Fig 1.
Development of POWV-SEV vaccine.
(A) A schematic representation of the POWV-SEV construct. (B) POWV antigenic protein expressions by SDS-PAGE using Western blot analyses. POWV-SEV vaccine construct- or pMV101(empty vector) -transfected 293T cell lysates (CL) were incubated with anti-Flavivirus group antigen antibody (1:500). (C) Immunofluorescence Analysis (IFA) of POWV-SEV expression in vitro. IFA analysis for POWV-SEV protein expression in vero CCL-81 cells. Fourty-eight hours after transfection, immunofluorescence labeling was performed by incubating transfected cells with the anti-Flavivirus group antigen antibody (1:500) followed by the addition of goat anti-mouse IgG-AF 488 antibody (1:4000) for fluorescence conjugation. Nuclear DNA is stained with DAPI.
Fig 2.
POWV-SEV vaccine induces functionally binding antibodies in mice.
(A) A POWV-SEV-immunization schedule of C57BL/6 mice over 21 days. (B) Evaluation of antigen-specific antibody responses following immunization of mice with POWV-SEV vaccine at day 21. C57BL/6 mice were immunized two times at day 0 and 14, where 25μg of POWV-SEV DNA or pMV101 empty vector was delivered intramuscularly (i.m.) using EP-enhanced delivery system. Sera were collected one week after the 2nd immunization at day 21. Half-log dilutions of day 21 immune sera from individual mice (n = 4) were evaluated for their binding capacity to a recombinant POWV-envelope protein at 1μg/ml concentration. The binding capacity of day 21 sera from pMV101-vaccinated mice were depicted as a single group called pMV101 (unfilled circles). (C) The endpoint titer of POWV-SEV-specific IgG post one and two immunizations. The antibody endpoint titer was defined as the highest dilution of a serum sample with OD values > (mean ±SD; n = 4) of vector vaccinated mice. Samples with a titer <50 were given an endpoint titer of 1. (D) Western blot analysis of POWV-SEV immunized murine sera. Pooled day 21 immune sera from the aforementioned experiment was used as a primary antibody to probe POWV-envelope protein (rPOWV-ENV in μg). An irrelevant recombinant gp120 protein (1.0μg) was used as a negative control. (E) Indirect immunofluorescence assay of POWV-SEV transfected Vero CCL-81 cells probed with POWV-SEV sera. POWV-SEV immune sera (day 21) obtained from vaccinated mice and pMV101-sera obtained from the vehicle-vaccinated group were diluted at 1:50 and placed on to slide chambers with POWV-SEV-transfected Vero CCL-81 cells. Further probing with goat anti-mouse IgG-AF-488 illuminates areas where POWV-envelop antigens are identified by POWV-SEV murine sera. DAPI nuclear staining is shown as blue. (F) Non-cross-reactivity of POWV-SEV immune sera against flavivirus envelope antigens-WNV, ZIKV, DENV 1–4, POWV. Day 21 POWV-SEV murine immune sera (1:50) is probed against flavivirus envelope antigens.
Fig 3.
POWV-SEV -induced immune sera are comparable to POWV convalescent patient serum.
Detection of IgG antibodies and IgG avidity in POWV- convalescent patient sera and POWV-SEV immunized sera. (A) Antibody mapping of sera from POWV- patients with a recent primary infection POWV with POWV-SEV envelop peptides spanning the entire length of the protein were evaluated specific IgG antibodies detected by ELISA. POWV-SEV pooled murine immune sera (day 21), pMV101 vector control-immunized sera and POWV convalescent patient sera were probed over 74 individual POWV envelope peptides (15-mers with 9 overlapping amino acid sequences). Assessment performed in duplicate. (B-C) IgG avidity in POWV-SEV immunized sera (n = 4) avidity compared to POWV convalescent sera. Antibody responses were assessed by ELISA.
Fig 4.
POWV-SEV vaccine elicits antigen-specific CD4+ and CD8+ T cell responses in mice.
(A-B) ELISpot analysis of splenocytes secreting IFN-γ in response to POWV-SEV immunizations measured in spot-forming units (SFUs) per 106 splenocytes. Harvested splenocytes of POWV-SEV vaccinated C57BL/6 mice (n = 5) were stimulated ex vivo with POWV envelope peptides spanning the entire length of the protein. (A) IFN-γ-secreting SFU per 106 stimulated with linear peptide pools or (B) matrix peptide pools. The immunodominant epitope matched with the H2-Db-restricted prediction of dominant epitope is highlighted in arrows. (C) Percent intracellular cytokine population for antigen-specific CD4+ and CD8+ T cells. Splenocytes that were stimulated with POWV-envelope peptides spanning the entire length of the protein were evaluated for CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α via flow cytometry. (D) Polyfunctionality of antigen-specific CD4+ and CD8+ T cells. Frequency of total CD4+ and CD8+ T cells expressing double- or triple- positive cytokines (IFN-γ, IL-2, and/or TNF-α) using Boolean gating strategy.
Fig 5.
Immunizations with POWV-SEV vaccine confer protective efficacy in mice.
C57BL/6 mice (seven per group) were immunized with POWV-SEV (blue circles) or mMV101 vector (red filled circles) and challenged with POWV virus. (A) The percent change from initial body weight, (B) Kaplan-Meier survival curve, (C) and organ viral loads (FFU/μg RNA) post POWV viral challenge are shown. (C) Powassan viral burden in organs of pMV101- or POWV-SEV vaccinated mice at endpoint or 14 days post infection, whichever came later. The average end point for POWV-challenged pMV101-control group was 8 dpi. POWV-challenged POWV-SEV group were sacrificed at day 14 post infection. (D) Kinetics of circulating peripheral blood viral load of pMV101 or POWV-SEV-vaccinated C57BL/6 mice upon challenge (n = 7) depicting average FFU/μg RNA of each cohort. The dotted line indicates the average survival in days for control pMV101 vaccinated mice upon challenge. Error bars (C) and (D) depict the standard error of the mean. Results were compared by using Student’s t test.