Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guérin

Abstract

The development of novel vaccine strategies to replace or supplement bacille Calmette-Guérin (BCG) is urgently required. Here we study, in cattle, the use of heterologous prime-boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A). Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5). Vaccine-induced levels of cellular immunity were assessed by determining interferon-gamma (IFN-gamma) responses in vitro. Prime-boost protocols, using recombinant MVA and BCG in combination (groups 1-3), resulted in significantly higher frequencies of Ag85-specific IFN-gamma-secreting cells than the two viral vectors used in combination (P=0.0055), or BCG used alone (groups 2 and 3, P=0.04). The T-cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization. In a second experiment, the effects of BCG\MVA85A heterologous prime-boost vaccination were compared with BCG\BCG homologous revaccination. The results suggested a higher Ag85A-specific response with a wider T-cell repertoire in the MVA85A-boosted calves than in the BCG\BCG-vaccinated calves. In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime-boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis.

Figure 1

Interferon-γ (IFN-γ) responses after heterologous prime/boost vaccination. (a) Animals were vaccinated with Modified vaccinia virus Ankara (MVA85A) followed by boosting with bacille Calmette–Guérin (BCG) (group 1).(b)Calves were vaccinated with BCG followed by boosting with MVA85A (group 2).(c)Calves were vaccinated with BCG followed by boosting with attenuated fowlpox strain FP9 (FP85A) and MVA85A (group 3).(d)Calves were vaccinated with MVA85A followed by boosting with MVA85A and FP85A (group 4).(e)Calves were vaccinated with FP85A followed by boosting with FP85A and MVA85A (group 5). Ag85-specific IFN-γ-secreting spot-forming cells (SFC) were determined by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) at the time-points indicated. Data of individual calves are expressed as SFC/10⁶ peripheral blood mononuclear cells (PBMCs) (filled symbols); open diamonds connected by a line represent the median values. Arrows indicate time of vaccinations; B, BCG vaccination; M, MVA85A vaccination; F, FP85A vaccination.

Figure 2

Proportion of Ag85-specific responses within total purified protein derivative-B (PPD-B) responses. Three weeks after bacille Calmette–Guérin (BCG) priming (BCG1), and 3 weeks after MVA85A (a) (MVA) or BCG (b) (BCG2) boosting, Ag85-specific and PPD-B-specific interferon-γ (IFN-γ)-secreting cells were determined by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). Data are expressed as the ratio of spot-forming cells (SFC) stimulated with Ag85/SFC stimulated with PPD-B.

Figure 3

Frequencies of peptide-specific T cells after stimulation with Ag85A peptide. Peripheral blood mononuclear cells (PBMCs), prepared 3 weeks after boosting with MVA85A (BCG/MVA85A) or bacille Calmette–Guérin (BCG/BCG), were stimulated with synthetic peptides (15 µg/ml each), and interferon-γ (IFN-γ) enzyme-linked immunosorbent spot-forming cell assays (ELISPOTs) were performed. Results are presented as the sum of the number of cytokine-releasing cells per 2 × 10⁵ cells responding to peptides, after correcting for responses in medium-only control wells. *P = 0·05 (unpaired, one-way Mann–Whitney test).

Figure 4

CD8⁺ T cells producing interferon-γ (IFN-γ). The percentage of IFN-γ-producing CD8⁺ T cells was determined by intracellular cytokine staining and flow cytometry. Peripheral blood mononuclear cells (PBMCs), isolated 3 weeks postbooster vaccination, were stimulated with purified protein derivative-B (PPD-B) (10 µg/ml). Boxed area: CD8⁺ IFN-γ⁺ cells (percentage of total indicated above the box). (a) Result from a bacille Calmette–Guérin (BCG)/BCG-vaccinated animal. (b) Result from a BCG/MVA-vaccinated animal. CD8⁺ IFN-γ⁺ cells in two remaining BCG/BCG-vaccinated calves: 0 and 0·05%; remaining two BCG/MVA-vaccinated calves: 0 and 1·34% (data not shown).

Figure 5

Epitope specificity of the T-cell repertoire after vaccination with bacille Calmette–Guérin (BCG)/MVA and BCG/BCG. Short-term T-cell lines, established 3 weeks after vaccination with MVA85A or BCG, were prepared from all six calves and incubated in the presence of 20 μg/ml synthetic peptides and autologous CD4⁺ T-cell-depleted, mitomycin C-treated peripheral blood mononuclear cells (PBMCs) as a source of antigen-presenting cells (APCs). After 4 days of culture, proliferative responses were determined. Results are expressed as stimulation indices (SI) (counts per minute with peptide ÷ counts per minute of medium control). Positive result: SI > 3 (indicated by the horizontal line). Boxes indicate peptides to which T cells from at least two calves responded.