Fig 1.
Enterovirus Capsid Conservation Across Various Species.
A. Phylogenetic tree generated using Geneious Prime software and the capsid protein multiple sequence alignment. The reference sequence was set to EV-D68 US/MO/18947 (in green) which was used to generate the molecular structure below. B. Molecular structure of Enterovirus D68 capsid pentamer (PDB: 6CRR) colored based on conservation score calculated by ChimeraX software using a multiple sequence alignment of 40 different enterovirus capsid sequences. Residues range from red to white to blue from least conserved to most conserved across the alignment as calculated by the AL2CO algorithm.
Fig 2.
Enterovirus and enterovirus-like-particle preparations contain contaminating host-cell proteins.
A-C. Transmission electron microscopy of purified EV-D68 VLPs (A), purified EV-A71 VLPs (B) and concentrated IPV particles (C). D. SDS-Polyacrylamide gel showing the protein content of enterovirus and VLP preps. The identity of each sample is listed above the lane. Bands representing VLP proteins or IPV proteins are highlighted by a black box.
Fig 3.
Immunization of mice with enterovirus VLPs or IPV fails to generate detectable cross-reactive binding antibody responses using a sandwich ELISA.
A. Study design showing immunization and blood sampling schedule along with immunization groups. B. Sandwich ELISA using an EV-D68 monoclonal antibody and EV-D68 VLP demonstrated that only animals immunized with EV-D68 VLP generated EV-D68 binding antibody. C. Sandwich ELISA using and EV-A71 monoclonal antibody and EV-A71 VLP demonstrated that only animals immunized with EV-A71 VLP generated EV-A71 binding antibody. D. Indirect Polio ELISA against all three poliovirus serotypes demonstrated that only animals that received IPV generated binding antibody. Data are shown as individual points with mean, error bars indicate standard deviation, n = 10 per group. There were no significant differences between groups (p<0.05) when compared by one-way ANOVA. Diagram in Fig 3A was created with BioRender.com.
Fig 4.
Homologous immunization is necessary to generate enterovirus neutralizing antibodies in mice.
Endpoint serum neutralization against EV-D68 (A) and EV-A71 (B) show that immunization with the corresponding VLP is required to generate neutralizing antibodies. We observed similar neutralization titers in groups that receive monovalent or bivalent EV-D68 and EV-A71 VLP formulations. EV-A71 neutralizing antibodies were only detected after a second dose of EV-A71 VLP in both the monovalent and bivalent groups. Data are shown as individual points with mean, error bars indicate standard deviation, n = 10 per group. There were no significant differences between groups (p<0.05) when compared by one-way ANOVA.
Fig 5.
Immunization of baboons with IPV fails to generate cross-reactive neutralizing enterovirus antibody responses.
A. Indirect ELISA against EV-D68 or EV-A71 VLP using serum from baboons before and after three doses of IPV. Data are shown as individual points with mean, error bars indicate standard deviation, n = 24. Mean titers were compared by paired t-test with p = 0.6677 for EV-D68 indirect ELISA and p = 0.0002 for EV-A71 indirect ELISA. B. Sandwich ELISA for EV-D68 or EV-A71 VLP using serum from baboons before and after three doses of IPV. Data are shown as individual points with mean, error bars indicate standard deviation, n = 24. C. EV-A71 endpoint neutralization assay using serum samples from baboons before and after IPV immunization. D. Some baboons possessed IPV binding antibodies before immunization, but all animals exhibited a robust boost response following IPV immunization. Mean titers were compared by paired t-test with p = <0.0001. Data are shown as individual points with mean, error bars indicate standard deviation, n = 24. Asterisks indicate p value with *** indicating p < 0.001 and **** indicating p < 0.0001, ns indicates a p value > 0.05 determined by paired t-test.