Recombinant soluble, multimeric HA and NA exhibit distinctive types of protection against pandemic swine-origin 2009 A(H1N1) influenza virus infection in ferrets

Abstract

The emergence and subsequent swift and global spread of the swine-origin influenza virus A(H1N1) in 2009 once again emphasizes the strong need for effective vaccines that can be developed rapidly and applied safely. With this aim, we produced soluble, multimeric forms of the 2009 A(H1N1) HA (sHA(3)) and NA (sNA(4)) surface glycoproteins using a virus-free mammalian expression system and evaluated their efficacy as vaccines in ferrets. Immunization twice with 3.75-microg doses of these antigens elicited strong antibody responses, which were adjuvant dependent. Interestingly, coadministration of both antigens strongly enhanced the HA-specific but not the NA-specific responses. Distinct patterns of protection were observed upon challenge inoculation with the homologous H1N1 virus. Whereas vaccination with sHA(3) dramatically reduced virus replication (e.g., by lowering pulmonary titers by about 5 log(10) units), immunization with sNA(4) markedly decreased the clinical effects of infection, such as body weight loss and lung pathology. Clearly, optimal protection was achieved by the combination of the two antigens. Our observations demonstrate the great vaccine potential of multimeric HA and NA ectodomains, as these can be easily, rapidly, flexibly, and safely produced in high quantities. In particular, our study underscores the underrated importance of NA in influenza vaccination, which we found to profoundly and specifically contribute to protection by HA. Its inclusion in a vaccine is likely to reduce the HA dose required and to broaden the protective immunity.

FIG. 1.

Design and expression of soluble, multimeric HA (sHA) and NA (sNA) proteins of 2009 A(H1N1) influenza virus. (A) Schematic representation of the recombinantly expressed sHA and sNA protein constructs. For sHA, the HA ectodomain (aa 17 to 522) is expressed with an N-terminal CD5 signal peptide and a C-terminal trimerization (GCN4-pII) GCN4 domain and Strep-Tag (ST). For sNA, the NA head domain (aa 75 to 469) is expressed with an N-terminal CD5 signal peptide, a OneSTrEP (OS) peptide, and a tetramerization (GCN4-pLI) GCN4 domain. (B) Coomassie blue stained reducing SDS-PAGE of affinity-purified sHA and sNA proteins.

FIG. 2.

Antibody response to vaccination with multimeric 2009 A(H1N1) influenza virus HA and NA antigens. Ferrets were immunized on day 0 and day 20 with 3.75 μg sHA₃ + 3.75 μg sNA₄ (sHA+sNA), 3.75 μg sHA₃ in adjuvant (ISCOM Matrix M [IMM]) (sHA+IMM), 3.75 μg sNA₄ in adjuvant (sNA+IMM), 3.75 μg sHA₃ + 3.75 μg sNA₄ in adjuvant (sHA+sNA+IMM), PBS, or IMM, as indicated. The antibody response to the 2009 A(H1N1) influenza virus was evaluated by hemagglutination inhibition (HI) (upper panel), virus neutralization (VN) (second panel), and neuraminidase inhibition (NI) assays (lower two panels). Each dot represents the result for one ferret. Horizontal lines represent means. The horizontal gray bar indicates the detection limit of the assay.

FIG. 3.

Clinical effects after challenge inoculation with 2009 A(H1N1) influenza virus. Ferrets immunized as described in the legend to Fig. 2 were inoculated intratracheally on day 52 with 10⁶ TCID₅₀ of virus. Body weight losses are expressed as percentage of body weight before infection (upper panel). Lung weights are expressed as percentage of body weight, as an indicator of lung consolidation (middle panel). Lungs were observed macroscopically and scored for lung area percentage displaying consolidated areas (bottom panel). Mean values are displayed; error bars indicate standard deviations. The horizontal gray bar indicates the detection limit of the assay.

FIG. 4.

Examples of histopathological findings in lungs of ferrets after inoculation. (A) Inflammatory infiltrates and loss of epithelial cells in the bronchiolar walls and cellular debris in the bronchiolar lumen observed in the lungs of unprotected ferrets mock vaccinated with PBS or adjuvant only (IMM) or vaccinated with the nonadjuvanted sHA₃+sNA₄. (B) Proteinaceous fluid (edema) and infiltrate of inflammatory cells in the alveoli of lungs of ferrets mock vaccinated with PBS or adjuvant only (IMM) or vaccinated with the nonadjuvanted sHA₃+sNA₄. (C) Peribronchiolar infiltrate and cellular debris in the bronchiole of a ferret vaccinated with sHA+IMM. (D) Inflammatory infiltrate in the alveolar septa and hypertrophy and hyperplasia of type II pneumocytes in lungs of ferrets vaccinated with sHA+IMM. (E) Peribronchiolar infiltrate observed in lungs of ferrets of the sNA+IMM and sHA+sNA+IMM groups. (F) Absence of inflammatory cells and hyperplasia of type II pneumocytes in alveoli of lungs of ferrets of the sNA+IMM and sHA+sNA+IMM groups. H&E staining was used. Magnifications, ×20 (bronchioles) and ×40 (alveoli).

FIG. 5.

Viral titers in lungs, noses, and throats of challenge-inoculated animals. Virus replication in the ferrets immunized and challenged as described in the legend to Fig. 3 was analyzed at 4 days after inoculation. Virus titers were determined in lung homogenates (upper panel), nose swaps (middle panel), and throat swaps (bottom panel). Titers were assayed by means of endpoint titration in MDCK cells. Each dot represents the result for one ferret. Horizontal lines represent means. The horizontal gray bar indicates the detection limit of the assay.

FIG. 6.

Induction of cross-neutralizing antibodies by vaccination with multimeric 2009 A(H1N1) influenza virus sHA₃ and sNA₄ antigens. (A) Sera of ferrets immunized twice with sHA₃ or sHA₃+sNA₄, both in adjuvant, as described in the legend to Fig. 2 were tested in an HI assay for activity toward different influenza viruses, including A/Swine/shope/1/56, A/Italy/1443/76, A/NL/386/86, A/Iowa/15/30, A/NL/25/80, A/New Jersey/8/76, A/PR/8/34, and IVR/148 influenza H1N1. Mean titers are displayed; error bars indicate standard deviations. (B) Sera of ferrets immunized once or twice with sNA₄ or sHA₃+sNA₄, both in adjuvant, were pooled and tested in a NI assay for activity against the sNA₄ of A/Kentucky/UR06-0258/2007(H1N1) and A/turkey/Turkey/1/2005(H5N1) influenza viruses. The NA of A/California/04/2009(H1N1) was used as a positive control. Positive-control sera specific for A/NL/602/09(H1N1) or A/turkey/Turkey/1/2005(H5N1) influenza virus were obtained from a ferret infected with these viruses. Average titers from two replicates are displayed; error bars indicate standard deviations. The horizontal gray bar indicates the detection limit of the assay.