Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever

Abstract

Background: Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable.

Methodology/principal findings: A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5) TCID(50). Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles.

Conclusions/significance: The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.

Figure 1. Plasmid transfer vectors (A, C) and genome structures of MVA-YF (B) and dVV-YF (D).

The plasmid vector pd3-lacZ-mH5-YFprMEco (A) targets the deletion III insertion site in the MVA genome. To obtain recombinant virus (B) without any auxiliary sequences, the transient lacZ/gpt screening marker in the plasmid is flanked by a 220 bp self repeat (R) of one of the MVA flanks that mediates removal of the marker cassette by homologous recombination. The insertion site for the plasmid vector pDW-mH5-YFprMEco (C) is the region between the ORFs D3R and D5R in the wild-type Lister/Elstree virus. The lacZ/gpt marker cassette is located between tandem DNA repeats (R, hatched boxes) to achieve eventual removal of the marker cassette. The resulting recombinant defective virus (D) lacks the uracil DNA glycosylase gene (D4R), and still contains one tandem repeat . Both plasmids contain the human codon-optimized YFV prM and E coding region under the control of the early/late vaccinia virus mH5 promoter.

Figure 2. Double Immunostaining of infected chicken cells (DF-1).

MVA-YF (A), wild-type MVA (B) and MVA-YF/MVA spike control (C). After 4 days infected cells were fixed, incubated with guinea pig anti YFV-17D antiserum and anti-guinea pig IgG conjugated to peroxidase. Expressors of prME were visualized as black plaques staining with DAB solution with nickel. To detect MVA without prME expression, cells were incubated with rabbit-anti-vaccinia virus serum and anti-rabbit peroxidase-conjugated IgG antibody and subsequent staining with DAB solution without nickel, resulting in brown plaques (prME non-expressors).

Figure 3. YFV prME protein expression under permissive (A and B) and non-permissive (C and D) conditions.

(A) Western blot of lysates from chicken cells (DF-1) infected with MVA-YF or the corresponding controls. MVA-YF (Lane 1), wild-type MVA (Lane 2), non-infected DF-1 cells (Lane 3), positive control YFV-17D infected DF-1 cells (17D, Lane 4), YFV-17D prepared from infected HeLa cells (17D control, Lane 5). (B) Western blot of lysates from cVero22 cells infected with dVV-YF or the corresponding controls. dVV-YF (Lane 1), wild-type dVV (Lane 2), non-infected cVero22 cells (Lane 3), positive control YFV-17D infected cVero22 (17D, Lane 4), 17D control (Lane 5). Western blot of mouse muscle cells (Sol8, C) or human cells (HeLa, D) infected with the recombinants or the corresponding controls. MVA-YF (Lane 1), dVV-YF (Lane 2), wild-type MVA (Lane 3), wild-type dVV (Lane 4), non-infected Sol8 (C) HeLa (D) cells (Lane 5), cells infected with YFV-17D (17D, Lane 6), 17D control (Lane 7). The band around 55 kDa marked “E” indicates the YFV envelope protein.

Figure 4. Protection studies in Balb/c mice.

Animals were vaccinated i.m. in a single dose scheme with the indicated doses of (A) MVA-YF, (B) dVV-YF or with (C) the positive control YFV-17D (17D) and the negative controls wild-type MVA, defective vaccinia virus (dVV) or buffer (PBS). Mice were challenged i.c. 21 days later with 1×10⁵ TCID₅₀ of YFV-17D vaccine strain and monitored for 14 days. Results are the average of 3 individual experiments.

Figure 5. Cellular immune response elicited against YFV E-antigen.

(A) FACS analysis of the number of IFN-γ secreting CD4⁺ T-cells after two immunizations with MVA-YF, dVV-YF or the corresponding YFV-17D (17D) positive or wild-type MVA and dVV negative controls. Splenocytes from mice were stimulated with 15mer peptides of the YFV E-protein, E57–71 (E4; black bars), E129–143 (E5; grey bars) and E133–147 (E6; white bars). (B) FACS analysis of the number of IFN-γ secreting CD8⁺ T cells after the two immunizations as indicated above. Splenocytes from mice were stimulated with 9-mer peptides of the YFV E-protein, E60–68 (E1; black bars), E330–338 (E2; grey bars), E332–340 (E3 white bars); ** p<0.001. (C) FACS analysis of cytotoxic killing of peptide-pulsed target cells by specific CD8⁺ T cells. Target cells were loaded with 9mer peptides of the YFV E-protein, E60–68 (E1; black bars), E330–338 (E2; grey bars), E332–340 (E3 white bars). The data are mean values (+/− SD) of two independent experiments.

Figure 6. Safety of recombinant candidate vaccines in BALB/c mice.

(A) Animals were injected i.c. with 1×10⁵ to 1×10⁷ TCID₅₀ (only 1×10⁷ TCID₅₀ dose shown) of MVA-YF (bright grey line), dVV-YF (grey line) and the corresponding controls wild-type MVA (dotted line) and dVV (black line) and monitored for 21 days. (B) Mice were injected i.c. with YFV-17D vaccine at doses of 1×10¹ (bright grey line), 1×10² (grey line) or 1×10³ (dotted line), and monitored for 21 days.