Introduction of Cysteines in the Stalk Domain of Recombinant Influenza Virus N1 Neuraminidase Enhances Protein Stability and Immunogenicity in Mice

Abstract

Influenza virus surface glycoproteins represent the main targets of the immune system during infection and vaccination. Current influenza virus vaccines rely mostly on the hemagglutinin, requiring a close match between the vaccine and circulating strains. Recently, the neuraminidase (NA) has become an attractive target; however low immunogenicity and stability in vaccine preparations remain an obstacles. Here, we took advantage of the hypervariable stalk domain of the NA to introduce cysteines at different positions and to produce more stable multimeric forms of the protein. We generated 11 N1 constructs and characterized the proteins by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and by testing their enzymatic activity and representation of antigenic epitopes. Moreover, we evaluated their potential to induce a protective immune response in vivo and characterized the polyclonal antibody responses of immunized mice. We observed that the introduction of cysteines at certain positions led to the formation of stable N1 dimers, which are capable of inducing a strong antibody response characterized by neuraminidase inhibiting activity and protection of mice from high dose viral challenge. Overall, our results provide evidence for the feasibility of introducing stalk modifications to enhance the stability and immunogenicity of NA-based recombinant antigens.

Influenza Neuraminidase Characteristics and Potential as a Vaccine Target

“…Cysteine mutations in the stalk led to more efficient dimer formation of recombinant N1, resulting in enhanced enzymatic activity and immune protection. The cysteine-stabilized dimers were however still outperformed by a VASP-stabilized tetrameric NA (148). It was only recently recognized that recombinant NA proteins can adopt an open conformation in addition to the closed conformation that is found on the surface of influenza virions.…”

“…Mutations in the NA stalk ( 148 ) or the interface between protomers ( 149 ) can also enhance the stability and immunogenicity of recombinant NA. Cysteine mutations in the stalk led to more efficient dimer formation of recombinant N1, resulting in enhanced enzymatic activity and immune protection.…”

A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model

“…NA tetramerization is usually mediated by the transmembrane domain, disulfide bond formation in the variable stalk domain, and interprotomer interactions in the head domain ( 40 , 41 ). Importantly, multimerization of NA has been shown to be required for induction of protective immune responses ( 25 , 42 ).…”

Recent Progress in Recombinant Influenza Vaccine Development Toward Heterosubtypic Immune Response

“…Crucial to the formation of tetrameric structures are cysteine residues along the NA stalk. The introduction of cysteines along the NA stalk were found to generate recombinant NAs with enhanced enzymatic activities, protection of mice from weight loss and mortality, and higher NA-inhibiting antibody titers in mice ( 206 ). NA dose dependency in recombinant protein, DNA, or VLP vaccines has also been demonstrated, wherein higher concentrations relate to increased NA inhibition titers, antibody-secreting B cells, IgG titers, and survival rates ( 109 , 207 ).…”