title: Protocol for recombinant RBD-based SARS vaccines: protein preparation, animal vaccination and neutralization detection.
abstract: Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.
authors.affiliation: Lindsley F. Kimball Research Institute, New York Blood Center, USA.
authors.forename: Lanying
authors.initials: L
authors.lastname: Du
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Zhang
authors.forename: Jixiang
authors.initials: J
authors.lastname: Liu
authors.forename: Shibo
authors.initials: S
authors.lastname: Jiang
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology; D005260:Female; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology*; D064370:Spike Glycoprotein, Coronavirus; D014162:Transfection; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000097:blood / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014765:Viral Vaccines / Q000008:administration & dosage* / Q000097:blood / Q000235:genetics* / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus