EV-D68 virus-like particle vaccines elicit cross-clade neutralizing antibodies that inhibit infection and block dissemination

Figures

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  Fig. 1.. B1 EV-D68 VLPs are antigenically similar to inactivated B1 EV-D68 viral particles. (A) Plasmid constructs expressing P1 and 3CD polypeptides based on the sequences from the EV-D68 US/CO/2014-93 isolate for VLP production. (B) Protein gel of EV-D68–purified VLP (VP0, VP1, and VP3) and β-propiolactone–inactivated EV-D68 US/MO/2014-18947 viral particles (InVP; VP1, VP2, and VP3). M, protein marker. (C) Western blot (WB) probed with a commercial anti–EV-D68 VP1 (α-VP1) monoclonal antibody. (D) EV-D68 monoclonal antibody detection of VLP and InVP antigens by direct ELISA. Open symbols, plates coated with 100 ng of VLP; closed symbols, plates coated with 100 ng of InVP. OD450, optical density at 450 nm. Human monoclonal antibodies (mAb) 228 and 219 were a gift from J. Crowe at Vanderbilt University and are described in (16). (E and F) Negative-stain electron microscopy of EV-D68 VLP (E) and InVP (F). Scale bars, 20 nm.

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  Fig. 2.. B1 VLP and B1 InVP elicit potent neutralizing antibody that blocks EV-D68 in cell culture and abrogates lung replication and dissemination in vivo. (A) Vaccination schema. Serum was obtained on weeks 2, 6, and 12. IM, intramuscularly. (B and C) Neutralization of EV-D68 US/MO/2014-18947 at week 2 (B) or week 6 (C). Dotted lines indicate limits of detection; error bars indicate ±SD. Ordinary one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was used to compare neutralizing titers at each time point. Light blue and dark blue asterisks indicate comparison of the VLP to 0.5 μg of InVP and 2.0 μg of InVP, respectively. (D) Antibody transfer study schema. IgG from VLP- or InVP-vaccinated mice at week 12 were purified and injected intraperitoneally (IP) into AG129 mice at the indicated concentration (n = 3 to 5 mice per group). Serum was taken to confirm the transfer of neutralizing antibodies. IN, intranasally. (E) B1 subclade EV-D68 US/MO/2014-18947 end point neutralization titers before virus challenge. Dotted lines indicate limits of detection; error bars indicate ±SD. Mice were challenged intranasally with 104.4 median tissue culture infectious dose…

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  Fig. 3.. B1 VLP and InVP vaccination elicited antibodies with reduced capacity to neutralize heterologous EV-D68 strains in cell culture and require a higher transfer dose to abrogate virus replication in vivo. (A) Cross-subclade neutralization of EV-D68 with serum antibodies elicited by B1 subclade immunogens. Week 6 serum samples from mice vaccinated with the indicated B1 immunogen (described in Fig. 2A) were used to neutralize the indicated EV-D68 viruses. n = 5 to 10 mice per group. Each serum sample was tested in duplicate in two assays. Dotted lines indicate the limits of detection in the assay; error bars indicate ±SD. (B) Passive transfer schema. AG129 mice were administered the indicated antibody at the indicated dose via intraperitoneal injection. Eighteen hours later, serum was taken to confirm the transfer of neutralizing antibodies against B3 subclade EV-D68. Four hours later, the mice were challenged intranasally with 104.2 TCID50 B3 subclade mouse adapted EV-D68 Mp9. Two days postinfection, lungs, spleen, and serum were taken for assessment of viral load. (C) B3 subclade EV-D68 US/2018-23087 pre-challenge endpoint neutralization titers. Dotted lines indicate upper and lower…

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  Fig. 4.. B3 EV-D68 VLP vaccine formulations elicit robust and durable neutralizing antibody. (A) B3 VLP vaccination schema. Ten mice per group were vaccinated and boosted intramuscularly with the indicated formulation of adjuvant and B3 VLP. Sera were sampled 2, 6, and 18 weeks after prime vaccination (−2, 2, and 14 weeks after boost vaccination). (B) 20% Adjuplex-formulated B3 VLP stimulates a balanced TH response, indicated by the ratio of IgG1 to IgG2a determined by an IgG subclass–specific ELISA. Fourteen days after boost sera were used to bind to EV-D68 VLP coated on ELISA plates and reacted with anti-IgG1 or anti-IgG2a secondary antibodies as described in Materials and Methods. Samples with undetectable IgG2a were omitted from the comparison; see fig. S4 for details. (C) Magnitude and durability of neutralizing antibody elicited from B3 VLP vaccine formulations. The indicated sera were used to neutralize EV-D68 US/2018-23087. Serum samples were tested in duplicate in two individual assays. At each time point, ANOVA with Tukey’s multiple comparisons test was used to compare the significance between the neutralizing titers elicited from each B3 VLP formulation. Horizontal dotted lines…

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  Fig. 5.. B3 VLP Adjuplex formulations elicit neutralizing antibody that blocks B3 virus replication and dissemination in vivo. (A) Passive transfer schema. Week 18 sera from mice vaccinated with 2 and 20% Adjuplex/B3 VLP formulations were pooled, and the IgG was purified. AG129 (n = 5) mice were administered IgG at the indicated dose via intraperitoneal injection. Serum was obtained to confirm antibody transfer and then the mice were challenged with 104.5 TCID50 B3 EV-D68 Mp20. (B) Pre-challenge serum neutralization titers against EV-D68 B3 2018-23087. The dotted line indicates the limit of detection; error bars indicate ±SD. Red symbols represent mice that were omitted from the analysis because of very low neutralization titer in the pre-challenge sera. (C) Dose-dependent inhibition of EV-D68 replication in mouse lung. ANOVA with Dunnett’s multiple comparisons test was used to compare the virus recovered from lung in each group compared to the naïve IgG negative control group. The green dotted line indicates the limit of virus detection in the lung; error bars indicate ±SD. (D) Pearson correlation demonstrates that serum α–EV-D68 VLP antibody titer is indirectly proportional to virus…

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  Fig. 6.. Cross-clade neutralizing antibody elicited from B3 VLP vaccination in mice and NHPs. (A) Cross-neutralization of EV-D68 viruses with mouse sera raised against B3 VLP formulated with 2 or 20% Adjuplex from the experiment described in Fig. 4A. Sera were used to neutralize the following viruses: B3 USA/2018-23087 (blue boxes), B1 US/MO/2014-18947 (black boxes), and A2 US/KY/2014-18953 (red boxes). Dashed lines indicate the upper and lower limits of detection; error bars indicate ±SD. (B) NHP vaccination schema. Two rhesus macaques were primed (week 0) and boosted (week 4) with 50 μg of B3 VLP formulated with 20% Adjuplex. Serum samples were acquired on the indicated weeks after prime vaccination and used to neutralize the homologous B3 subclade USA/2018-23087 virus (C) (blue), the B1 subclade heterologous US/MO/2014-18947 virus (D) (black), and the A2 subclade heterologous US/KY/2014-18953 virus (E) (red) as described in Materials and Methods. Each point represents the mean neutralization titer from two assays, and error bars reflect the range of the individual replicates. Blue asterisks on horizontal axes indicate the dates of vaccination.