Fig. 2.

Immunogenicity of triSpike. Sera collected at indicated time points from vaccinated mice or hamsters were analyzed for reactivity with triSpike. (A) Western Blot analysis of pooled sera from immunized mice with or without alum adjuvant. Sera were collected and used at 1/1000 dilution against S-FLAG that was separated under conditions that allowed simultaneous detection of mono-, di-, and trimers of S-protein. A SARS patient serum (SARS), a rabbit serum against S1 and M2 monoclonal antibody against the FLAG peptide was used as control. FLAG-tagged bacterial alkaline phosphatase (BAP-FLAG) was used to assess the presence of antibodies against the FLAG tag. Immune complexes were detected with HRP-conjugated goat anti-mouse, -human or -rabbit IgG polyclonal antibody. Sizes of molecular weight markers are indicated on the right. (B) Reactivity of immune sera from pooled immunized hamster with live BHK-21 cells expressing S-protein at the plasma membrane using FACS analysis. Values were expressed as mean ± standard deviations. (C) Effect of alum adjuvant on longevity of neutralizing response in mouse sera against SARS-CoV (determined on FRhK-4 cells). Values were expressed as mean ± standard deviations. (D) Dose effect of triSpike immunization with alum adjuvant on neutralizing response against SARS-CoV in hamsters (determined on FRhK-4 cells). Values were expressed as mean ± standard deviations. (E) Inhibition of S-protein binding to ACE2 by sera from immunized mice. S-protein coated beads were pre-incubated with sera prior to incubation with soluble ACE2 (sACE2) and detection of the receptor was performed with a polyclonal goat-anti human ACE2 antibody. BAP-FLAG coated beads were used as control.