Delivery of foreign antigens by engineered outer membrane vesicle vaccines

Abstract

As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.

Fig. 1.

The immunogenicity of model antigen GFP in mice is significantly enhanced when administered in fusion with ClyA. Scatter dot plots (with median lines) show individual host anti-GFP IgG responses in serum diluted 1:12,800. Groups of five BALB/c mice were s.c. immunized with 2.5 μg GFP (group I), 2.5 μg ClyA (group II), 5 μg ClyA-GFP fusion (group III), and 2.5 μg ClyA mixed with 2.5 μg GFP (group IV). Mice were immunized on day 0 and day 28, which is marked by the arrowheads in each chart. The daggers (†) represent statistical significance (P < 0.05; Wilcoxon rank-sum test) of antibody titers in group III compared with titers in groups II and IV.

Fig. 2.

The model antigen GFP is functionally localized in engineered OMVs when fused with ClyA. (A) Electron micrograph of empty OMVs stained by uranyl acetate. (Scale bar: 200 nm.) This image is also representative of engineered OMVs containing ClyA-GFP (cf. reference 22). (B) Fluorescence micrograph (100× objective) of ClyA-GFP in association with engineered OMVs. (C) Western blot with anti-GFP antibodies of cell-free culture supernatants and OMV suspensions from cultures of E. coli expressing the empty plasmid vector or ClyA-GFP. (D) Z-average hydrodynamic diameter of empty and engineered OMV suspensions in PBS, as measured by dynamic light scattering. (E) LPS content in empty and engineered OMV suspensions is normalized by total protein content. The asterisk (*) denotes statistical significance (P < 0.05; Student’s t test).

Fig. 3.

ClyA-GFP localized in engineered OMVs retains its immunogenicity in mice. Scatter dot plots (with median lines) show individual host anti-GFP IgG titers in serum diluted 1:12,800. Groups of five BALB/c mice were immunized with purified ClyA-GFP fusion protein mixed with empty OMVs (group V) and ClyA-GFP fusion in association with engineered OMVs (group VI). The effective dose of ClyA-GFP in both treatment groups was 2.5 μg. Mice were immunized on day 0 and day 28, which is marked by the arrowheads in each chart. The asterisks (*) denote statistically significant differences (P < 0.05; Wilcoxon rank-sum test) compared with antibody titers in the purified ClyA-GFP treatment group (group III) on the corresponding day.

Fig. 4.

Head-to-head comparison of engineered OMVs with the clinical standard adjuvant, aluminum hydroxide (alum). Scatter dot plots show individual host anti-GFP IgG titers in serum diluted 1:1.600 at day 56. Groups of five BALB/c mice were immunized with purified GFP absorbed to alum, ClyA-GFP absorbed to alum, ClyA-GFP mixed with empty OMVs, or ClyA-GFP fusion in engineered OMVs. The effective dose for each group was 2.5 μg. Mice were immunized on day 0 and 28. No statistical differences in antibody titers were observed among the groups (Wilcoxon rank-sum test).