RESUMO
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.
RESUMO
In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.