antibodytiters against the more conserved NA would be boosted as well inthe absence of an immunodominant globular-head domain (69,70). These antibodies would then also contribute to broad protec-tion. Furthermore, conserved internal proteins like the nucleo-protein induce strong protective T-cell responses that contributeto protection as well (71–74). We have conclusively shown thatsuch a vaccination strategy based on the H1 HA stalk domain isable to broadly protect against group 1 HA-expressing viruses in

mice but was unable to protect against an H3N2 challenge virus(21). We therefore believe that a successful human vaccinationstrategy would need to contain a group 1, a group2, and an influ-enza B virus stalk component to induce broadly neutralizing stalkantibodies.

In summary, we have shown that treatment of ferrets with astalk-reactive antibody and vaccination by a stalk-based vaccina-tion strategy are efficacious in protecting against an influenza vi-rus challenge. We believe that both strategies are valuable addi-tions to the armamentarium for fighting seasonal and pandemicinfluenza virus infections in the human population.

ACKNOWLEDGMENTS

We thank Chen Wang and Richard Cadagan for excellent technical assis-tance.

This study was partially funded by a National Institutes of HealthNational Institute of Allergy and Infectious Diseases program project

FIG 5 The HA stalk-based vaccination strategy confers protection against a challenge infection with A/Netherlands/602/09 virus. Ferrets (n � 5) were vaccinatedwith B-cH9/1 HA, boosted with VSV-cH5/1 HA, and boosted a second time with an adenovirus 5 vector expressing the cH6/1 protein. Control ferrets (n � 4)were vaccinated with wild-type influenza B virus or VSV (expressing GFP) and adenovirus (empty) vectors. (A) Titers of challenge virus in nasal wash samplescollected on day 1 or 3 after the challenge infection were determined by plaque assay. On day 4 after the challenge infection, titers of influenza virus in nasalturbinate (B), olfactory bulb (C), and lung (D) samples were assessed by plaque assay. n.s., not significant.

Hemagglutinin Stalk Antibodies in the Ferret Model

March 2014 Volume 88 Number 6 jvi.asm.org 3439

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

grant (P01AI097092), by PATH, and by R01-AI080781. Florian Krammerwas supported by an Erwin Schrödinger fellowship (J 3232) from theAustrian Science Fund (FWF). Matthew S. Miller was supported by aCanadian Institutes of Health Research postdoctoral fellowship.

REFERENCES1. Molinari NA, Ortega-Sanchez IR, Messonnier ML, Thompson WW,

Wortley PM, Weintraub E, Bridges CB. 2007. The annual impact ofseasonal influenza in the US: measuring disease burden and costs. Vaccine25:5086 –5096. http://dx.doi.org/10.1016/j.vaccine.2007.03.046.

2. Centers for Disease Control and Prevention. 2010. Estimates of deathsassociated with seasonal influenza—United States, 1976-2007. MMWRMorb. Mortal. Wkly. Rep. 59:1057–1062. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5933a1.htm.

3. Centers for Disease Control and Prevention. 2011. Interim results:state-specific influenza vaccination coverage—United States, August2010-February 2011. MMWR Morb. Mortal. Wkly. Rep. 60:737–743.http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6022a3.htm.

4. Williams WW, Lu PJ, Lindley MC, Kennedy ED, Singleton JA, Centersfor Disease Control and Prevention. 2012. Influenza vaccination cover-age among adults—National Health Interview Survey, United States,2008-09 influenza season. MMWR Morb. Mortal. Wkly. Rep. 61(Suppl):65–72. http://www.cdc.gov/mmwr/preview/mmwrhtml/su6102a11.htm.

5. Wang TT, Palese P. 2011. Biochemistry. Catching a moving target. Sci-ence 333:834 – 835. http://dx.doi.org/10.1126/science.1210724.

6. Centers for Disease Control and Prevention. 2009. Swine influenza A(H1N1) infection in two children—Southern California, March-April2009. MMWR Morb. Mortal. Wkly. Rep. 58:400 – 402. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5815a5.htm.

7. Centers for Disease Control and Prevention. 2009. Update on influenzaA (H1N1) 2009 monovalent vaccines. MMWR Morb. Mortal. Wkly. Rep.58:1100 –1101. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5839a3.htm.

8. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, XuK, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, ZhangZ, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Yang L, Guo J, DongJ, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Han J, Yu H,Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y. 2013. Human infectionwith a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med.368:1888 –1897. http://dx.doi.org/10.1056/NEJMoa1304459.

9. Centers for Disease Control and Prevention. 2012. Notes from the field:highly pathogenic avian influenza A (H7N3) virus infection in two poultryworkers—Jalisco, Mexico, July 2012. MMWR Morb. Mortal. Wkly. Rep.61:726 –727. http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6136a4.htm.

10. Kayali G, Barbour E, Dbaibo G, Tabet C, Saade M, Shaib HA, Debeau-champ J, Webby RJ. 2011. Evidence of infection with H4 and H11 avianinfluenza viruses among Lebanese chicken growers. PLoS One 6:e26818.http://dx.doi.org/10.1371/journal.pone.0026818.

11. Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H,Vennema H, Meijer A, van Steenbergen J, Fouchier R, Osterhaus A,Bosman A. 2004. Transmission of H7N7 avian influenza A virus to hu-man beings during a large outbreak in commercial poultry farms in theNetherlands. Lancet 363:587–593. http://dx.doi.org/10.1016/S0140-6736(04)15589-X.

12. Peiris M, Yuen KY, Leung CW, Chan KH, Ip PL, Lai RW, Orr WK,Shortridge KF. 1999. Human infection with influenza H9N2. Lancet 354:916 –917. http://dx.doi.org/10.1016/S0140-6736(99)03311-5.

13. Tweed SA, Skowronski DM, David ST, Larder A, Petric M, Lees W, LiY, Katz J, Krajden M, Tellier R, Halpert C, Hirst M, Astell C, LawrenceD, Mak A. 2004. Human illness from avian influenza H7N3, BritishColumbia. Emerg. Infect. Dis. 10:2196 –2199. http://dx.doi.org/10.3201/eid1012.040961.

14. Yuan J, Zhang L, Kan X, Jiang L, Yang J, Guo Z, Ren Q. 2013. Originand molecular characteristics of a novel 2013 avian influenza A(H6N1)virus causing human infection in Taiwan. Clin. Infect. Dis. 57:1367–1368.http://dx.doi.org/10.1093/cid/cit479.

15. Bommakanti G, Citron MP, Hepler RW, Callahan C, Heidecker GJ,Najar TA, Lu X, Joyce JG, Shiver JW, Casimiro DR, ter Meulen J, LiangX, Varadarajan R. 2010. Design of an HA2-based Escherichia coli ex-pressed influenza immunogen that protects mice from pathogenic chal-

lenge. Proc. Natl. Acad. Sci. U. S. A. 107:13701–13706. http://dx.doi.org/10.1073/pnas.1007465107.

16. Sagawa H, Ohshima A, Kato I, Okuno Y, Isegawa Y. 1996. The immu-nological activity of a deletion mutant of influenza virus haemagglutininlacking the globular region. J. Gen. Virol. 77(Pt 7):1483–1487. http://dx.doi.org/10.1099/0022-1317-77-7-1483.

17. Steel J, Lowen AC, Wang TT, Yondola M, Gao Q, Haye K, Garcia-SastreA, Palese P. 2010. Influenza virus vaccine based on the conserved hem-agglutinin stalk domain. mBio 1:e00018 –10. http://dx.doi.org/10.1128/mBio.00018-10.

18. Goff PH, Eggink D, Seibert CW, Hai R, Martínez-Gil L, Krammer F,Palese P. 2013. Adjuvants and immunization strategies to induce influ-enza virus hemagglutinin stalk antibodies. PLoS One 8:e79194. http://dx.doi.org/10.1371/journal.pone.0079194.

19. Hai R, Krammer F, Tan GS, Pica N, Eggink D, Maamary J, Margine I,Albrecht RA, Palese P. 2012. Influenza viruses expressing chimeric hem-agglutinins: globular head and stalk domains derived from different sub-types. J. Virol. 86:5774 –5781. http://dx.doi.org/10.1128/JVI.00137-12.

20. Krammer F, Margine I, Tan GS, Pica N, Krause JC, Palese P. 2012. Acarboxy-terminal trimerization domain stabilizes conformational epitopeson the stalk domain of soluble recombinant hemagglutinin substrates. PLoSOne 7:e43603. http://dx.doi.org/10.1371/journal.pone.0043603.

21. Krammer F, Pica N, Hai R, Margine I, Palese P. 2013. Chimerichemagglutinin influenza virus vaccine constructs elicit broadly protectivestalk-specific antibodies. J. Virol. 87:6542– 6550. http://dx.doi.org/10.1128/JVI.00641-13.

22. Margine I, Krammer F, Hai R, Heaton NS, Tan GS, Andrews SA,Runstadler JA, Wilson PC, Albrecht RA, Garcia-Sastre A, Palese P.2013. Hemagglutinin stalk-based universal vaccine constructs protectagainst group 2 influenza A viruses. J. Virol. 87:10435–10446. http://dx.doi.org/10.1128/JVI.01715-13.

23. Wang TT, Tan GS, Hai R, Pica N, Ngai L, Ekiert DC, Wilson IA,Garcia-Sastre A, Moran TM, Palese P. 2010. Vaccination with a syntheticpeptide from the influenza virus hemagglutinin provides protectionagainst distinct viral subtypes. Proc. Natl. Acad. Sci. U. S. A. 107:18979 –18984. http://dx.doi.org/10.1073/pnas.1013387107.

24. Wei CJ, Boyington JC, McTamney PM, Kong WP, Pearce MB, Xu L,Andersen H, Rao S, Tumpey TM, Yang ZY, Nabel GJ. 2010. Inductionof broadly neutralizing H1N1 influenza antibodies by vaccination. Science329:1060 –1064. http://dx.doi.org/10.1126/science.1192517.

25. Wei CJ, Yassine HM, McTamney PM, Gall JG, Whittle JR, BoyingtonJC, Nabel GJ. 2012. Elicitation of broadly neutralizing influenza antibod-ies in animals with previous influenza exposure. Sci. Transl. Med.4:147ra114. http://dx.doi.org/10.1126/scitranslmed.3004273.

26. Kanekiyo M, Wei CJ, Yassine HM, McTamney PM, Boyington JC,Whittle JR, Rao SS, Kong WP, Wang L, Nabel GJ. 2013. Self-assemblinginfluenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibod-ies. Nature 499:102–106. http://dx.doi.org/10.1038/nature12202.

27. Krammer F, Palese P. 2013. Influenza virus hemagglutinin stalk-basedantibodies and vaccines. Curr. Opin. Virol. 3:521–530. http://dx.doi.org/10.1016/j.coviro.2013.07.007.

28. Krammer F, Palese P. 2013. Universal influenza virus vaccines: need forclinical trials. Nat. Immunol. 15:3–5. http://dx.doi.org/10.1038/ni.2761.

29. Corti D, Suguitan AL, Jr, Pinna D, Silacci C, Fernandez-Rodriguez BM,Vanzetta F, Santos C, Luke CJ, Torres-Velez FJ, Temperton NJ, WeissRA, Sallusto F, Subbarao K, Lanzavecchia A. 2010. Heterosubtypicneutralizing antibodies are produced by individuals immunized with aseasonal influenza vaccine. J. Clin. Invest. 120:1663–1673. http://dx.doi.org/10.1172/JCI41902.

30. Ekiert DC, Friesen RH, Bhabha G, Kwaks T, Jongeneelen M, Yu W,Ophorst C, Cox F, Korse HJ, Brandenburg B, Vogels R, Brakenhoff JP,Kompier R, Koldijk MH, Cornelissen LA, Poon LL, Peiris M, KoudstaalW, Wilson IA, Goudsmit J. 2011. A highly conserved neutralizing epitopeon group 2 influenza A viruses. Science 333:843– 850. http://dx.doi.org/10.1126/science.1204839.

31. Krammer F, Pica N, Hai R, Tan GS, Palese P. 2012. Hemagglutininstalk-reactive antibodies are boosted following sequential infection withseasonal and pandemic H1N1 influenza virus in mice. J. Virol. 86:10302–10307. http://dx.doi.org/10.1128/JVI.01336-12.

32. Li GM, Chiu C, Wrammert J, McCausland M, Andrews SF, Zheng NY,Lee JH, Huang M, Qu X, Edupuganti S, Mulligan M, Das SR, YewdellJW, Mehta AK, Wilson PC, Ahmed R. 2012. Pandemic H1N1 influenzavaccine induces a recall response in humans that favors broadly cross-

Krammer et al.

3440 jvi.asm.org Journal of Virology

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

reactive memory B cells. Proc. Natl. Acad. Sci. U. S. A. 109:9047–9052.http://dx.doi.org/10.1073/pnas.1118979109.

33. Miller MS, Gardner TJ, Krammer F, Aguado LC, Tortorella D, Basler CF,Palese P. 2013. Neutralizing antibodies against previously encountered influ-enza virus strains increase over time: a longitudinal analysis. Sci. Transl. Med.5:198ra107. http://dx.doi.org/10.1126/scitranslmed.3006637.

34. Miller MS, Tsibane T, Krammer F, Hai R, Rahmat S, Basler CF, PaleseP. 2013. 1976 and 2009 H1N1 influenza virus vaccines boost anti-hemagglutinin stalk antibodies in humans. J. Infect. Dis. 207:98 –105.http://dx.doi.org/10.1093/infdis/jis652.

35. Pica N, Hai R, Krammer F, Wang TT, Maamary J, Eggink D, Tan GS,Krause JC, Moran T, Stein CR, Banach D, Wrammert J, Belshe RB,García-Sastre A, Palese P. 2012. Hemagglutinin stalk antibodies elicitedby the 2009 pandemic influenza virus as a mechanism for the extinction ofseasonal H1N1 viruses. Proc. Natl. Acad. Sci. U. S. A. 109:2573–2578. http://dx.doi.org/10.1073/pnas.1200039109.

36. Thomson CA, Wang Y, Jackson LM, Olson M, Wang W, LiavonchankaA, Keleta L, Silva V, Diederich S, Jones RB, Gubbay J, Pasick J, PetricM, Jean F, Allen VG, Brown EG, Rini JM, Schrader JW. 2012. PandemicH1N1 influenza infection and vaccination in humans induces cross-protective antibodies that target the hemagglutinin stem. Front. Immu-nol. 3:87. http://dx.doi.org/10.3389/fimmu.2012.00087.

37. Wrammert J, Koutsonanos D, Li GM, Edupuganti S, Sui J, MorrisseyM, McCausland M, Skountzou I, Hornig M, Lipkin WI, Mehta A,Razavi B, Del Rio C, Zheng NY, Lee JH, Huang M, Ali Z, Kaur K,Andrews S, Amara RR, Wang Y, Das SR, O’Donnell CD, Yewdell JW,Subbarao K, Marasco WA, Mulligan MJ, Compans R, Ahmed R, WilsonPC. 2011. Broadly cross-reactive antibodies dominate the human B cellresponse against 2009 pandemic H1N1 influenza virus infection. J. Exp.Med. 208:181–193. http://dx.doi.org/10.1084/jem.20101352.

38. Schnell MJ, Buonocore L, Whitt MA, Rose JK. 1996. The minimalconserved transcription stop-start signal promotes stable expression of aforeign gene in vesicular stomatitis virus. J. Virol. 70:2318 –2323.

39. Lawson ND, Stillman EA, Whitt MA, Rose JK. 1995. Recombinantvesicular stomatitis viruses from DNA. Proc. Natl. Acad. Sci. U. S. A.92:4477– 4481. http://dx.doi.org/10.1073/pnas.92.10.4477.

40. Fuerst TR, Niles EG, Studier FW, Moss B. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizesbacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U. S. A. 83:8122– 8126. http://dx.doi.org/10.1073/pnas.83.21.8122.

41. Evans JD, Hearing P. 2003. Distinct roles of the adenovirus E4 ORF3 proteinin viral DNA replication and inhibition of genome concatenation. J. Virol.77:5295–5304. http://dx.doi.org/10.1128/JVI.77.9.5295-5304.2003.

42. Schmid SI, Hearing P. 1999. Adenovirus DNA packaging, p 47–59. InWold WSM (ed), Adenovirus methods and protocols. Humana Press,Totowa, NJ.

43. Tan GS, Krammer F, Eggink D, Kongchanagul A, Moran TM, Palese P.2012. A pan-H1 anti-hemagglutinin monoclonal antibody with potentbroad-spectrum efficacy in vivo. J. Virol. 86:6179 – 6188. http://dx.doi.org/10.1128/JVI.00469-12.

44. Moran T, Liu YC, Schulman JL, Bona CA. 1984. Shared idiotopes amongmonoclonal antibodies specific for A/PR/8/34 (H1N1) and X-31(H3N2)influenza viruses. Proc. Natl. Acad. Sci. U. S. A. 81:1809 –1812. http://dx.doi.org/10.1073/pnas.81.6.1809.

45. Margine I, Palese P, Krammer F. 2013. Expression of functional recom-binant hemagglutinin and neuraminidase proteins from the novel H7N9Influenza virus using the baculovirus expression system. J. Vis. Exp. 81:e51112. http://dx.doi.org/10.3791/51112.

46. Baker SF, Guo H, Albrecht RA, Garcia-Sastre A, Topham DJ, Martínez-Sobrido L. 2013. Protection against lethal influenza with a viral mimic. J.Virol. 87:8591– 8605. http://dx.doi.org/10.1128/JVI.01081-13.

47. Martínez-Romero C, de Vries E, Belicha-Villanueva A, Mena I,Tscherne DM, Gillespie VL, Albrecht RA, de Haan CA, Garcia-Sastre A.2013. Substitutions T200A and E227A in the hemagglutinin of pandemic2009 influenza A virus increase lethality but decrease transmission. J. Vi-rol. 87:6507– 6511. http://dx.doi.org/10.1128/JVI.00262-13.

48. Seibert CW, Kaminski M, Philipp J, Rubbenstroth D, Albrecht RA,Schwalm F, Stertz S, Medina RA, Kochs G, Garcia-Sastre A, Staeheli P,Palese P. 2010. Oseltamivir-resistant variants of the 2009 pandemic H1N1influenza A virus are not attenuated in the guinea pig and ferret transmis-sion models. J. Virol. 84:11219 –11226. http://dx.doi.org/10.1128/JVI.01424-10.

49. Seibert CW, Rahmat S, Krause JC, Eggink D, Albrecht RA, Goff PH,

Krammer F, Duty JA, Bouvier NM, Garcia-Sastre A, Palese P. 2013.Recombinant IgA is sufficient to prevent influenza virus transmission inguinea pigs. J. Virol. 87:7793–7804. http://dx.doi.org/10.1128/JVI.00979-13.

50. Zhu Z, Bossart KN, Bishop KA, Crameri G, Dimitrov AS, McEachernJA, Feng Y, Middleton D, Wang LF, Broder CC, Dimitrov DS. 2008.Exceptionally potent cross-reactive neutralization of Nipah and Hendraviruses by a human monoclonal antibody. J. Infect. Dis. 197:846 – 853.http://dx.doi.org/10.1086/528801.

51. Dreyfus C, Laursen NS, Kwaks T, Zuijdgeest D, Khayat R, Ekiert DC,Lee JH, Metlagel Z, Bujny MV, Jongeneelen M, van der Vlugt R,Lamrani M, Korse HJ, Geelen E, Sahin O, Sieuwerts M, Brakenhoff JP,Vogels R, Li OT, Poon LL, Peiris M, Koudstaal W, Ward AB, WilsonIA, Goudsmit J, Friesen RH. 2012. Highly conserved protective epitopeson influenza B viruses. Science 337:1343–1348. http://dx.doi.org/10.1126/science.1222908.

52. Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, Thro-sby M, Goudsmit J, Wilson IA. 2009. Antibody recognition of a highlyconserved influenza virus epitope. Science 324:246 –251. http://dx.doi.org/10.1126/science.1171491.

53. Heaton NS, Leyva-Grado VH, Tan GS, Eggink D, Hai R, Palese P. 2013.In vivo bioluminescent imaging of influenza a virus infection and charac-terization of novel cross-protective monoclonal antibodies. J. Virol. 87:8272– 8281. http://dx.doi.org/10.1128/JVI.00969-13.

54. Sui J, Hwang WC, Perez S, Wei G, Aird D, Chen LM, Santelli E, Stec B,Cadwell G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, CoxNJ, Bankston LA, Donis RO, Liddington RC, Marasco WA. 2009.Structural and functional bases for broad-spectrum neutralization ofavian and human influenza A viruses. Nat. Struct. Mol. Biol. 16:265–273.http://dx.doi.org/10.1038/nsmb.1566.

55. Throsby M, van den Brink E, Jongeneelen M, Poon LL, Alard P,Cornelissen L, Bakker A, Cox F, van Deventer E, Guan Y, Cinatl J, terMeulen J, Lasters I, Carsetti R, Peiris M, de Kruif J, Goudsmit J. 2008.Heterosubtypic neutralizing monoclonal antibodies cross-protectiveagainst H5N1 and H1N1 recovered from human IgM� memory B cells.PLoS One 3:e3942. http://dx.doi.org/10.1371/journal.pone.0003942.

56. Wang TT, Tan GS, Hai R, Pica N, Petersen E, Moran TM, Palese P. 2010.Broadly protective monoclonal antibodies against H3 influenza viruses fol-lowing sequential immunization with different hemagglutinins. PLoS Pathog.6:e1000796. http://dx.doi.org/10.1371/journal.ppat.1000796.

57. DeVries A, Wotton J, Lees C, Boxrud D, Uyeki T, Lynfield R. 2012.Neuraminidase H275Y and hemagglutinin D222G mutations in a fatalcase of 2009 pandemic influenza A (H1N1) virus infection. InfluenzaOther Respir. Viruses 6:e85– 8. http://dx.doi.org/10.1111/j.1750-2659.2011.00329.x.

58. Hu Y, Lu S, Song Z, Wang W, Hao P, Li J, Zhang X, Yen HL, Shi B, LiT, Guan W, Xu L, Liu Y, Wang S, Tian D, Zhu Z, He J, Huang K, ChenH, Zheng L, Li X, Ping J, Kang B, Xi X, Zha L, Li Y, Zhang Z, Peiris M,Yuan Z. 2013. Association between adverse clinical outcome in humandisease caused by novel influenza A H7N9 virus and sustained viral shed-ding and emergence of antiviral resistance. Lancet 381:2273–2279. http://dx.doi.org/10.1016/S0140-6736(13)61125-3.

59. Hurt AC, Leang SK, Tiedemann K, Butler J, Mechinaud F, Kelso A,Downie P, Barr IG. 2013. Progressive emergence of an oseltamivir-resistant A(H3N2) virus over two courses of oseltamivir treatment in animmunocompromised paediatric patient. Influenza Other Respir. Viruses7:904 –908. http://dx.doi.org/10.1111/irv.12108.

60. Iioka F, Sada R, Maesako Y, Nakamura F, Ohno H. 2012. Outbreak ofpandemic 2009 influenza A/H1N1 infection in the hematology ward: fatalclinical outcome of hematopoietic stem cell transplant recipients andemergence of the H275Y neuraminidase mutation. Int. J. Hematol. 96:364 –369. http://dx.doi.org/10.1007/s12185-012-1139-1.

61. Shetty AK, Ross GA, Pranikoff T, Gubareva LV, Sechrist C, GuirandDM, Abramson J, Lin JJ. 2012. Oseltamivir-resistant 2009 H1N1 influ-enza pneumonia during therapy in a renal transplant recipient. Pediatr.Transplant. 16:E153–E157. http://dx.doi.org/10.1111/j.1399-3046.2011.01489.x.

62. van der Vries E, Stittelaar KJ, van Amerongen G, Veldhuis Kroeze EJ,de Waal L, Fraaij PL, Meesters RJ, Luider TM, van der Nagel B, KochB, Vulto AG, Schutten M, Osterhaus AD. 2013. Prolonged influenzavirus shedding and emergence of antiviral resistance in immunocompro-mised patients and ferrets. PLoS Pathog. 9:e1003343. http://dx.doi.org/10.1371/journal.ppat.1003343.

63. van Kampen JJ, Bielefeld-Buss AJ, Ott A, Maaskant J, Faber HJ, Lutisan

Hemagglutinin Stalk Antibodies in the Ferret Model

March 2014 Volume 88 Number 6 jvi.asm.org 3441

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from

JG, Boucher CA. 2013. Case report: oseltamivir-induced resistant pan-demic influenza A (H1N1) virus infection in a patient with AIDS andPneumocystis jirovecii pneumonia. J. Med. Virol. 85:941–943. http://dx.doi.org/10.1002/jmv.23560.

64. Sangster MY, Baer J, Santiago FW, Fitzgerald T, Ilyushina NA, Sunda-rarajan A, Henn AD, Krammer F, Yang H, Luke CJ, Zand MS, WrightPF, Treanor JJ, Topham DJ, Subbarao K. 2013. The B cell response andhemagglutinin stalk-reactive antibody production in different age cohortsfollowing 2009 H1N1 influenza vaccination. Clin. Vaccine Immunol. 20:867– 876. http://dx.doi.org/10.1128/CVI.00735-12.

65. Ellebedy AH, Fabrizio TP, Kayali G, Oguin TH, Brown SA, Rehg J,Thomas PG, Webby RJ. 2010. Contemporary seasonal influenza A (H1N1)virus infection primes for a more robust response to split inactivated pan-demic influenza A (H1N1) virus vaccination in ferrets. Clin. Vaccine Immu-nol. 17:1998–2006. http://dx.doi.org/10.1128/CVI.00247-10.

66. Hossain MJ, Bourgeois M, Quan FS, Lipatov AS, Song JM, Chen LM,Compans RW, York I, Kang SM, Donis RO. 2011. Virus-like particlevaccine containing hemagglutinin confers protection against 2009 H1N1pandemic influenza. Clin. Vaccine Immunol. 18:2010 –2017. http://dx.doi.org/10.1128/CVI.05206-11.

67. Baras B, de Waal L, Stittelaar KJ, Jacob V, Giannini S, Kroeze EJ, vanden Brand JM, van Amerongen G, Simon JH, Hanon E, Mossman SP,Osterhaus AD. 2011. Pandemic H1N1 vaccine requires the use of anadjuvant to protect against challenge in naïve ferrets. Vaccine 29:2120 –2126. http://dx.doi.org/10.1016/j.vaccine.2010.12.125.

68. Kreijtz JH, Süzer Y, Bodewes R, Schwantes A, van Amerongen G,Verburgh RJ, de Mutsert G, van den Brand J, van Trierum SE, KuikenT, Fouchier RA, Osterhaus AD, Sutter G, Rimmelzwaan GF. 2010.Evaluation of a modified vaccinia virus Ankara (MVA)-based candidatepandemic influenza A/H1N1 vaccine in the ferret model. J. Gen. Virol.91:2745–2752. http://dx.doi.org/10.1099/vir.0.024885-0.

69. Kilbourne ED, Cerini CP, Khan MW, Mitchell JW, Ogra PL. 1987.Immunologic response to the influenza virus neuraminidase is influencedby prior experience with the associated viral hemagglutinin. I. Studies inhuman vaccinees. J. Immunol. 138:3010 –3013.

70. Palese P, Wang TT. 2011. Why do influenza virus subtypes die out? A hy-pothesis. mBio 2:e00150–11. http://dx.doi.org/10.1128/mBio.00150-11.

71. Bodewes R, Kreijtz JH, van Amerongen G, Hillaire ML, Vogelzang-vanTrierum SE, Nieuwkoop NJ, van Run P, Kuiken T, Fouchier RA,Osterhaus AD, Rimmelzwaan GF. 2013. Infection of the upper respira-tory tract with seasonal influenza A(H3N2) virus induces protective im-munity in ferrets against infection with A(H1N1)pdm09 virus after intra-nasal, but not intratracheal, inoculation. J. Virol. 87:4293– 4301. http://dx.doi.org/10.1128/JVI.02536-12.

72. Hillaire ML, van Trierum SE, Kreijtz JH, Bodewes R, Geelhoed-MierasMM, Nieuwkoop NJ, Fouchier RA, Kuiken T, Osterhaus AD, Rimmel-zwaan GF. 2011. Cross-protective immunity against influenza pH1N12009 viruses induced by seasonal influenza A (H3N2) virus is mediated byvirus-specific T-cells. J. Gen. Virol. 92:2339 –2349. http://dx.doi.org/10.1099/vir.0.033076-0.

73. Kreijtz JH, Bodewes R, van den Brand JM, de Mutsert G, Baas C, vanAmerongen G, Fouchier RA, Osterhaus AD, Rimmelzwaan GF. 2009.Infection of mice with a human influenza A/H3N2 virus induces protec-tive immunity against lethal infection with influenza A/H5N1 virus. Vac-cine 27:4983– 4989. http://dx.doi.org/10.1016/j.vaccine.2009.05.079.

74. Wilkinson TM, Li CK, Chui CS, Huang AK, Perkins M, Liebner JC,Lambkin-Williams R, Gilbert A, Oxford J, Nicholas B, Staples KJ, DongT, Douek DC, McMichael AJ, Xu XN. 2012. Preexisting influenza-specific CD4� T cells correlate with disease protection against influenzachallenge in humans. Nat. Med. 18:274 –280. http://dx.doi.org/10.1038/nm.2612.

Krammer et al.

3442 jvi.asm.org Journal of Virology

on June 28, 2014 by EA

ST

CA

RO

LINA

UN

IVhttp://jvi.asm

.org/D

ownloaded from