Research ArticleEMERGING INFECTIONS

A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates

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Science Translational Medicine  15 May 2019:
Vol. 11, Issue 492, eaav3113
DOI: 10.1126/scitranslmed.aav3113
  • Fig. 1 Characterization of WEEV, EEEV, and VEEV VLP vaccines.

    (A) Schematic representation of the alphavirus genome and WEEV, EEEV, and VEEV VLP expression vectors. The alphavirus genome consists of the following genes: nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and structural proteins capsid (C); envelope glycoproteins E3, E2, and E1; and the 6K protein. The NLS sequences in WEEV, EEEV, VEEV, and CHIKV are shown under the schematic representation. Expression of wild-type (WT) and NLS mutant VLPs in normal (−), basic pH buffers (+) (bottom). The expression was analyzed by Western blot using mouse sera against WEEV, EEEV, or VEEV. (B) Buoyant density gradient analysis of NLS mutant VLPs using OptiPrep. Each vector was transfected into 293F cells, and supernatants were collected after 4 days. VLPs were purified by density gradient centrifugation. (C) Surface rendering of three-dimensional (3D) cryogenic electron microscopy maps. The triangle in the central panel corresponds to the icosahedral symmetry unit, and the numbers mark the positions of the two-, three-, and fivefold symmetry axes. Scale bar, 10 nm.

  • Fig. 2 Monovalent and trivalent VLPs protected against matched WEEV, VEEV, or VEEV challenges in mice.

    BALB/c mice (n = 5 per group) were immunized intramuscularly twice with 5 μg of monovalent WEEV, EEEV, or VEEV VLPs or with 15 μg of trivalent VLPs (5 μg each of WEEV, EEEV, and VEEV VLPs) at a 3-week interval. (A) Vaccination, neutralization assay, and challenge time course. (B and C) Neutralization titers of sera from the immunized mice were evaluated by WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporter neutralization assays (B) and PRNT (C). Data are representative of at least two independent experiments. Each data point represents the means ± SEM of the values from five animals per group. Overall significance of multigroups was evaluated by Kruskal-Wallis analysis of variance (ANOVA). LOD, limit of detection. (D) Mice immunized with the indicated monovalent WEEV, EEEV, or VEEV VLPs or trivalent VLPs were challenged at week 8 with aerosolized WEEV CBA87, and Kaplan-Meier survival curves are shown over time after the challenge. (E) Mice immunized with trivalent VLPs (n = 5 per group) were challenged with aerosolized virus. Kaplan-Meier survival curves are shown after lethal WEEV CBA87, EEEV FL93-939, or VEEV Trinidad challenge.

  • Fig. 3 Monovalent VLP vaccine induced robust NAb responses and conferred complete protection against homologous virus challenge in NHPs.

    Cynomolgus macaques (WEEV VLPs, n = 3; EEEV VLPs, n = 4) were immunized intramuscularly twice with 20 μg of monovalent WEEV or EEEV VLPs at a 4-week interval. (A) Vaccination, neutralization assay, and challenge time course. (B and C) Neutralization titers of sera from the immunized macaques were evaluated by WEEV and EEEV Env-pseudotyped lentiviral reporter neutralization assay (B) and PRNT (C). Data are representative of at least two independent experiments. Each data point represents the means ± SEM of the values from three to four animals per group. Overall significance at multiple time points was evaluated by Friedman ANOVA. *P < 0.05, Mann-Whitney test. (D) The immunized macaques were aerosol challenged with homologous WEEV Fleming (left) or EEEV FL93-939 (right) with which they were immunized, and Kaplan-Meier survival curves are shown over time after the challenges.

  • Fig. 4 The trivalent VLP vaccine protected against virus challenge in NHPs and IgG from immunized NHPs protected against EEV challenge in mice.

    Cynomolgus macaques (n = 8 per challenge and n = 24 in total) were immunized intramuscularly twice with 60 μg of trivalent VLPs (20 μg each of WEEV, EEEV, and VEEV VLPs) vaccine at a 4-week interval. (A) Vaccination, neutralization data, and challenge time course. (B and C) NAb responses were evaluated against WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporter viruses (B) and PRNT (C). Data are representative of at least two independent experiments. Each data point represents the means ± SEM of the values from eight animals per group. Overall significance at multiple time points was evaluated by Friedman ANOVA. *P < 0.05, Mann-Whitney test. (D and E) Immunized monkeys were challenged separately with aerosolized WEEV, EEEV, or VEEV (n = 8 per challenge virus group) at 4 weeks after boost. Blood viremia was evaluated after VEEV Trinidad donkey challenge. P values were calculated by Fisher’s exact test (D). Kaplan-Mayer survival curves are shown over time after WEEV Fleming (left) and EEEV FL93-939 (right) challenges. P values were calculated by log-rank test (E).

  • Fig. 5 The trivalent VLP vaccine protected against clinical sequalae of viral challenge in NHPs.

    Cynomolgus macaques (n = 24) were immunized intramuscularly twice with 60 μg of trivalent VLPs (20 μg each of WEEV, EEEV, and VEEV VLPs) at 4-week intervals. Immunized monkeys were challenged separately with aerosolized WEEV, EEEV, or VEEV at 4 weeks after boost (n = 8 or 9 per each challenge in test or control group, respectively). PBS was administered to control monkeys. (A) Hallmarks of human disease including number of days of lymphopenia (left) and fever duration (right) were evaluated after VEEV Trinidad donkey challenge. (B) The days of lymphopenia (left) and fever duration (right) are shown after WEEV Fleming challenge. (C) The blood viremia (left) and fever duration (right) analyses are shown after EEEV FL93-939 challenge. P values were calculated by unpaired two-tailed Student’s t test. For lymphopenia and fever analyses, survivors in the control group are designated as circles, whereas nonsurvivors are designated as squares.

  • Fig. 6 IgG from immunized NHPs is sufficient to protect against EEV challenge in mice.

    IgG was purified from the pooled serum of trivalent VLP–immunized or naïve cynomolgus macaques using Protein A. BALB/c mice (n = 10 mice per challenge and n = 30 in total) were administered 2 mg of IgG by intraperitoneal injection twice on days −1 and +3. The mice were exposed separately by aerosol to WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey on day 0. Kaplan-Meier survival curves are shown. P values were calculated by log-rank test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/492/eaav3113/DC1

    Materials and Methods

    Fig. S1. Alignment and similarity of Env amino acid sequences of WEEV, EEEV, and VEEV.

    Fig. S2. WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporters have similar luciferase activity.

    Fig. S3. Transmission electron microscopy and cryogenic electron microscopy images of monovalent VLPs.

    Fig. S4. Monovalent VLP vaccine conferred complete protection against homologous virus challenge in cynomolgus macaques.

    Fig. S5. The trivalent VLP vaccine protected against virus challenge and eliminated clinically significant neuropathology and persistent viral infection in NHPs.

    Table S1. Observation of clinical signs and viremia after WEEV challenge in mice.

    Data file S1. Primary data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Alignment and similarity of Env amino acid sequences of WEEV, EEEV, and VEEV.
    • Fig. S2. WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporters have similar luciferase activity.
    • Fig. S3. Transmission electron microscopy and cryogenic electron microscopy images of monovalent VLPs.
    • Fig. S4. Monovalent VLP vaccine conferred complete protection against homologous virus challenge in cynomolgus macaques.
    • Fig. S5. The trivalent VLP vaccine protected against virus challenge and eliminated clinically significant neuropathology and persistent viral infection in NHPs.
    • Table S1. Observation of clinical signs and viremia after WEEV challenge in mice.

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    Other Supplementary Material for this manuscript includes the following:

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