A Novel Subnucleocapsid Nanoplatform for Mucosal Vaccination against Influenza Virus That Targets the Ectodomain of Matrix Protein 2

Abstract

In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.

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“…The native M2 is tetrameric while native HA2 is trimeric in the viral particles (48, 49). However, several reports, including work from our research groups, have shown that non-tetrameric M2e and non-trimeric HA2 AGs can also elicit antibodies that are protective against a viral challenge (8, 27, 28, 50). Importantly, the mechanism of protection induced by such non-native M2e and HA2 structures was shown to involve ADCC (9, 51) and not the classical neutralization by antibodies.…”

“…The entire coding sequence of the NP gene was PCR-amplified from the cDNA template using specific primers NPfor (5′- TAGCTAGCACTAGTAGTGACATCGAAGCCATGG- 3′) and NPrev (5′- TACTCGAGCTAGAATTCACTGTCATACTCCTCTGCAT- 3′). Codon-optimized “Strings™ DNA Fragments” (Thermo Fisher Scientific) synthetic genes were used to clone the genes encoding residues 76–130 of HA2 (RIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNA), or encoding three consecutive copies of the M2e (3M2e) AG (SLLTEVETPTRSEWE S R S SDSSDAAASLLTEVETPTRSEWESRSSDSSDAAASLLTEVETPTRSEWESRSSDSSDAAA) where cysteines 17 and 19 of the original sequence have been replaced by serine residues (underlined), to improve immunogenicity and avoid unwanted disulfide bounds in the chimera, based on previously published works (27, 28). The sequence-confirmed AG fused to streptavidin (SA–AG) fusion proteins were produced in Escherichia coli Bl21 λDE3 at 37°C following IPTG induction (0.5 mM).…”

“…The antigenicity of the SA–AG was analyzed by Western blot after under reducing conditions (Figure S1 in Supplementary Material). The M2e–nucleoprotein carrier from the respiratory syncytial virus was used as a positive control (28). After transfer onto nitrocellulose membrane, the blots were blocked in PBS 0.3% Tween 20 and 5% non-fat dry milk, and respectively, reacted with mAb anti-M2e (0.3 µg/ml), rabbit polyclonal IgG anti-NP (0.3 µg/ml), or anti-HA2 (5 µg/ml), followed by washing and decoration with HRP-conjugated anti-mouse IgG (1/10,000) or HRP-conjugated anti-rabbit IgG (1/10,000), respectively.…”

“…The working stocks of the recombinant viruses were prepared by two successive amplifications in MDCK cells at a multiplicity of infection (MOI) of 10 −3 for 3 days at 35°C in MEM. Virus titer was determined on MDCK cells using plaque assay procedure as previously described (28). The A/PARIS/2590/2009 (H1N1) viral production and titration was performed in the BSL3 facilities of the VIM-INRA laboratory in Jouy-en-Josas, France.…”

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“…Then, the virus in the allantoic fluid was inoculated into the MDCK (Madin-Darby canine kidney) cell line (ATCC CCL-34) for plaque assay [18]. Cells were cultured in DMEM supplemented with 10% fetal bovine serum.…”