Enhancing the protective immune response against botulism

Abstract

The need for a vaccine against botulism has increased since the discontinuation of the pentavalent (ABCDE) botulinum toxoid vaccine by the Centers for Disease Control and Prevention. The botulinum toxins (BoNTs) are the primary virulence factors and vaccine components against botulism. BoNTs comprise three domains which are involved in catalysis (LC), translocation (HCT), and host receptor binding (HCR). Recombinant HCR subunits have been used to develop the next generation of BoNT vaccines. Using structural studies and the known entry properties of BoNT/A, an HCR subunit vaccine against BoNT/A that contained the point mutation W1266A within the ganglioside binding pocket was designed. HCR/A(W1266A) did not enter primary neurons, and the crystal structure of HCR/A(W1266A) was virtually identical to that of wild-type HCR/A. Using a mouse model, experiments were performed using a high-dose vaccine and a low-dose vaccine. At a high vaccine dose, HCR/A and HCR/A(W1266A) elicited a protective immune response to BoNT/A challenge. At the low-dose vaccination, HCR/A(W1266A) was a more protective vaccine than HCR/A. α-HCR IgG titers correlated with protection from BoNT challenge, although titers to block HCR/A entry were greater in serum in HCR/A-vaccinated mice than in HCR/A(W1266A)-vaccinated mice. This study shows that removal of receptor binding capacity enhances potency of the subunit HCR vaccine. Vaccines that lack receptor binding capacity have the added property of limited off-target toxicity.

Fig 1

HCR/A(W1266A) structure. (A) Linear schematic of HCR domains and color-coded crystal structures with ganglioside modeled into the binding site. (B) Ganglioside binding pocket of HCR/A, with tryptophan 1266 colored fuchsia, cocrystallized ganglioside in gray, oxygen atoms in red, and nitrogen atoms in blue. (C) Overlay of wild-type HCR/A from holotoxin structure (W1266 in fuchsia) and HCR/A(W1266A) in orange (alanine 1266 in green). RMSD = 0.4Å.

Fig 2

W1266 is required for HCR entry into neurons. (Top) HCR/A or HCR/A(W1266A) at 40 nM was incubated with rat primary cortical neurons for 5 min in either low potassium (5.6 mM) as a resting condition or high potassium (56 mM) to stimulate synaptic vesicle cycling. Cells were stained for 3×FLAG (HCR) and synaptophysin1. (Bottom) Ten fields were imaged, and the average intensity of 3×FLAG-HCR was normalized to the synaptic vesicle marker synaptophysin1.

Fig 3

IgG titers to HCR/A and HCR/A(W1266A) following high- and low-dose HCR/A vaccination. (A) A total of 0.5 μg of HCR/A(W1266A) (black boxes) or HCR/A (gray boxes) was spotted, in triplicate, onto nitrocellulose strips. Each strip was individually probed with serum (1:20,000) from mice vaccinated with 1.0 μg (A1 vaccination) or HCR/A(W1266A) (W1266A vaccination) from mice who survived BoNT/A challenge. Strips were incubated with goat α-mouse IgG-HRP and incubated in Super Signal, and light emission was imaged. Backgrounds were subtracted using 3×FLAG-HCR/TeNT for FLAG epitope tag reactivity and LC/TeNT as a control protein. Results are presented as arbitrary units of HRP reactivity using the mean and standard error of the mean (SEM). Significance was not observed between IgG titers. Output signals were proportional to serum dilutions in the dose range and time of imaging used in this analysis, allowing relative HCR-specific IgG titers to be determined. (B) Experiment was performed as in panel A except HCR was probed with sera from mice receiving 0.1 μg HCR/A or HCR/A(W1266A) vaccine. In the HCR/A vaccination, 13 of 20 mice survived BoNT/A challenge. Sera from animals that did not survive challenge were assayed separately (columns labeled “Dead”). All animals immunized with HCR/A(W1226A) survived BoNT/A challenge. In HCR/A(W1266A) vaccination, 20 of the 20 mice survived 0.1-μg challenge. ns, not significant; *, P value of 0.05.

Fig 4

HCR vaccine neutralization of HCR-receptor interactions. HA-tagged HCR/A at 40 nM in high-potassium buffer was incubated with serial dilutions of vaccine sera from mice receiving 1 μg HCR/A or HCR/A(W1266A) or preimmune sera at the highest dilution for 30 min at 4°C and incubated with rat primary cortical neurons for 5 min (37°C). Cells were fixed and HCR detected with α-HA antibody and secondary Alexa 488. Intensity was normalized to synaptophysin1. ***, significance between HCR/A signals from HCR/A vaccine sera and HCR/A(W1266A) vaccine sera.