Recombinant Soluble, Multimeric HA and NA Exhibit Distinctive Types of Protection against Pandemic Swine-Origin 2009 A(H1N1) Influenza Virus Infection in Ferrets^▿

Influenza A challenge virus.

Influenza virus A/Netherlands/602/2009 was isolated from the first case of a laboratory-confirmed 2009 A(H1N1) infection in the Netherlands by inoculation of 11-day-old embryonated chicken eggs (32). Virus stocks of influenza virus A/Netherlands/602/2009(H1N1) were prepared by infecting confluent Madin-Darby canine kidney (MDCK) cells. After cytopathological changes were complete, culture supernatants were cleared by low-speed centrifugation and stored at −70°C. Infectious virus titers were determined in MDCK cells as described previously (36). All experiments with these viruses were performed under biosafety level 3 (BSL-3) conditions.

Preparation of HA and NA antigens.

Human codon-optimized sequences encoding the soluble hemagglutinin ectodomain (sHA, amino acids [aa]17 to 522) and the neuraminidase head domain (sNA, aa 75 to 469) of influenza virus A/California/04/2009(H1N1) were synthesized (GenScript) and cloned into a derivative of expression plasmid pS1-Ig (30) for expression in HEK293T cells. The HA gene was preceded by a sequence encoding an N-terminal CD5 signal peptide and followed by sequences encoding a C-terminal artificial GCN4 trimerization domain (GCN4-pII) (12) and a Strep tag for affinity purification (IBA GmbH) as described recently (4, 9). The NA gene was preceded by sequences successively coding for an N-terminal CD5 signal peptide, a double Strep tag (One-STrEP; IBA GmbH), and an artificial GCN4 tetramerization domain (GCN4-pLI) (12).

Protein expression and purification.

HEK293T cells were transfected with the sHA and sNA expression plasmids using polyethyleneimine (PEI) in a 1:5 ratio (μg DNA to μg PEI). After 6 h of incubation, the transfection medium was replaced by 293 SFM II expression medium (Invitrogen) supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 units/ml), streptomycin (100 μg/ml), glutaMAX (Gibco), and 1.5% dimethyl sulfoxide (DMSO). Tissue culture supernatants were harvested at 5 to 6 days posttransfection, and sHA and sNA proteins were purified from the culture medium using Strep-Tactin affinity chromatography (IBA GmbH). sHA and sNA protein expression and purification were confirmed by Western blotting using a Strep-Tactin-horseradish peroxidase (HRP) conjugate (IBA GmbH) (data not shown) and SDS-PAGE analysis. Oligomerization of the proteins was determined by gel filtration chromatography and by blue-native PAGE analysis. Quantification of protein amounts was done using bovine serum albumin (BSA) as a reference.

Ferrets.

Healthy young adult outbred female ferrets (Mustela putorius furo; between 6 and 12 months old) were purchased from a commercial breeder. The animals were checked for the absence of antibodies against circulating seasonal A/H1N1 and A/H3N2 influenza viruses and against the swine-origin influenza A/NL/602/09 virus by hemagglutination inhibition (HI) assay. An independent animal ethics committee approved the experimental protocol before the start of the experiment.

Immunizations and infections.

Thirty-six seronegative ferrets were divided into six groups of six ferrets each and vaccinated twice with the following formulations: 3.75 μg sHA₃ plus 3.75 μg sNA₄ in phosphate-buffered saline (PBS) (group 1), 3.75 μg sHA₃ in ISCOM Matrix M (IMM) (Isconova, Uppsala, Sweden) (group 2), 3.75 μg sNA₄ in IMM (group 3), 3.75 μg sHA₃ plus 3.75 μg sNA₄ in IMM (group 4), PBS (group 5), and IMM (group 6). Vaccinations were performed with an interval of 20 days under anesthesia with ketamine in the quadriceps muscles of the hind leg in a total volume of 1 ml. Ferrets were housed in groups and received food and water ad libitum. At 32 days after the last vaccination, the animals were anesthetized with ketamine-medetomidine (reversed with atipamezole), weighed, and subsequently challenged intratracheally with 1 × 10⁶ 50% tissue culture infective doses (TCID₅₀) of influenza A/NL/602/09(H1N1) in a volume of 3 ml PBS (7, 42). Ferrets were subsequently monitored three times daily for the development of clinical signs. Before infection and 2 and 4 days after infection, nose and throat swabs of each ferret were collected while ferrets were anesthetized with ketamine. Four days after inoculation, animals were weighed and subsequently euthanized by exsanguination while under anesthesia with ketamine and medetomidine. Necropsies were performed according to standard procedures. One ferret in group 1 died between the first and second vaccinations due to reasons unrelated to the experiment.

Serology.

Serum samples were collected before vaccination, at the day of the second vaccination (day 20), and at the day of challenge (day 52). Sera were stored at −20°C until use. Sera were tested for the presence of anti-HA antibodies using a hemagglutination inhibition (HI) assay with 1% turkey erythrocytes and for the presence of virus-neutralizing antibodies using a micro-virus neutralization (VN) assay as described previously (11, 33). Sera were tested for the presence of antibodies reactive with influenza A/NL/602/09(H1N1) virus. For this purpose, reverse genetics viruses were produced. The titers obtained with these viruses were comparable to those against the wild-type strains (data not shown). Positive control serum specific for influenza A/NL/602/09(H1N1) virus was obtained from a ferret infected with this virus (32). Other H1N1 influenza viruses used in the HI assay were A/Netherlands/386/86 (NL/86), A/Netherlands/25/80 (NL/80), A/New Jersey/8/76 (NJ/76), A/Swine/shope/1/56 (Sw/56), A/Italy/1443/76 (It/76), A/Iowa/15/30 (Io/30), A/Puerto Rico/8/34 (Pr/34), and A/Brisbane/59/07 (IVR-148 vaccine strain; IVR/148). Serum samples from ferrets infected with these viruses were used as a positive control in this assay (6).

Sera were also tested for the presence of neuraminidase-inhibiting (NI) antibodies using a previously described fetuin-based assay (29). Briefly, 96-well Nunc MaxiSorp plates were coated overnight at 4°C with 100 μl of 5-μg/ml fetuin. Sixty-microliter volumes of serially diluted serum samples were incubated for 30 min at 37°C with an equal volume of sNA₄-containing culture supernatant (prediluted in PBS with Ca [0.901 mM]-Mg [0.493 mM] to give a half-maximum optical density at 450 nm [OD₄₅₀] of 1.5), after which 100 μl of the mixture was added to the fetuin-coated wells. After 1 h of incubation at 37°C, the plates were washed and neuraminidase activity was subsequently measured by adding peroxidase-labeled peanut agglutinin (2.5 μg/ml; Sigma), incubating for 1 h at room temperature, washing the plates, and adding 100 μl of peroxidase substrate (TMB) to each well. After 5 min, the reaction was stopped by the addition of 100 μl of 0.3 M phosphoric acid, and OD values were measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (EL-808 [BioTEK]). To test the sera for cross-reactive NI antibodies, sNA₄ expression constructs similar to the ones described above for A/California/04/2009(H1N1) were also made for the head domains of A/Kentucky/UR06-0258/2007(H1N1) (aa 75 to 470) and A/turkey/Turkey/1/2005(H5N1) influenza virus (aa 55 to 449). Sera specific for influenza A/NL/602/09(H1N1) and A/turkey/Turkey/1/2005(H5N1) viruses obtained from a ferret infected with these viruses were used as a positive control.

Virus replication in the upper and lower respiratory tracts.

Samples of all lobes of the right lung and of the accessory lobe were collected from the infected ferrets, snap-frozen on dry ice with ethanol, and stored at −70°C until further processing. Lung samples were weighed and subsequently homogenized with a FastPrep-24 (MP Biomedicals, Eindhoven, Netherlands) in Hanks' balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/ml streptomycin, 100 U/ml polymyxin B sulfate, 250 μg/ml gentamicin, and 50 U/ml nystatin (ICN Pharmaceuticals, Zoetermeer, Netherlands) and centrifuged briefly. Nose and throat swabs were stored directly at −70°C in the same medium used to homogenize the lung samples. Quadruplicate 10-fold serial dilutions of throat, nose, and lung samples were used to infect MDCK cells as described previously (36). The HA activity of the culture supernatants collected at 5 days postinoculation (dpi) was used as an indicator of infection. The titers were calculated according to the Spearman-Karber method and expressed as log TCID₅₀ per gram for lung tissue or per ml for swabs (22).

Histopathology.

At 4 postinoculation (dpi) with influenza A/NL/602/09 virus, ferrets were euthanized and lungs were observed macroscopically and weighed before samples from the right lungs were collected to determine the virus titers. Subsequently, left lung lobes were inflated with 10% neutral buffered formalin. After fixation and embedding in paraffin, lungs were sectioned at 4 μm and tissue sections were examined by staining with hematoxylin and eosin (H&E).

Statistical analysis.

Significance among animal groups was analyzed by one-way analysis of variance (ANOVA) and Tukey test after ANOVA. Differences were considered significant at a P value of <0.05.

Production of sHA₃ and sNA₄ antigens.

Constructs were designed to express the trimeric HA ectodomain (aa 17 to 522) and the tetrameric NA head domain (aa 75 to 469) of the 2009 A(H1N1) influenza virus, as pictured in Fig. 1A. The sHA₃ and sNA₄ proteins were produced by expression in HEK293T cells and purified from the culture medium by affinity chromatography, yielding glycoproteins of the expected size (Fig. 1B). Gel filtration analysis indicated the trimeric and tetrameric oligomeric natures of the HA and NA subunits, respectively (data not shown). The multimeric complexes were also biologically active, further confirming their native state, as judged by their sialic acid binding (sHA₃) (unpublished data) and neuraminidase activity (sNA₄) (see below).

Antibody responses induced by immunization with sHA₃ and sNA₄.

The glycoproteins were tested for their ability to induce protective immunity against homologous virus challenge. Ferrets were immunized at days 0 and 20 with sHA₃ plus sNA₄ without adjuvant (sHA+sNA), with sHA₃ plus sNA₄ adjuvanted with ISCOM Matrix M (IMM) (sHA+sNA+IMM), or with similarly adjuvanted sHA₃ (sHA+IMM) or sNA₄ (sNA+IMM). Sera were collected at the day of the second immunization and prechallenge (days 20 and 52), and antibody responses were measured by HI and VN assays against the homologous virus and by NI assay (Fig. 2). No responses with any of these assays were observed in control animals vaccinated with PBS or with adjuvant only but also not in the animals immunized with the nonadjuvanted mixture of sHA₃ and sNA₄. In contrast, the immunizations with adjuvanted sHA₃ (sHA+IMM) induced high HI titers, with a geometric mean titer of 91 at the day of challenge. Interestingly, the additional inclusion of sNA₄ (sHA+sNA+IMM) significantly increased the average HI titers to 468 (P < 0.05 by one-way ANOVA and Tukey test). Also, the NI titrations revealed the adjuvant-dependent induction of NA antibodies, which were low after one immunization (Fig. 2) but strongly boosted after two immunizations (Fig. 2). However, in this case no clear augmentation of these titers due to the coadministration of sHA₃ was observed. Consistent with the observed HI titers, high VN titers were found both in the sHA+IMM- and in the sHA+sNA+IMM-vaccinated animals (Fig. 2). Also here, the coadministration of the sNA₄ antigen with the sHA+sNA+IMM vaccine resulted in an increase in the mean VN titer, with average values ranging from 1:202 to 1:468 in the sHA+IMM and sHA+sNA+IMM groups, respectively.

Protection against clinical signs after infection with 2009 A(H1N1) influenza virus.

Vaccinated ferrets were challenged with 10⁶ TCID₅₀ 2009 A(H1N1) at 5 weeks after the second vaccination. From day 2 after inoculation onwards, clinical signs were observed in inoculated ferrets, which included breathing difficulties, lethargy, decreased appetite, and weight loss. In general, only mild clinical signs were observed in ferrets of groups 2, 3, and 4, while more severe symptoms were observed in ferrets of groups 1, 5, and 6. Loss of body weight became obvious in the PBS- and IMM-vaccinated control groups as well as in the nonadjuvanted sHA+sNA vaccine group (Fig. 3). Interestingly, the animals immunized with sHA+IMM showed nearly similar weight losses, while body weights were not significantly affected after vaccination with both the sNA₄-containing formulations (groups sNA+IMM and sHA+sNA+IMM). More or less consistently, the lung weights of the ferrets determined postmortem showed a corresponding tendency, with adjuvanted sNA₄-vaccinated animals having the least disease-related increase due to lung consolidation (Fig. 3).

Gross pathological and histopathological findings in the lungs of ferrets.

Four days after inoculation with influenza 2009 A(H1N1) virus, the lungs of the ferrets were examined macroscopically and weighed before samples were taken for assessing virus replication and histopathological changes. Dark red and firm consolidated areas were observed macroscopically in lungs of inoculated ferrets. The percentage of affected lung tissue was estimated and was found to vary between groups. Mean percentages of affected areas in the lungs of about 50% were observed in ferrets of groups 1, 5, and 6, while the extent of consolidation was less pronounced in ferrets of groups 2, 3, and 4, which showed less than 25% of the lung area being affected (Fig. 3). The relative lung weight also was lower in these groups than in ferrets of groups 1, 5, and 6 (Fig. 3).

The histopathological changes observed at day 4 postinoculation in the lungs of ferrets mock vaccinated with PBS or adjuvant (IMM) only or vaccinated with the nonadjuvanted sHA₃+sNA₄ were characteristic for a moderate to severe necrotizing broncho-interstitial pneumonia. Multifocally, many neutrophils and macrophages and variable numbers of erythrocytes, edema fluid, and fibrin were present in the alveoli of the lungs of these ferrets. In addition, inflammatory infiltrates were present in the alveolar septa, in the bronchioles, in the bronchi, and in the walls of bronchi and bronchioles. A dramatic reduction in histopathological changes was observed in the adjuvanted sNA₄-vaccinated animals (sNA+IMM and sHA+sNA+IMM groups), while ferrets immunized with sHA+IMM were partially protected from developing pathology (Fig. 4).

Protection against virus replication in the upper and lower respiratory tracts.

To measure the effect of vaccination on the virus replication in the respiratory tract, virus titers in lungs, throat, and nose were determined at 4 days after inoculation. As shown in Fig. 5 the challenge virus replicated efficiently in the lungs of the control ferrets (PBS and IMM groups) and in the animals immunized with the nonadjuvanted mixture of sHA₃ and sNA₄ (sHA+sNA group), with mean viral titers of approximately 10⁷ to 10⁸ TCID₅₀/gram tissue. These viral loads were reduced by about 5 log₁₀ units in the animals immunized with the sHA₃ protein in adjuvant (sHA+IMM group) and in animals coimmunized with sHA₃ and sNA₄ in adjuvant (sHA+sNA+IMM group). Mean viral loads were reduced by 2 to 3 log₁₀ units in animals immunized with adjuvanted sNA₄ antigen (sNA+IMM group).

High viral loads in the nose were observed in the control animals (PBS and IMM groups) (Fig. 5) at day 4 after the challenge. Though not statistically significant due to the large variations in titers within groups, these viral loads appeared to be somewhat lower in the animals immunized with adjuvanted sHA₃ or adjuvanted sNA₄ or with the nonadjuvanted sHA₃+sNA₄ combination. The highest reduction of nose viral titers was found in animals immunized with the adjuvanted combination of sHA₃ and sNA₄ antigens. Viral titers in the throat were generally high and were not significantly affected by vaccination, except in the animals vaccinated with the adjuvanted combination of sHA₃ and sNA₄. These ferrets did not have detectable titers in the throat.

Cross-reacting antibody responses induced by immunization with sHA₃ and sNA₄.

To investigate whether the antibodies induced by the sHA₃ and sNA₄ antigens could cross-react with other H1N1 influenza viruses, we performed additional HI and NI assays with the postvaccination sera. As expected, the highest HI titers were measured against the homologous virus, while various extents of cross-reactivity were observed with a range of other H1 strains (Fig. 6A). Thus, no cross-reactivity was detected for A/Swine/shope/1/56, A/Italy/1443/76, A/Iowa/15/30, A/PR/8/34, and IVR/148, whereas significant cross-reactivity was measured against A/NL/25/80 and A/New Jersey/8/76 and, particularly, against A/NL/386/86, more or less consistent with the sequence similarities of their antigenic domains (Table 1). This was the case with the sera from both the sHA₃+IMM- and the sHA₃+sNA₄+IMM-vaccinated animals. Consistent with the earlier observed differences in HI activity against the homologous virus (Fig. 2), the levels of cross-reactivity were markedly higher with the sera from ferrets immunized with sHA₃+sNA₄+IMM than with those from sHA₃+IMM-immunized animals, confirming again the enhancing effect of the sNA₄ antigen. HI titers against each strain were detected in control sera of ferrets infected with the homologous influenza A/H1N1 virus (data not shown).

To investigate the cross-reactivity of the NA antibodies, we produced sNA₄ glycoprotein complexes of two other N1 influenza viruses, the human H1N1 strain A/Kentucky/UR06-0258/2007 and the avian H5N1 strain A/turkey/Turkey/1/2005. When tested in our NI assay, there was a strong neuraminidase-inhibiting activity with the pooled sera of the sNA₄+IMM- and sHA₃+sNA₄+IMM-immunized animals against the avian H5N1 virus sNA₄ protein, while some inhibition of the seasonal H1N1 virus sNA₄ protein was observed (Fig. 6B). Of note, a control serum derived from an H5N1 virus-infected chicken tested negative against both human H1N1 virus sNA₄ proteins.