Abstract

Results

The Immunogenicity of GFP in Mice Is Significantly Enhanced when Administered in Fusion with ClyA.

ClyA, GFP, and ClyA-GFP were overexpressed in E. coli DH5α and purified from the soluble fractions of the bacteria (SI Text and Fig. S1). The immunostimulatory effects of these soluble proteins were then tested in mice; s.c. immunization of BALB/c mice with ClyA-GFP elicited GFP-reactive antibody responses that were significantly higher than immunization with ClyA mixed with GFP (Fig. 1). A GFP-specific IgG response was detected beginning 2 weeks after priming in mice immunized with ClyA-GFP; this response was augmented by booster vaccination and sustained for 4 weeks after the booster injection. No detectable anti-GFP IgG antibodies were observed until after boosting in any of the other treatment groups. Interestingly, immunization with GFP elicited little to no detectable response at any time during the study period, whereas in two mice, immunization with ClyA alone triggered fluctuating levels of GFP cross-reactive antibody species after the booster. Antibody titers in the ClyA-GFP immunization group (group III) were significantly higher (P < 0.05) than antibody titers in mice vaccinated with unfused proteins (group IV). The difference was statistically significant beginning at day 14 of the study and remained so through the conclusion of the study period. GFP-specific antibody levels in the treatment group immunized with ClyA and GFP separately (group IV) were statistically similar to the antibody levels generated by ClyA immunization alone (group II) at all time points throughout the study.

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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.

ClyA-GFP Is Localized in Engineered OMV Vaccines.

OMVs were prepared from vesicle hyper-producing E. coli strain JC8031 transformed with plasmid pClyA-GFP-His6 (engineered OMVs) or the empty pBAD18-Cm cloning vector (empty, wild-type OMVs). Electron microscopic analysis after negative staining showed the spherical bilayered structure of OMVs (Fig. 2A), which have a diameter of ~100 nm. ClyA-GFP fluorescence was observed in association with the engineered OMVs (Fig. 2B), a finding confirmed by Western blotting with anti-GFP antibodies (Fig. 2C). Fluorescence-intensity measurements and SDS/PAGE gel-band densitometry indicated that ClyA-GFP comprises ~5% of the total protein content in OMVs. Because expression of clyA in E. coli is strongly repressed under normal laboratory conditions (24), no free ClyA was detected in the engineered or empty OMVs preparations. Consistent with earlier observations (22), the association of ClyA-GFP in engineered OMVs had no apparent effect on the size of vesicles (Fig. 2D). Bacterial lipopolysaccharide (LPS) content in empty and engineered OMVs was measured by a colorimetric assay to detect 2-keto-3-deoxyoctonate (KDO), a core sugar component of LPS. The KDO assay indicated that empty OMVs contain a slightly higher concentration of LPS, normalized by total protein content, than engineered OMVs (Fig. 2E).

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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).

The Immunogenicity of ClyA-GFP in Engineered OMV Vaccines Is Equivalent to the Immunogenicity of the Purified ClyA-GFP Fusion Protein Alone.

To examine the immunogenicity of ClyA-GFP in outer membrane vesicles, BALB/c mice were immunized with empty OMVs mixed with ClyA-GFP or engineered OMVs containing the ClyA-GFP fusion protein (Fig. 3). The effective dose of ClyA-GFP in the OMV formulations (2.5 μg) was one-half the amount used in these purified protein immunizations to observe if ClyA-GFP association with OMVs contributes any additional immunostimulatory effect. Immunization with empty OMVs mixed with soluble ClyA-GFP (group V) resulted in a GFP-specific response beginning at 2 weeks after priming and continuing for 4 weeks after the booster. Immunization with ClyA-GFP in engineered OMVs (group VI) elicited similar GFP-reactive IgG responses starting 2 weeks after the initial dose that then markedly increased after booster immunization at day 28. At all time points throughout the study, antibody titers between group V and group VI remained statistically indistinguishable. Additionally, antibody titers in OMV-immunized mice (both groups V and VI) were statistically equivalent to those in group III with an exception on day 56 for group V and on day 35 for group VI, when titers were significantly higher in group III (P < 0.05). Despite the presence of LPS detected in the OMV formulations, no significant inflammatory responses were observed in animal subjects. In addition, local reactions to vaccination, as determined by histological analysis of excised tissue, were indistinguishable between groups treated with the formulations with or without depletion of LPS from vaccine preparations, indicating that the adjuvant effect was a function of the OMV and not LPS (SI Text and Table S1 and Figs. S2 ‒ S5).

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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.

Antibody Titers Elicited by ClyA-GFP in Engineered OMV Vaccines Are Equivalent to Those Elicited by the Adjuvant Aluminum Hydroxide (AlOH₃, alum).

To directly compare the level of immune response elicited by the engineered OMV vaccines with a standard adjuvant, mice were immunized with the OMV formulations or with GFP or ClyA-GFP absorbed to aluminum hydroxide. As shown in Fig. 4, the antibody responses to all four preparations were statistically equivalent, suggesting that engineered OMV preparations are potentially viable candidates for further clinical development and evaluation.

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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).

Discussion

Vaccination remains one of the most cost-effective strategies for preventing infectious disease (25, 26). The safety of protein subunit vaccines makes them particularly attractive for administration to wide swaths of the human population, healthy and immunocompromised individuals alike. A major limiting factor in the further development of subunit vaccines, however, remains the poor immunogenicity of purified antigens when administered alone. Despite ongoing research to novel immune potentiators, the only compounds currently licensed for human use in North America remain aluminum salts (27). In addition to increasing the cost of vaccine production, these aluminum adjuvants may sometimes result in severe adverse events postadministration (28, 29). With ongoing disease threats and the promise of emerging immunotherapies on the horizon, there clearly is strong demand for new vaccine technologies.

Owing largely to their size, plasticity, and safety profile in humans, OMVs are attractive vehicles for vaccine delivery. After the first reports of OMV vaccines for serogroup B meningococcal disease, vesicles from Salmonella thyphimurium and Pseudomonas aeruginosa containing surface antigens native to the pathogens have also been shown to exhibit immunogenic properties (30, 31). The prospects of using OMVs to deliver antigens that are nonnative to the parental bacteria are not yet realized, largely because of challenges in transporting heterologous proteins to vesicles. In a recent demonstration of the remarkable tunability of OMVs by our group, heterologous proteins fused with the native bacterial protein ClyA are efficiently transported in their native functional forms to vesicles (22). Previous work also points to the utility of ClyA in enhancing the immunogenicity of antigens secreted from a live attenuated Salmonella vector, further suggesting the possibility that antigens may be exported from live vectors in the form of OMVs (23).

The work presented here illustrates the potential of engineered OMVs to act as a potent adjuvant and carrier system for poorly immunogenic subunit antigens. Specifically, an OMV vaccine engineered to contain a model GFP antigen elicited strong GFP-specific humoral responses in immunized mice, whereas immunization with GFP alone failed to result in any significant antibody responses. GFP was chosen as the model antigen, because it constitutes a relatively weak antigen in and by itself (3). A salient advantage in the production of recombinant OMV overpurified proteins lies in vesicle purification by facile ultracentrifugation, which effectively eliminates the need for costly infrastructure to support a priori antigen purification. The results from this study introduce engineered OMVs as a versatile strategy for delivering poorly immunogenic protein antigens, while simultaneously sidestepping the complex purification processes of traditional subunit vaccines.

Expression of the engineered ClyA-GFP fusion protein in E. coli resulted in a 61-kDa product that retained the native biological activities of ClyA and GFP (Fig. S1). The specific fluorescence and hemolysis activities of ClyA-GFP under the specified assay conditions were diminished relative to the unfused constituent proteins, which were similar to previous observations made with other ClyA fusion partners (32). The retention of protein activity indicates that ClyA-antigen fusions maintain their conformations and suggests that ClyA may be an amenable carrier for both linear epitopes and conformational antigens.

Naïve mice were immunized with ClyA, GFP, the soluble ClyA-GFP fusion protein, and ClyA mixed with GFP. The results in Fig. 1, depicting antibody titers in mice immunized with the purified proteins, show ClyA to be an effective carrier protein for enhancing the immunogenicity of GFP. The immunogenicity of ClyA fusion proteins was first shown in a study by Galen et al. (23), where the observed enhancement in the immune response to the antigens was postulated to be largely dependent on export from a live Salmonella vector. Here, the results show that antigen fusion with ClyA is directly responsible for enhanced immunogenicity. Residual LPS was not detectable with the KDO assay in any of the purified protein preparations, suggesting that the enhanced immune response to GFP was a result of fusion with the ClyA moiety. As a bacterial cytolytic protein from enterobacteria (33), ClyA joins the ranks of other known toxins with adjuvant properties such as diphtheria toxin or heat-labile enterotoxin (34). Although no pathological effects were observed in mice immunized with ClyA in this study, detoxification of ClyA through mutation (35), truncation, or chemical methods may attenuate any possible toxicity while retaining its immunomodulatory capabilities.

Unlike other known bacterial toxin adjuvants, however, enhancement in the immune response to GFP was not observed when mice were immunized with a mixture of soluble ClyA and soluble GFP. Whereas the specific mechanisms that drive the immunostimulatory behavior of ClyA remain to be investigated, physical or chemical conjugation to protein carriers has frequently improved the immunogenicity of otherwise poorly immunogenic antigens by increasing the antigen size or improving antigen organization to optimize the B cell response (2, 3, 36). In general, the ability for antigens to be presented in the proper structure to activate B cell proliferation requires efficient antigen processing by-antigen presenting cells and costimulation by T helper cells. Further studies on ClyA and its interaction with the immune system are necessary to more fully characterize the immune-enhancing activity of ClyA and to explore the potential to simulate T cell, especially cytotoxic, responses.

Leveraging the secretion of ClyA to OMVs, engineered OMVs were purified from the nonpathogenic E. coli strain JC8031 expressing ClyA-GFP (Fig. 2). Fluorescence microscopy and Western blotting with anti-GFP antibodies verified the presence of ClyA-GFP in engineered OMVs; association of the active fusion protein with vesicles occurred through secretion and thus required no separate antigen purification, unlike the cases of other established particulate-delivery systems.

To examine the humoral immune response to ClyA-GFP when administered with OMVs, mice were immunized with empty (wild-type) OMVs mixed with soluble ClyA-GFP (Fig. 3, group V) or with the engineered OMV formulation containing ClyA-GFP (Fig. 3, group VI). It is generally observed that particulate antigens are more efficiently recognized by the immune system than soluble proteins (37). Therefore, to examine if ClyA-GFP association with OMVs contributes additional immunostimulatory effect over the purified protein alone, the effective dose of ClyA-GFP in the OMV formulations (2.5 μg) was one-half the amount used in the purified protein immunizations. The results show that the immunostimulatory behavior of ClyA-GFP is retained when delivered in vesicles. On day 35 of the study, anti-GFP titers in the recombinant OMV group (Fig. 3, group VI) were lower than in the group immunized with purified ClyA-GFP (Fig. 1, group III); however, the titers in these two treatment groups were statistically indistinguishable from each other beginning at day 42. The decreased ClyA-GFP dosage in the OMV vaccine formulation may be a factor in the slightly delayed immune response to engineered OMVs (at day 35) compared with the purified fusion protein.

The anti-GFP antibody titers observed in response to the engineered OMV immunizations are remarkable, especially when taking into account that the effective dose of the ClyA-GFP fusion protein in the OMV formulations was one-half that of the purified protein treatments. The results suggest that OMVs contribute additional immune-stimulating activities. As judged from the effect of the OMV preparations in inoculated mice, the low levels of residual LPS in OMVs at the administered doses did not cause a local inflammatory response (SI Text, Table S1 and Figs. S2 ‒ S5). However, because LPS can be counterproductive in vaccines, future work will examine the immunogenic effect of total LPS depletion from vesicles (38). As the immune-potentiation capabilities of ClyA become better understood, the combination of engineered OMVs with additional adjuvants or immunostimulatory compounds may also be of considerable interest in directing a specific immune response.

Comparisons between animals treated with the wild-type and engineered OMV formulations (Fig. 3) suggest that anti-GFP titers are largely independent of the direct association of ClyA-GFP with OMVs. There remains a possibility that the nonspecific association of purified ClyA-GFP with empty vesicles may render the two tested OMV formulations virtually indistinguishable to the immune system. An exception may be that the engineered OMV treatments seemed to result in a prolonged antibody response. At day 56, the antibody titers in group V (empty OMVs mixed with ClyA-GFP) were statistically decreased relative to group III (purified ClyA-GFP), whereas the titers in the engineered OMV treatment group exhibited no such decrease. Analogous to other particulate-delivery systems, the colocalization of antigens in the lumen of OMVs may protect antigens from protease degradation in vivo, resulting in more efficient B cell activation.

Importantly, a direct comparison of the engineered OMV preparations to a standard adjuvant, aluminum hydroxide (AlOH₃ or alum), revealed that the antibody responses generated in both cases were equivalent. Given that alum is the basis of the only FDA-approved adjuvants and that new adjuvants continuously are sought, we conclude that the concept of engineered OMV vaccines may hold clinical promise. Work testing the broader applicability of OMV vaccines is currently ongoing in our laboratories.

The results here show that engineered OMVs are unique particulate vaccine-delivery vectors with adjuvant and carrier activity that combine features borrowed from both synthetic and living antigen-delivery systems. Whereas OMVs are derived from live bacterial cultures, vesicles themselves are nonreplicating entities that avoid the potential safety concerns associated with attenuated living bacteria or viruses. As nanoscale spherical structures comprising mainly protein and lipid, OMVs may share some structural or compositional similarity with liposome or proteosome carriers; but in the most significant departure from these particulate systems or other subunit vaccines, where as many as three components—antigen, adjuvant, and delivery system—are produced separately before formulated into a final product, engineered OMVs may not require these extensive purification and formulation procedures.

This work illuminates the potential of engineered OMV technology to overcome the significant safety and economic limitations that often arise in the course of vaccine development. A model green-fluorescent protein fused with the bacterial hemolysin ClyA was secreted in OMVs of E. coli, while maintaining the biological activity of both components. These engineered vesicles were administered to mice and found to be highly immunogenic, eliciting high anti-GFP antibody titers, whereas immunization with GFP alone failed to elicit any significant humoral response. Additionally, the engineered OMV formulation elicited a humoral response that is on par with the standard adjuvant aluminum hydroxide. Combining adjuvant and carrier activity, engineered OMVs enhance the response to an otherwise poorly immunogenic antigen and circumvent the protein-purification requirements of traditional subunit vaccines and particulate antigen-delivery modalities. With the wide assortment of heterologous proteins that are functionally localized to OMVs by fusion with ClyA, including enzymes, fluorescent proteins, and single-chain antibody fragments (22), and with additional possibilities of further engineering of vesicle luminal or surface features, OMVs are a uniquely tunable platform with ease of manufacture for the delivery of a wide spectrum of poorly immunogenic subunit antigens for vaccines against infectious disease or other therapeutic targets.

Materials and Methods

Supplementary Material

Acknowledgments

Footnotes

References