Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia

Abstract

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.

Fig. 1.

Effects of adjuvant on the resulting immune response generated from PA+spore (BclA, ExsFA and p5303) vaccinations. (a) Changes in the anti-PA IgG titres of each vaccine group during the 12-week vaccination (pre-challenge) period. The vaccines were delivered at weeks 0, 4 and 8. Sera were collected just prior to each vaccine dose. (b, c) Spore antibody ELISAs using plates coated with Ames spores (b) or ΔbclA spores (c) and sera from mice collected at week 12 post-vaccination/pre-challenge. Representative data are shown. Sera collected from mice vaccinated with a prime and boost (2×) or a prime followed by two boosts (3×) were analysed.

Fig. 2.

ELISA titrations of rabbit anti-spore IgGs specific for the three spore exosporium antigens BclA (▴), ExsFA (▪) and p5303 (•) were carried out on plates coated with wild-type (WT) Ames spores (a) or the BclA deletion mutant of Ames, ΔbclA (b). The negative control (○) was rabbit antibody specific for the unrelated vegetative protein CylI.

Fig. 3.

Survival results of mice vaccinated with PA alone (▪) or PA+spore antigens (▴) and challenged i.p. (a) or i.n. (b) with ΔAmes spores. All vaccines were given as two immunizations each separated by 4 weeks and all contained ~25 µg PA or 25 µg PA+10 µg BclA+10 µg ExsFA+10 µg p5303 and ~125 µg AL. Mice challenged by i.p. injection (a) received ~3×10⁵ spores delivered in 200 µl sterile water for injections (WFI) (representing ~15 LD₅₀ equivalents). Mice challenged through i.n. instillations (b) received ~5.2×10⁷ spores delivered in 40 µl WFI (representing ~15 LD₅₀ equivalents). The results from the i.p. challenge indicated that the addition of spore antigens to the vaccine afforded significant protection against ΔAmes spores (P = 0.0004 for the survival curve and P = 0.004 for TTD). The results after i.n. challenge were not statistically significant.

Fig. 4.

ELISAs to detect anti-PA and anti-spore antibodies in sera from vaccinated BALB/c mice. (a) Mice were vaccinated twice with PA with (+Sp) or without spore antigens on day 0 and week 4 and challenged at week 8 with ΔAmes spores. Sera were collected on weeks 2, 4, 6 and 8 and assayed on plates coated with PA. There were no statistically significant differences noted in the week 2 samples. There were significant differences between vaccines consisting of PBS or PA alone (P = 0.0009, week 4; P<0.0001, weeks 6 and 8), PBS or PA+Sp (P<0.0001 for weeks 4, 6 and 8) and PA or PA+Sp (P = 0.0009, week 2; P<0.0001, week 6). (b–d) Anti-spore antibody titres were determined for sera collected on week 2 (b, c) and week 6 (d, e) of the vaccination period and assayed on plates coated with wild-type Ames (b, d) or ΔbclA Ames (c, e) spores. The responses (b–e) of the PA+Sp vaccine group were significantly greater than those of the PBS control groups, with a mean reciprocal end-point dilution titre of 800 (P = 0.0347) for (b) and (c) and 12 800 (P = 0.007) for (d) and (e), as determined by t tests with stepdown Bonferroni adjustment for multiple comparisons. The symbols for the PBS control and PA-only groups overlapped in the graphs depicted in (c–e).

Fig. 5.

Survival results of guinea pigs vaccinated with PA alone (▪) or PA+spore antigens (▴) and challenged i.d. (a) or i.n. (b) with Ames spores. All vaccines were given as three immunizations each separated by 4 weeks and all contained ~30 µg PA or 30 µg PA+10 µg BclA+10 µg ExsF+10 µg p5303 and ~500 µg AL. The control groups (•) received PBS and AL alone. The guinea pigs challenged by i.d. injection (a) received ~156 spores delivered in 200 µl WFI and the guinea pigs challenged through i.n. instillations (b) received ~1.35×10⁷ spores delivered in 100 µl WFI. The guinea pigs challenged i.d. were significantly protected with PA alone (P = 0.0062 for percentage survival and 0.0002 for the survival curve) and the vaccine including spore antigens was also protective (P<0.0001 for percentage survival and 0.0002 for the survival curve). The P value comparing the survival curves of PA alone with PA+spore antigens was 0.067. When challenged i.n., the animals receiving PA alone were significantly protected (P = 0.013 for TTD and P = 0.0002 for the survival curve), as were the animals receiving the PA vaccine with the addition of the spore antigens (P = 0.012 for TTD and P = 0.0002 for the survival curve). There were 10 animals per group with the exception of the negative-control groups, which contained nine animals in each group.

Fig. 6.

Titres of anti-PA and anti-spore antibodies in guinea pigs before challenge with Ames spores. The animals were vaccinated three times at 4-week intervals with PA, PA+spore antigens (PA+Sp) or PBS alone (controls). Sera were collected from each animal 4 weeks after the first dose (a, c, e) and 4 weeks after the third dose (b, d, f, just before challenge with wild-type Ames spores at week 12). ELISAs were carried out on plates coated with PA (a, b), wild-type Ames spores (c, d) or ΔbclA spores (e, f). The results are the geometric mean titres (±sem) in µg PA ml⁻¹ and anti-spore antibody levels (mean A₄₀₅ and sd). The anti-PA titres (a, b) of the PA-only group were significantly higher than those of the PA+Sp group at week 4 (P = 0.0044), but the reverse was true for sera collected just prior to challenge at week 12 (P = 0.0054). Anti-spore antibody titres in sera collected at week 4 (c, e) and week 12 (d, f) were determined in ELISAs against wild-type (c, d) or ΔbclA (e, f) spores of Ames. The responses (c–f) of the PA+Sp vaccine group were significantly greater than those of the PA-only groups with mean reciprocal end-point dilution titres of up to 51 200 (P = 0.0053) for (c), ≤12 800 (P<0.033) for (d), through all reciprocal dilutions tested (P≤0.0007) for (e) and up to 51 200 (P<0.017) for (f), as determined by t tests with stepdown Bonferroni adjustment for multiple comparisons.