A recombinant Modified Vaccinia virus Ankara expressing prME of tick-borne encephalitis virus affords mice full protection against TBEV infection

Abstract

Introduction: Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause a serious disease involving the central nervous system (tick-borne encephalitis, TBE). Although approved inactivated vaccines are available, the number of TBE cases is rising, and breakthrough infections in fully vaccinated subjects have been reported in recent years.

Methods: In the present study, we generated and characterized a recombinant Modified Vaccinia virus Ankara (MVA) for the delivery of the pre-membrane (prM) and envelope (E) proteins of TBEV (MVA-prME).

Results: MVA-prME was tested in mice in comparison with a licensed vaccine FSME-IMMUN® and proved to be highly immunogenic and afforded full protection against challenge infection with TBEV.

Discussion: Our data indicate that MVA-prME holds promise as an improved next-generation vaccine for the prevention of TBE.

Keywords: FSME-IMMUN®; MVA; T cells; TBEV; protection; vaccination; virus-neutralizing antibodies.

Figure 1

Generation of MVA-prME and in vitro characterization. (A) Homologous recombination within deletion site III of MVA-GFP and pIIIsynIIred-TBEV prME as well as intragenomic homologous recombination (marker gene deletion) generated recombinant MVA expressing prME of TBEV (MVA-prME). Created with BioRender.com. (B) Separation of DNA on 1% agarose TBE gel amplified by PCRs targeting the six major deletion sites of MVA (I: 291 bp, II: 354 bp, III: 447 bp, IV: 502 bp, V: 603 bp, VI: 702 bp). Successful integration of prME in deletion site III was verified (III: 2,490 bp). (C) Expression of TBEV E protein demonstrated by Western blot on MVA-prME-infected HeLa cells (MOI 5, 24 hpi). For controls, anti-GAPDH and anti-D8 antibodies were used. (D) Immunostaining of MVA- or MVA-prME-infected HeLa cells (MOI 0.1, 24 hpi) stained against VACV D8 protein or TBEV E protein. Cells were non-permeabilized (surface) or treated with Triton X^®-100 (intracellular). Images were taken with ×20 objective.(E) Growth curves of MVA- (gray) or MVA-prME (black)-infected permissive primary CEF (dotted lines) or non-permissive HeLa (solid lines) cells (MOI 0.05).

Figure 2

Humoral and cellular immune response in vaccinated mice. (A) Virus-neutralizing titer (VNT₁₀₀) against TBEV Neudoerfl of murine sera samples obtained 56 days after prime immunization (samples of immunogenicity and protective efficacy study, n = 16). Samples with ≤40 VNT₁₀₀ (dotted line, lowest serum dilution) are considered negative. The FSME-IMMUN^®-vaccinated mouse that displayed signs of disease is highlighted with a diamond symbol. n.s., not significant (p>0.05). (B) Displayed are IFN-γ spot-forming cells (SFC) per one million splenocytes after background subtraction. Only significance between MVA-prME to other treatment groups is indicated (gray) and for E_1-255 versus E_245-496 of MVA-prME-vaccinated mice (black) (*p≤0.05, **p≤0.01, ****p≤0.0001). (C–G) Frequency of CD3⁺ subpopulations gated on CD4⁺IFN-γ⁺ (C), CD4⁺Granzyme B⁺ (D), CD4⁺CD69⁺ (E), CD8⁺IFN-γ⁺ (F), or CD8⁺Granzyme B⁺ (G) after background substraction. n.d., not determined. For all graphs, bars show the mean with standard deviation. Mice were either immunized with TBS (gray circle), MVA (non-filled triangle), FSME-IMMUN^® (non-filled blue circle), or MVA-prME (red triangle).

Figure 3

Body weight change and survival curves of vaccinated and challenge infected mice. (A–D) Percentage of body weight change compared with initial body weight on the day of challenge during the entire course of TBEV infection for PBS- (A), MVA- (B), FSME-IMMUN^®- (C), and MVA-prME-vaccinated (D) mice. (E) Kaplan–Meier curves showing the percentage of survival of mice vaccinated with PBS (gray), MVA (black), FSME-IMMUN^® (blue, dotted), or MVA-prME (red) (n = 6, ***p≤0.001). p = 0.0007: FSME-IMMUN^®/MVA-prME versus PBS, p = 0.0004: FSME-IMMUN^®/MVA-prME versus MVA.

Figure 4

Quantification of TBEV RNA copies in the periphery, CNS, and GIT of vaccinated and infected mice. Cleared organ homogenates collected on the day of sacrifice were tested for the presence of TBEV RNA by real-time RT-qPCR. Mice were either sacrificed at (A) 8 dpi or (B) stayed in the experiment until the study endpoint (16 dpi). The median values of the respective treatment groups are indicated in the graphs. Mice were immunized with PBS (gray circle), MVA (non-filled triangle), FSME-IMMUN^® (non-filled blue circle), or MVA-prME (red triangle).

Figure 5

Histological and immunohistochemical analyses of the cerebral cortex at 8 dpi. (A–C) H&E-stained sections of the cerebral cortex of TBEV-infected mice which were either treated with PBS (A) or vaccinated with FSME-IMMUN^® (B) or MVA-prME (C). (A) The cerebral cortex of the PBS-treated mouse displays cellular necrosis with karyorrhectic, karyolytic, and pyknotic cells (insert) and shrunken, hypereosinophilic, triangular-shaped necrotic neurons (arrows and insert) as well as inflammatory cell infiltrates in destructed vascular walls (necrotizing vasculitis; arrowheads) and the perivascular space (arrowheads). Microgliosis and hypertrophy of microglia/macrophages are present. (B, C) In FSME-IMMUN^®- (B) or MVA-prME-vaccinated (C) mice, no significant microscopic lesions within the cerebral cortex parenchyma are visible. (D–F) IHC for the TBEV E antigen of the cerebral cortex of TBEV-infected mice which were either treated with PBS (D) or vaccinated with FSME-IMMUN^® (E) or MVA-prME (F). (D) Immunohistochemically, a cytoplasmic TBEV immunoreactivity is present in multiple cells representing neurons of the cerebral cortex from a PBS-treated mouse. (E, F) The cerebral cortex of FSME-IMMUN^®- (E) and MVA-prME-vaccinated (F) mice lack immunoreactivity. Scale bars: 50 µM.

Figure 6

Histological and immunohistochemical analyses of the jejunum at 8 dpi. (A–C) H&E-stained sections of the jejunum of TBEV-infected mice which were either treated with PBS (A) or vaccinated with FSME-IMMUN^® (B) or MVA-prME (C). (A) Marked hypercellularity/inflammatory cell infiltrates in the submucosal plexus (asterisk) and, to a lesser extent, in the myenteric plexus (arrowhead) as well as cellular necrosis with karyorrhectic, karyolytic, and pyknotic cells (arrowhead) are present. (B, C) No significant microscopic lesions within the submucosal (asterisk) and myenteric plexus (arrowhead) are present in vaccinated mice. (D–F) IHC for TBEV E antigen of the jejunum of TBEV-infected mice which were either treated with PBS (D) or vaccinated with FSME-IMMUN^® (E) or MVA-prME (F). (D) Immunohistochemically, TBEV-positive neurons are observed in the myenteric plexus (arrowhead) but not in the submucosal plexus (asterisk). (E, F) No immunopositive cells are present in the submucosal (asterisk) or myenteric plexus (arrowhead). Band-like, not cell-associated, brownish discoloration in (E) represents a staining artifact due to the unspecific binding of the antibody to serum components. Scale bars: 20 µM.

Figure 7

Histological and immunohistochemical analyses of the cerebral cortex and jejunum at 16 dpi. (A, B, E, F) H&E-stained sections of the cerebral cortex (A, B) and jejunum (E, F) of TBEV-infected mice which were either vaccinated with FSME-IMMUN^® (A, E) or MVA-prME (B, F). (C, D, G, H) IHC for TBEV E antigen of the cerebral cortex (C, D) and jejunum (G, H) of TBEV-infected mice either vaccinated with FSME-IMMUN^® (C, G) or MVA-prME (D, H). (A, B) No significant microscopic lesions within the cerebral cortex parenchyma are visible. (C, D) TBEV immunoreactivity was absent within the parenchyma of the cerebral cortex. (E, F) The jejunum shows mild hypercellularity of the submucosal plexus (asterisk), while the myenteric plexus (arrowhead) reveals no significant findings. (G) No specific TBEV immunoreactivity is detectable in the submucosal (asterisk) or the myenteric plexus (arrowhead). (H) A single cell in the submucosal plexus (asterisk) is immunolabeled, while no immunoreactivity is present in the myenteric plexus (arrowhead). Scale bars: (A–D) 50 µM, (E–H): 20 µM.