Article Figures & Data
Figures
- FIG. 1.
Comparison of full-length S (OPT9) glycoprotein versus the deletion SΔ(422-463) mutant. (A) Western blot expression of the full-length codon-optimized (OPT9) S glycoprotein (left), mutant SΔ(422-463) (middle), and 293T cell lysate (right). (B) 293T cell surface staining of mutant SΔ(422-463) expression (left) and 293T cells (right). Expression was measured with a polyclonal antibody to the first 400 amino acids of the S glycoprotein at a 1:100 dilution. (C) Neutralization assay of mice sera immunized with two injections of OPT9 or the mutant SΔ(422-463). Mutant SΔ(422-463) failed to induce neutralization activity. (D) Entry assay of the OPT9 set at 100% efficiency, compared with the lack of entry with mutant SΔ(422-463) pseudotyped virus. Neg., negative.
- FIG. 2.
Construction of eight individual S glycoprotein point mutations and comparative expression. (A) Schematic diagram of the ACE2 receptor binding region with arrows indicating the residue outside the SΔ(422-463) deletion region. The eight positively charged residues are annotated (+) above each corresponding residue, and the individual mutations are shown below as a change to an alanine. (B) Similar levels of expression of each of the mutants (1 to 8), OPT9, and cell lysate (−) are detected in Western blots stained with a polyclonal antibody to the first 400 amino acids of the N terminus. WT, wild-type; Mut-, mutant.
- FIG. 3.
SARS-CoV-specific neutralization antibody response in BALB/c mice after EP DNA vaccinations. Each group (2 mice per group) was immunized with 20 μg of mutants (1 to 8) or OPT9 plasmid twice, 3 weeks apart. Serum was collected and diluted from 1:100 to 1:72,900 and measured for the ability to neutralize OPT9 pseudovirus. The average values are plotted.
- FIG. 4.
Detection of binding antibodies to S glycoprotein in vaccinated mice. Sera (1:50 dilution) of mice vaccinated with OPT9, mutant 3, 8, or SΔ(422-463) were used to stain 293T cells transiently transfected with OPT9 plasmid. Binding antibodies were present in each vaccinated mouse. Neg., negative.
- FIG. 5.
Induction of SARS-CoV neutralizing antibodies with OPT9 in mice prevaccinated with mutant 3 (R441A). Nonresponsive mice immunized with mutant 3 were subsequently immunized with two EP injections of OPT9, 3 weeks apart. Serum was collected after each injection (1st, 2nd) and analyzed for the ability to neutralize OPT9 pseudovirus at dilutions of 1:10 to 1:7,290. The average NAb titer of two mice is presented.
- FIG. 6.
Functional analysis of mutant pseudoviruses. Ten nanograms (measured by p24) of each pseudovirus was used to infect HEK293T-ACE2 cells. Luciferase activity was measured 72 h postinfection and normalized with OPT9 infection (100%). Five replicates were tested in each experiment. The average values and standard errors are presented. This experiment was repeated three times.
- FIG. 7.
Susceptibility of mutant pseudoviruses to neutralization with sera derived from immunized mice and human convalescent-phase sera. BALB/c mice vaccinated twice with OPT9 (DD131) or ADS-MVA (MM3) were analyzed for the ability to neutralize the functional mutants (1, 2, 4, 5, 6, and 7) and OPT9 pseudoviruses. Serum dilutions ranged from 1:600 to 1:48,600. Human convalescent-phase sera from patients exposed to SARS-CoV (patients numbered 14 and 48) were analyzed for the ability to neutralize the functional mutants (1, 2, 4, 5, 6, and 7) and OPT9 pseudoviruses. Serum dilutions ranged from 1:300 to 1:24,300.
