Assessment of Influenza Virus Hemagglutinin Stalk-Based Immunity in Ferrets

ABSTRACT

Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus hemagglutinin and stalk-based universal 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 divergent H1N1 strains in the mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret model of influenza disease. Furthermore, we recently developed a universal influenza virus vaccine strategy based on chimeric hemagglutinin constructs that focuses the immune response on the conserved stalk domain of the hemagglutinin. Here we set out to test this vaccination strategy in the ferret model. Both strategies, pretreatment of animals with a stalk-reactive monoclonal antibody and vaccination with chimeric hemagglutinin-based constructs, were able to significantly reduce viral titers in nasal turbinates, lungs, and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal wash viral titers. In summary, both strategies 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 neutralize seasonal and pandemic influenza virus strains. The ferret model was used to assess the potential of hemagglutinin stalk-based immunity to provide protection against influenza virus infection. The novelty and significance of the findings described in this report support the development of vaccines stimulating stalk-specific antibody responses.

INTRODUCTION

In the United States, epidemics of seasonal influenza cause substantial morbidity (1) and significant mortality (2). Despite the proven ability of inactivated and live attenuated influenza virus vaccines to reduce the impact of influenza, the potential of currently licensed influenza vaccines is not fully manifested because of several factors. First, influenza vaccination coverage rates remain low (3). In particular, a recent survey of 11,963 adults (18 to 64 years of age) revealed that only 28.2% reported receiving the 2008-2009 influenza vaccine (4). Second, influenza vaccines induce immune responses that specifically neutralize influenza viruses that are closely related to the vaccine strain, yet the potency of these neutralizing responses diminishes with antigenic drift. Thus, annual influenza vaccination is required to maintain protective immune responses against a “moving target” (5). Third, the emergence of pandemic influenza virus strains is difficult to predict, and once an influenza pandemic emerges, it is even more difficult to redirect vaccine production in a timely fashion to respond to a pandemic, as happened during the 2009 H1N1 influenza pandemic (6, 7). Predictions of influenza pandemics is further complicated by the realization that several influenza virus subtypes possess pandemic potential, as evidenced by the emergence of avian influenza A (H7N9) virus in March 2013 (8) and sporadic human infections with H4, H5, H6, H7, H9, and H10 avian influenza viruses (9–14).

Hemagglutinin (HA)-specific universal influenza vaccines have the potential to mitigate these limitations by focusing humoral immune responses on its antigenically conserved stalk region. Approaches to developing stalk-focused universal vaccines have included headless HA (15–17), recombinant soluble HA (18–22), synthetic polypeptides (23), prime-boost regimens (24, 25), nanoparticles (26), and recombinant influenza viruses expressing chimeric HA (cHA) (19, 21). Stalk-specific vaccines would shift the humoral immune responses away from the immunodominant globular-head domain to the more conserved stalk domain. Universal vaccines stimulating stalk-specific antibody responses would have several desirable aspects, including (i) conferring protection against homologous and drifted influenza virus strains, (ii) obviating the need for annual influenza vaccinations with reformulated H1, H3, and B virus strains that antigenically match prevalent circulating strains, and (iii) conferring increased protection against newly emerging influenza viruses with pandemic potential (27, 28). Importantly, stalk-reactive antibodies occur 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 influenza vaccine targeting the HA stalk would likely require three components to cover group 1 (H1, H2, H5, H6, H8, H9, H11, 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 protection conferred by group 1 HA stalk-specific antibodies against a challenge infection with pandemic H1N1 virus. Ferrets were passively immunized with stalk-reactive monoclonal antibodies (MAbs) or vaccinated with recombinant viral vectors expressing cHAs known to induce stalk-reactive antibodies in mice. These studies revealed that group 1 stalk-specific antibodies could reduce titers of infectious virus within the nasal cavity and also reduced pulmonary virus titers in immunized ferrets challenged with a pandemic H1N1 influenza virus that contains an HA head not present in the cHA vaccination regimen. These findings suggest that ferrets produce HA stalk-reactive antibodies following vaccination with cHAs and that stalk-reactive antibodies provide protection from heavy viral loads after a challenge infection in this influenza animal model.

MATERIALS AND METHODS

Cells and viruses.

Madin-Darby canine kidney (MDCK), 293T, 293, A549, and baby hamster kidney 21 (BHK-21) cells were propagated in Dulbecco's modified Eagle's medium (DMEM) or minimum essential medium (both from Gibco). A/Netherlands/602/09 pandemic H1N1 virus and the recombinant B-cH9/1 virus (a B/Yamagata/16/88 virus that expresses a cH9/1 HA as described in reference 35) were grown in embryonated chicken eggs, and titers were determined on MDCK cells in medium containing tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin as described before.

Generation of a VSV vector expressing cH5/1 protein.

The cH5/1 gene (an A/Viet Nam/1203/04 H5 head on top of an A/PR/8/34 H1 stalk domain [19, 21]) was amplified by PCR, and the SalI-NheI restriction enzyme-digested PCR product was then cloned into the XhoI and NheI sites of the pVSV-XN2 (38) vector to generate pVSV-cH5/1. Recombinant vesicular stomatitis virus (VSV) expressing cH5/1 HA (VSV-cH5/1) was recovered with the above plasmid with minor modifications to the previously described method (39). Briefly, BHK-21 cells were infected with the T7 polymerase-expressing vaccinia virus vTF7-3 (40) at a multiplicity of infection (MOI) of 20. At 1 h postinfection, the cells were transfected with the 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 collected, filtered through a 0.1-μm filter, and passaged onto BHK-21 cells. After a cytopathic effect (CPE) became evident, the culture medium was collected and virus was plaque purified and used to grow stocks. A VSV vector 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 globular-head domain on top of an H1/PR8 stalk domain [21, 35]) was cloned into a previously described transfer plasmid (pE1A-CMV, lacking the HA epitope tag) (41). For virus generation, 2.0 × 10⁶ human embryonic kidney 293 (HEK-293) cells (generously supplied by Patrick Hearing) were plated per well of a six-well dish and transfected the following day with a 3:1 ratio of X-tremeGENE 9 (Roche) to DNA according to the manufacturer's instructions. Cells were transfected with a total of 5.5 μg of DNA consisting of 5 μg of PvuI-linearized cH6/1 pE1A-CMX plasmid and 500 ng of dl309 viral DNA that had been digested with ClaI/XbaI to remove the left end of the adenoviral genome (bp 1 to 920). X-tremeGENE 9-transfected, ClaI/XbaI-digested viral DNA was used as a negative control. After 24 h of incubation, cells were overlaid with 2× DMEM-supplemented 1% agarose for plaque selection. Overlays were reapplied approximately every 3 days for 1 week, and then plaques were isolated for screening and used for 1⁰ lysate generation. Once a CPE was evident (2 to 3 days), cells were harvested and frozen at −80°C. Cells underwent four freeze-thaw cycles, and then viral DNA was prepared by an established method for sequencing (42). Once the cH6/1 sequence was confirmed, virus stocks were amplified on HEK-293 cells and purified by consecutive banding on step and equilibrium cesium chloride gradients. Expression of the cH6/1 protein was confirmed by immunofluorescence staining on A549-infected cells with anti-stalk MAb 6F12 (43), and virus titers were determined by standard plaque assay on HEK-293 cells. The empty control adenovirus vector (in the same genomic background) was kindly provided by Patrick Hearing.

Immunostaining.

MDCK cells were infected at a MOI of 1 with B-cH9/1 or wild-type B/Yamagata/16/88 and fixed (0.5% paraformaldehyde) at 24 h postinfection. A subset of cells was permeabilized with 0.1% Triton X-100 and stained with an anti-influenza B virus nucleoprotein antibody (Abcam; 1:1,000). The rest of the cells were stained with anti-H1 stalk antibody 6F12 (10 μg/ml) or anti-H9 head antibody G1-26 (BEI Resources NR-9485; 1:1,000). 293T and A549 cells were infected/transduced with empty or cH6/1-expressing adenovirus at an MOI of about 100. Cells were permeabilized with 0.5% Triton X-100 and stained with an anti-hexon antibody (Abcam; 1:1,000), an anti-H1 stalk antibody 6F12 (10 μg/ml), or an anti-H6 head antibody NatalieC (10 μg/ml). An Alexa 488-conjugated anti-mouse antibody (Life Technologies; 1:1,000) was used as the secondary antibody for immunofluorescence analysis. Vero cells 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-VSV serum (1:1,000), MAb 6F12 (10 μg/ml), or anti-H5 head antibody VN4-10 (BEI Resources NR-2737; 1:1,000). A horseradish peroxidase (HRP)-linked anti-mouse antibody (Santa Cruz; 1:3,000) was used as the secondary antibody, and stained cells were visualized with aminoethyl carbazole substrate solution (Millipore).

Antibodies and recombinant proteins.

Mouse MAbs 6F12 (H1 stalk reactive, IgG2b) (43) and XY102 (A/Hong Kong/1/68 HA head reactive, hemagglutination inhibition [HI] active, IgG2b) (44) were purified from supernatants of hybridoma cultures as described before. Briefly, the supernatants were passed over a column loaded with protein G-Sepharose (GE Healthcare), washed, eluted, and concentrated, and the buffer was exchanged for phosphate-buffered saline (PBS; pH 7.4) with Amicon Ultra centrifugation units (Millipore). Protein concentrations were determined by the A₂₈₀ method with a NanoDrop device. Recombinant HAs were expressed as ectodomains with a C-terminal trimerization domain and 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 circulating H1N1, H3N2, and B influenza viruses prior to purchase from Triple F Farms (Sayre, PA). Ferrets were housed in PlasLabs poultry incubators with free access to food and water (46–48). All of the animal experiments described here were conducted by using protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee. Animals were anesthetized by intramuscular administration of ketamine/xylazine for all of the procedures described here, including bleeding, nasal washes, vaccination, infection, and passive transfer.

For passive-transfer experiments, animals were bled to obtain baseline serum samples 2 weeks before the transfer. On day −1, 30 mg/kg of mouse MAb 6F12 or XY102 (n = 2 per group) was transferred intravenously via the vena cava (Fig. 1A). At 24 h postinoculation, animals were bled and infected with 10⁴ PFU of A/Netherlands/602/09 (pandemic H1N1) virus. Nasal washes were then taken on days 1 and 3 postinfection, and body weights were monitored daily. Animals were observed for approximately 30 min daily for signs of morbidity (e.g., sneezing). On day 4 postinfection, animals were sacrificed and exsanguinated and tissue samples were taken from the upper left and right lobes of the lungs, olfactory bulb, and nasal turbinates.

FIG 1.

Persistence and distribution of MAb 6F12 in two passively immunized ferrets. (A) Schematic representation of the passive immunization and challenge study. 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 collected on 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 MAb 6F12 in nasal wash samples collected on days 1 and 3 of passive immunization were measured by ELISA reactivity against baculovirus-produced H1 from A/California/04/09. (D) Titers of MAb 6F12 in lung homogenate samples collected on day 4 of passive immunization were measured by ELISA reactivity against baculovirus-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 ferrets passively immunized with MAb XY102, which specifically recognizes H3 of A/Hong Kong/1/1968, served as negative controls (n = 2 ferrets). OD, optical density.

For vaccination experiments, animals (n = 5) were intranasally infected with 2 × 10⁷ PFU (in 1 ml of PBS) of influenza B virus vector B-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 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]). A second boost consisting of a replication-deficient recombinant adenovirus 5 vector expressing the cH6/1 protein (an H6 globular-head domain on 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. Control group 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 (pandemic H1N1) virus. Nasal washes were then taken on days 1 and 3 postinfection, and weight was monitored daily. Animals were observed for approximately 30 min daily, and signs of morbidity (e.g., sneezing) were recorded. On day 4 postinfection, animals were sacrificed and tissue samples were taken from the lung (upper right lobe), olfactory bulb, and nasal turbinates.

FIG 4.

Ferrets develop HA stalk-specific humoral responses after repeated immunization with viral vectors expressing cHAs. (A) Schematic representation of the HA stalk-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, 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. Ferrets were then challenged by infection with 10⁴ PFU of A/Netherlands/602/09 virus. The development of broadly cross-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.

HI assays.

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

ELISAs.

Enzyme-linked immunosorbent assays (ELISAs) were performed as described before (20, 31). Briefly, plates were coated with 2 μg/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/Japan/305/57), or H17 (from A/yellow shouldered bat/Guatemala/06/10) HA protein (20). Wells were then incubated with serial (2-fold) dilutions of ferret sera, nasal washes, or lung homogenates for 1 h at room temperature. After extensive washes, plates were incubated with anti-mouse antibody (for MAbs; Santa Cruz) or anti-ferret (Alpha Diagnostics International) IgG HRP-labeled secondary antibody for another hour at room temperature. Plates were washed again and then developed with SigmaFast o-phenylenediamine dihydrochloride substrate and read on a Synergy H1 (BioTek) plate reader.

RESULTS

Persistence and tissue distribution of 6F12 in the ferret.

Previously, we have shown that HA head-reactive IgA but not IgG antibody is able to prevent transmission in the ferret and guinea pig models of influenza virus infection (49). We reasoned that at an especially low concentration (3 mg/kg), IgG is not efficiently transported to mucosal surfaces. This transport might be additionally inhibited by the lower Fc-Fc receptor interactions between mouse MAbs and the ferret host. In addition, the half-life of mouse IgG in ferrets has not been well characterized; however, a previous study that examined the therapeutic potential of a humanized MAb, m102, in the ferret model of Nipah virus infection reported an elimination half-life of 3.5 days following the intravenous administration of 25 mg of MAb (50). We were therefore curious if treatment with a large dose of MAb (30 mg/kg) would increase the Ab concentration on mucosal surfaces and protect from upper respiratory tract infection. Ferrets were passively immunized by the intravenous administration of 30 mg/kg of either H1 stalk-specific MAb 6F12 or H3-specific MAb XY102 (isotype control) (Fig. 1A). The persistence and tissue distribution of MAb 6F12 were examined by ELISA with baculovirus-produced H1 from 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 wash samples collected on day 1, but the level of MAb declined by day 3 after a challenge infection (Fig. 1C). MAb 6F12 was also detected by ELISA in lung homogenates on day 4 postchallenge (Fig. 1D). These results suggest that passive immunization by intravenous administration of MAb 6F12 would confer a window of protection against a challenge infection within the ferret respiratory tract.

Prophylactic administration of 6F12 reduces viral loads in lungs, olfactory bulbs, and nasal turbinates.

Mouse MAb 6F12 is an H1 stalk domain-specific antibody that potently inhibits viral replication of H1N1 virus isolates spanning from 1930 to 2009 and efficiently protects mice prophylactically and therapeutically from a viral challenge (43). In order to investigate whether 6F12 would also be efficacious prophylactically in the ferret model of influenza disease, a 30-mg/kg dose of this MAb was administered to ferrets intravenously 24 h prechallenge and the animals were then challenged with pandemic H1N1 strain A/Netherlands/602/09. Control group ferrets received the same amount of an isotype control antibody (Fig. 1A). Viral titers from nasal wash samples taken on days 1 and 3 were slightly lower in the 6F12-treated animals than in the control group (Fig. 2A). The effect was more pronounced on day 1 than on day 3, which matches the lower 6F12 titers found in nasal washes on day 3 postchallenge. Furthermore, the day 4 nasal turbinate titers of 6F12-treated ferrets were lower than those of control animals (Fig. 2B). A reduction of approximately 2 logs in 6F12-treated animals was also observed in the olfactory bulb (Fig. 2C), and lung titers were approximately 1 log lower than those of control animals (Fig. 2D). Weight loss was only minimal and similar in both groups (data not shown). In summary, prophylactic treatment of ferrets with MAb 6F12 reduced the viral loads in challenged animals in all of the analyzed tissues. The readouts established for this experiment were then also used to compare and analyze the efficacy of a cHA vaccine regimen in ferrets.

FIG 2.

Prophylactic administration of MAb 6F12 reduced viral titers following a challenge infection. Ferrets were passively immunized with MAb 6F12 (green bars; 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 PFU of 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 after the challenge infection, titers of influenza virus in nasal turbinate (B), olfactory bulb (C), and lung (D) samples were assessed by plaque assay.

Vaccination with cHAs induces stalk-reactive antibodies in the ferret.

We have previously shown that vaccination of inbred BALB/c mice with cHA constructs (HAs with a conserved stalk domain but divergent head domains) induces broadly neutralizing stalk-reactive antibodies (21). Here we wanted to test if vaccination of ferrets would also induce stalk-reactive antibodies. To this end, we used viral vectors expressing cHA constructs (Fig. 3). Prior to vaccination of ferrets with the viral vectors, the expression of the cHA was demonstrated by immunostaining. The expression of cHA by an influenza B virus vector expressing B-cH9/1 HA (21, 35) was demonstrated by immunofluorescence assay with infected MDCK cells (Fig. 3A). The expression of cHA by a VSV expressing cH5/1 HA (19, 21) was demonstrated by immunostaining of virus plaques in Vero cells (Fig. 3B). The expression of cHA by a replication-deficient adenovirus 5 vector expressing cH6/1 HA (an H6 head on top of an H1/PR8 stalk domain [19, 21]) was demonstrated by immunofluorescence assay with infected 293T and A549 cells (Fig. 3C).

FIG 3.

Expression of cHAs by viral vectors. (A) An engineered influenza B virus expresses cH9/1 HA (H9 head on top of an H1 stalk domain) instead of influenza B HA. Shown is staining of B-cH9/1- or B-wt (wild-type influenza B virus)-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 (H5 head on top of an H1 stalk domain) HA as a transgene. Shown is staining of VSV-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 antibody (anti-H5 head). (C) A replication-deficient adenovirus was engineered to express cH6/1 HA (H6 head on top of an H1 stalk domain). Shown are infected 293T cells stained for the presence of adenovirus (anti-hexon) and transduced A459 cells stained with anti-H1 stalk antibody 6F12 (anti-stalk) or an anti-H6 head antibody (anti-H6 head).

Ferrets were first vaccinated with B-cH9/1 HA and then boosted with VSV-cH5/1 HA (an H5 head on top of an H1 stalk domain [19, 21]) and then with a replication-deficient adenovirus 5 vector expressing cH6/1 HA (an H6 head on top of an H1/PR8 stalk domain [19, 21]) (Fig. 4A). This vaccination regimen was chosen in order to avoid the generation of antibodies against any antigen in pandemic H1N1 virus different from the HA stalk, which could also contribute to protection after a subsequent challenge. Vaccinated animals developed low seroreactivity against pandemic H1 HA after priming. This reactivity was boosted approximately 4-fold by the cH5/1 vaccination and then again 8-fold by the final cH6/1 vaccination. Sera from vector control animals exhibited only background reactivity that was comparable to the reactivity of pooled prevaccination sera of the ferrets used. Since cHA-vaccinated animals were naive to the H1 head domain and also tested HI negative against the pandemic H1N1 strain A/Netherlands/602/09, we conclude that any reactivity to H1 strains is based on cross-reactive antibodies to the conserved stalk domain. Furthermore, our cHA vaccine constructs are based on the stalk domain of A/PR/8/34 H1 HA. Therefore, reactivity to pandemic H1 HA already represents heterologous stalk reactivity within H1 HAs. We also tested reactivity to two more H1 HAs, the HA from prepandemic seasonal strain A/New Caledonia/20/99 and the HA from 1918 pandemic H1N1 strain A/South Carolina/1/18 (Fig. 4C and D). Sera from cHA-vaccinated animals reacted strongly with both proteins. In order to test if cHA vaccination induces cross-reactivity to other group 1 subtypes, we also tested reactivity against an H2 HA from A/Japan/305/57 virus (Fig. 4E) and against an H17 HA (from recently discovered bat H17N10 influenza virus strain 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 background reactivity (Fig. 4E and F). Cross-reactivity against group 2 HA was not expected, since earlier studies with mice have shown that group 1 stalk-based cHA vaccination regimens do not protect from a group 2 virus challenge and vice versa (21, 22). Importantly, we did not detect any H1 head-specific antibody responses against the challenge virus following the vaccination regimen as measured by HI assay (data not shown). As positive controls, convalescent-phase reference sera from two ferrets infected with A/California/7/2009 were included in the HI assay, and each reference 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 confer on ferrets, we challenged the animals with the pandemic H1N1 strain A/Netherlands/602/09 (Fig. 5A). The readouts were the same as for the passive-transfer experiment; we measured virus titers in day 1 and 3 nasal washes and in the lungs, olfactory bulb, and nasal turbinates on day 4 postinfection. Interestingly, nasal wash titers were lower in cHA-vaccinated ferrets than in control animals on day 1 (approximately 5-fold), as well on day 3 (more than 10-fold), when the difference was highly significant (P = 0.0005) (Fig. 5A). This result is not surprising since we expected that the intranasally applied priming and second boost would induce stalk-reactive mucosal IgA antibodies. The reduction of virus titers in the nasal washes is also reflected by a significant reduction of 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 of vector control animals (P = 0.0062) (Fig. 5C). In fact, we were unable to detect virus in the olfactory bulbs of four out of five cHA-vaccinated ferrets, whereas high virus titers were found in the olfactory bulbs of all four control ferrets. Finally, we also detected a reduction of approximately half a log of lung virus titers in cHA-vaccinated ferrets compared to those of vector control ferrets (Fig. 5D). In summary, the protective efficacy of the cHA vaccine was comparable to (nasal turbinates, olfactory bulbs, and lung titers) or better than (nasal wash titers) that of prophylactically administered MAb 6F12.

FIG 5.

The HA stalk-based vaccination strategy confers protection against a challenge infection with A/Netherlands/602/09 virus. Ferrets (n = 5) were vaccinated with 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 samples collected 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 nasal turbinate (B), olfactory bulb (C), and lung (D) samples were assessed by plaque assay. n.s., not significant.

DISCUSSION

In recent years, broadly neutralizing antibodies against the conserved stalk domain of the influenza virus HA have been isolated (30, 32, 36, 37, 43, 51, 52, 53–56). These antibodies can be used for prophylactic and therapeutic treatments of influenza virus infections. Although the large amount of MAb needed for treatment might preclude the use of the antibodies in the general population, this approach might be useful for the therapy of severe influenza cases, especially when drug-resistant viruses in an immunocompromised host are involved (57–63). We therefore wanted to evaluate MAb 6F12 in a prophylactic setting in the ferret model. This antibody has pan-H1 neutralizing activity in vitro and is able to protect mice from a challenge with H1N1 influenza viruses that span almost 100 years of antigenic drift (43). We show here that MAb 6F12 is indeed efficacious against a pandemic H1N1 strain in the ferret model as well. In particular, prophylactic administration of MAb 6F12 resulted in a more pronounced reduction of virus titers in olfactory bulbs and lungs. Unexpectedly, we could also detect this mouse IgG antibody at low titers in nasal wash samples from treated ferrets. These low levels of antibody found in the nasal washes correlated well with small reductions of nasal wash viral titers. Several factors could contribute to the pronounced reduction of virus titers in olfactory bulb and lung samples compared to the modest reduction of virus titers observed in nasal wash samples. On day 4 after intravenous injection, high levels of MAb 6F12 could be detected in serum and lung samples, which contrasts with the low level of MAb 6F12 detected in nasal wash samples. In addition, MAb 6F12 liberated by the homogenization of olfactory bulb and lung tissue samples would bind to and neutralize a small fraction of the virus present in the tissue samples prior to the determination of virus titers by plaque assay. We speculate that 6F12-like antibodies, if transported efficiently to mucosal surfaces (e.g., locally induced by intranasally administered vaccines) would be able to efficiently reduce nasal wash virus titers and possibly have an impact on transmission as well. We recently showed that this is the case for globular-head-reactive MAb 30D1, which efficiently blocks replication when administered to guinea pigs as IgA (efficiently transported to mucosal surfaces) but lacks efficacy when administered as IgG (not efficiently transported to mucosal surfaces) (49).

In an “antibody-guided” vaccine approach based on stalk-reactive antibodies, we have developed cHA vaccine constructs (19, 21). These constructs possess a conserved, structurally integrated stalk domain in combination with divergent globular-head domains from “exotic” subtypes (21). By sequentially immunizing mice with these constructs, we protected them from a challenge with heterologous (H1N1) and heterosubtypic (other group 1 HA-expressing viruses) influenza viruses (21). Here, we tested the efficacy of this vaccine approach in the ferret model. By immunizing ferrets with combinations of divergent globular heads and a conserved stalk domain, we hoped to get an immune response focused on broadly neutralizing epitopes in the stalk. This strategy is based on the observation that sequential infection/vaccination with seasonal H1N1 and pandemic H1N1 viruses (which have highly divergent globular-head domains and highly conserved stalk domains) induces high levels of stalk-reactive antibodies in humans (32, 35–37, 64). Similar findings were also obtained in the mouse model (31). Here, in the ferret model, we show that a cHA-based immunization strategy confers protection against a pandemic H1N1 challenge. The observed level of protection was similar to or better than that conferred by inactivated, antigenically matched, unadjuvanted split vaccine administered once (65, 66) or twice (67) or an antigenically matched experimental vaccinia virus-vectored construct (68). It is of note that the cHA-based vaccine did not induce any HI-active antibodies, but vaccinated ferrets were able to produce a broadly reactive anti-stalk response against divergent group 1 HA subtypes. This proof-of-principle study focused on protection afforded by the stalk domain of HA. A human vaccine candidate based on the same principle would most likely consist of inactivated or attenuated cHA-expressing viruses that also have a neuraminidase (NA). We believe that the antibody titers against the more conserved NA would be boosted as well in the absence of an immunodominant globular-head domain (69, 70). These antibodies would then also contribute to broad protection. Furthermore, conserved internal proteins like the nucleoprotein induce strong protective T-cell responses that contribute to protection as well (71–74). We have conclusively shown that such a vaccination strategy based on the H1 HA stalk domain is able 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 vaccination strategy would need to contain a group 1, a group2, and an influenza B virus stalk component to induce broadly neutralizing stalk antibodies.

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