Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase

Abstract

Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA. IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

Broadly Protective Strategies Against Influenza Viruses: Universal Vaccines and Therapeutics

“…An important challenge in inducing NA directed immune responses involves overcoming the immunodominance of HA over NA. In an attempt to subvert the HA immunodominance, a recent study generated two recombinant influenza viruses based on the H1N1 stain A/Puerto Rico/8/1934 (PR8) in which the NA stalk domain was extended by 15-30 amino acids (Broecker et al, 2019b). Using formalin-inactivated viruses expressing wildtype NA or the extended NA, the authors demonstrated that the extended NA stalk induced higher anti-NA IgG responses than the unmodified NA in mice (Broecker et al, 2019b).…”

“…In an attempt to subvert the HA immunodominance, a recent study generated two recombinant influenza viruses based on the H1N1 stain A/Puerto Rico/8/1934 (PR8) in which the NA stalk domain was extended by 15-30 amino acids (Broecker et al, 2019b). Using formalin-inactivated viruses expressing wildtype NA or the extended NA, the authors demonstrated that the extended NA stalk induced higher anti-NA IgG responses than the unmodified NA in mice (Broecker et al, 2019b). Similarly, extension of the NA stalk from H3N2 virus increased NA-specific antibody responses (Broecker et al, 2019b).…”

“…Using formalin-inactivated viruses expressing wildtype NA or the extended NA, the authors demonstrated that the extended NA stalk induced higher anti-NA IgG responses than the unmodified NA in mice (Broecker et al, 2019b). Similarly, extension of the NA stalk from H3N2 virus increased NA-specific antibody responses (Broecker et al, 2019b). While additional challenge studies need to be performed, the NA stalk extension approach offers an interesting solution to improving the immunogenicity of NA.…”

Host immune response–inspired development of the influenza vaccine

“…Therefore, many groups are working to increase the immunogenicity of NA through development of new vaccine formulations and strategies. [31][32][33] Similarly, antibodies that target the conserved ectodomain of the influenza matrix 2 protein (M2e) were protective in preclinical and clinical settings. 34 Because of its abundance on the surface of the virus, HA has been the most attractive vaccine target.…”

Structure-based design of stabilized recombinant influenza neuraminidase tetramers

“…The antigenicity of NA has long been detailed using monoclonal antibodies (mAbs) isolated from immunized and/or infected mice, which provided an initial understanding of its distinct antigenic regions-some of which span adjacent protomers-as well as mechanisms of both enzymatic inhibition and protection [21][22][23][24][25] . Although an NA-based vaccine, extracted from viral membranes and inactivated by formalin, improved humoral NA-directed inhibition compared to commercial vaccines in humans in a Phase I clinical trial more than 25 years ago 26,27 , only preclinical studies of NA-based vaccines have been reported since [28][29][30][31][32] . More recently, studies comparing antibodies elicited by infection or immunization with split virus vaccines in humans as well as mice have clearly demonstrated that infection results in substantially more robust NA-directed humoral responses 33 .…”