Outer Membrane Vesicles Displaying Engineered Glycotopes Elicit Protective Antibodies

Outer membrane vesicles displaying engineered PNAS PLUS glycotopes elicit protective antibodies

Linxiao Chena,1, Jenny L. Valentinea,1, Chung-Jr Huanga, Christine E. Endicotta, Tyler D. Moellera, Jed A. Rasmussenb, Joshua R. Fletcherc, Joseph M. Bolld,e, Joseph A. Rosenthalf, Justyna Dobruchowskag, Zhirui Wangg, Christian Heissg, Parastoo Azadig, David Putnama,f, M. Stephen Trentd,e, Bradley D. Jonesb,c, and Matthew P. DeLisaa,f,2

aRobert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853; bDepartment of Microbiology, University of Iowa, Iowa City, IA 52242; cGenetics Program, University of Iowa, Iowa City, IA 52242; dDepartment of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712; eDepartment of Infectious Diseases, The University of Georgia College of Veterinary Medicine, Athens, GA 30602; fNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853; and gComplex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602

Edited by Carolyn R. Bertozzi, Stanford University, Stanford, CA, and approved April 26, 2016 (received for review September 15, 2015) The O-antigen polysaccharide (O-PS) component of lipopolysaccha- IgM-to-IgG switching, memory B-cell development, and long- rides on the surface of gram-negative bacteria is both a virulence lived T-cell memory (5, 8–11). Such glycoconjugates have proven to factor and a B-cell antigen. Antibodies elicited by O-PS often confer be a highly efficacious and safe strategy for protecting against vir- protection against infection; therefore, O-PS glycoconjugate vaccines ulent pathogens, including Haemophilus influenzae, Neisseria men- have proven useful against a number of different pathogenic ingitidis, and Streptococcus pneumoniae (10, 12, 13), with several bacteria. However, conventional methods for natural extraction or already licensed and many others in clinical development (9, 12). chemical synthesis of O-PS are technically demanding, inefficient, and Despite their effectiveness, traditional conjugate vaccines are expensive. Here, we describe an alternative methodology for producing not without their drawbacks. Most notable among them is the com- glycoconjugate vaccines whereby recombinant O-PS biosynthesis plex, multistep process required for the purification, isolation, and Escherichia is coordinated with vesiculation in laboratory strains of conjugation of bacterial polysaccharides, which is expensive, time coli to yield glycosylated outer membrane vesicles (glycOMVs) dec- consuming, and low yielding (14). A greatly simplified and cost- orated with pathogen-mimetic glycotopes. Using this approach, effective alternative, known as protein glycan coupling technology glycOMVs corresponding to eight different pathogenic bacteria were (PGCT), has been described recently (15). This approach is based generated. For example, expression of a 17-kb O-PS gene cluster from on engineered protein glycosylation in living Escherichia coli (16), the highly virulent Francisella tularensis subsp. tularensis (type A) wherein an O-antigen polysaccharide (O-PS), the outermost com- strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice ponent of bacterial LPS (2), is conjugated to a coexpressed carrier with glycOMVs elicited significant titers of O-PS–specific serum IgG protein by the Campylobacter jejuni oligosaccharyltransferase PglB (CjPglB). However, whereas PGCT has been used to make several

antibodies as well as vaginal and bronchoalveolar IgA antibodies. SCIENCES Importantly, glycOMVs significantly prolonged survival upon subse- novel protein/glycan combinations (15, 17, 18), it currently has a

quent challenge with F. tularensis Schu S4 and provided complete limited substrate specificity because the natural substrate specificity APPLIED BIOLOGICAL protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the Significance vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described Conjugate vaccines have proven to be an effective and safe here could be readily adapted for developing vaccines against many strategy for reducing the incidence of disease caused by bac- other bacterial pathogens. terial pathogens. However, the manufacture of these vaccines is technically demanding, inefficient, and expensive, thereby glycan | glycoconjugate vaccine | humoral immune response | O-antigen limiting their widespread use. Here, we describe an alternative polysaccharide | anti-glycan antibodies methodology for generating glycoconjugate vaccines whereby recombinant polysaccharide biosynthesis is coordinated with or decades, vaccines have served as an important pillar in pre- vesicle formation in nonpathogenic Escherichia coli, resulting in Fventative medicine, providing protection against a wide array of glycosylated outer membrane vesicles (glycOMVs) that can ef- disease-causing pathogens by inducing humoral and/or cellular im- fectively deliver pathogen-mimetic glycotopes to the immune munity. In the context of humoral immunity, carbohydrates are ap- system. An attractive feature of our approach is the fact that pealing vaccine candidates owing to their ubiquitous presence on the different plasmid-encoded polysaccharide biosynthetic pathways surface of diverse pathogens and malignant cells. For example, most can be readily transformed into E. coli, enabling a “plug-and- pathogenic bacteria are prominently coated with carbohydrate play” platform for the on-demand creation of glycOMV vaccine moieties in the form of capsular polysaccharides (CPSs) (1) and candidates that carry heterologous glycotopes from numerous lipopolysaccharides (LPSs) (2), which are often the first epitopes pathogenic bacteria. perceived by the immune system. However, a major impediment to the development of polysaccharide-based vaccines is the fact Author contributions: L.C., J.L.V., C.-J.H., J. A. Rasmussen, J.R.F., J.M.B., J.D., Z.W., C.H., P.A., M.S.T., B.D.J., and M.P.D. designed research; L.C., J.L.V., C.-J.H., C.E.E., T.D.M., that pure carbohydrates typically stimulate T cell-independent J. A. Rasmussen, J.R.F., J.M.B., J.D., Z.W., C.H., P.A., and B.D.J. performed research; L.C., immune responses (3–5), which are characterized by lack of IgM-to- J.L.V., C.-J.H., C.E.E., T.D.M., J. A. Rasmussen, J.R.F., J.M.B., J. A. Rosenthal, C.H., P.A., D.P., IgG class switching (6), failure to induce a secondary antibody re- M.S.T., B.D.J., and M.P.D. analyzed data; and L.C., J.L.V., C.H., P.A., D.P., M.S.T., B.D.J., and sponse after recall immunization, and no sustained T-cell memory (7). M.P.D. wrote the paper. A common strategy for enhancing the immunogenicity of carbo- Conflict of interest statement: M.P.D. has a financial interest in Glycobia, Inc. hydrates and evoking carbohydrate-specific immunological memory is This article is a PNAS Direct Submission. + to covalently couple a carbohydrate epitope to a CD4 Tcell- 1L.C. and J.L.V. contributed equally to this work. dependent antigen such as an immunogenic protein carrier. Indeed, 2To whom correspondence should be addressed. Email: [email protected]. conjugate vaccines composed of bacterial CPS- or LPS-derived glycans This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chemically bound to a carrier protein induce glycan-specific 1073/pnas.1518311113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1518311113 PNAS | Published online June 6, 2016 | E3609–E3618 Downloaded by guest on September 27, 2021 of the conjugating enzyme, CjPglB, restricts the diversity of glycans that can be transferred (19) and causes the conjugation efficiency between certain nonnative glycan and protein substrates to E. coli be very low (18). Additionally, it remains to be determined whether the carrier proteins used in licensed glycoconjugate vaccines, such as pO-PS the toxins from Clostridium tetani and Corynebacterium diphtheriae, are compatible with expression and CjPglB-mediated glycosyla- Pathogen-specific tion in E. coli. glycOMVs Here, we sought to create a new approach for the production of glycoconjugate vaccines that circumvents these problems by combining recombinant O-PS biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains of E. coli. OMVs are naturally occurring spherical nanostructures (∼20–250 nm) produced by all gram-negative bacteria. They are composed of Pathogen-specific proteins, lipids, and glycans, including LPS, derived primarily O-PS from the bacterial periplasm and outer membrane (20). In recent Core years, OMVs have garnered attention as a vaccine platform be- Lipid A cause they are nonreplicating, immunogenic mimics of their pa- OM rental bacteria that stimulate both innate and adaptive immunity and possess intrinsic adjuvant properties (21–23). These char- acteristics are exemplified by OMVs isolated directly from N. Peptidoglycan meningitidis, which induce potent protective immune responses and have been incorporated successfully into several commercial xn MsbA vaccine formulations for use in humans (22, 24, 25). To expand WaaL Wzx Wzy Wzz the vaccine potential of OMVs, several groups have used genetic IM engineering techniques to load OMVs with foreign protein antigens by targeting expression either to the outer membrane or the periplasm of an OMV-producing host strain (26–32). These Pathogen-specific O-PS Lipid A-core repeating unit OMV-associated recombinant proteins were internalized by eukaryotic cells (26, 27) and stimulated strong and specific im- Fig. 1. Assembly and display of pathogen-specific O-PS structures on OMVs. mune responses in mice (28–32). However, although efforts to Schematic of a pathway for biosynthesis of heterologous O-PS structures and their load OMVs with recombinant protein antigens are well docu- incorporation into OMVs in E. coli. Lipid-linked O-PS repeating units are assem- mented (33), an analogous strategy to engineer the polysaccha- bled on the cytoplasmic face of the inner membrane (IM) by glycosyltransferases ride component of OMVs for specific vaccine applications has encoded in plasmid pO-PS, after which translocation to the periplasmic face occurs yet to be demonstrated. by the action of endogenous flippase Wzx. Polymerization of O-PS repeating units We sought to engineer OMVs that efficiently deliver surface- on the periplasmic face of the inner membrane is then catalyzed by endogenous associated glycotopes to the immune system in a manner that in- Wzy polymerase in a block transfer mechanism that is regulated by endogenous Wzz. The resulting O-PS is then transferred to the lipid A-core polysaccharide by duces protective immunity. Toward this goal, heterologous O-PS endogenous O-antigen ligase, WaaL. The resulting lipopolysaccharide is shuttled structures were expressed in hypervesiculating E. coli cells, resulting to the outer membrane (OM), where it becomes incorporated in budding vesicles in glycosylated OMVs (glycOMVs) whose surfaces were remodeled to produce pathogen-specific glycOMVs. with pathogen-mimetic polysaccharides. A major advantage of this approach is that designer carbohydrates are directly conjugated to lipid A, which is a powerful adjuvant whose bioactivity and toxic- live vaccine strain (LVS) Iowa and LVS Rocky Mountain Lab- ity can be genetically modulated (34). One of these candidate oratories (RML) that display the same O-PS structure on their glycOMVs was subsequently evaluated for its ability to confer outer membrane (38). Overall, OMVs displaying designer gly- protection against highly virulent Francisella tularensis subsp. cotopes on lipid A, itself a strong adjuvant, represent a potent tularensis (type A) strain Schu S4, a gram-negative, facultative glycoconjugate vaccine design that, given the generality of the coccobacillus and the causative agent of tularemia. This bacte- approach, could be developed for numerous other bacterial rium is one of the most infectious agents known to man and is pathogens. categorized as a class A bioterrorism agent due to its high fatality rate, low dose of infection, and ability to be aerosolized (35). Results Although there is currently no available licensed vaccine, several Glycosylation of OMVs with Heterologous O-PS. LPS is found exclusively studies have confirmed the important role of antibodies directed in the outer leaflet of the gram-negative outer membrane and con- against F. tularensis LPS, specifically the O-PS repeat unit, in sists of three distinct regions: a hydrophobic domain known as “lipid providing protection against the highly virulent Schu S4 strain A,” a core oligosaccharide, and an O-PS (2) (Fig. 1). Laboratory (36–38). More recently, a purified recombinant vaccine comprising E. coli strains usually lack O-PS structures but do produce a com- the F. tularensis Schu S4 O-PS conjugated to the Pseudomonas plete lipid A-core that serves as an acceptor for O-PS if the genes for aeruginosa exotoxin A carrier protein was produced using PGCT its synthesis are supplied in trans (39). Because LPS is a major (17). This glycoconjugate boosted IgG levels and significantly in- component of released OMVs (20), we postulated that expression of creased the time to death upon subsequent pathogen challenge, heterologous O-PS pathways in hypervesiculating E. coli would result albeit with the less virulent F. tularensis subsp. holarctica (type B) in OMVs whose lipid A-core was glycosylated with desired O-PS strain HN63. Here, we show that immunization of mice with structures (Fig. 1). To test this notion, we introduced the gene cluster glycOMVs displaying F. tularensis Schu S4 O-PS induced high for the synthesis of F. tularensis Schu S4 O-PS into O-PS–deficient titers of functional serum IgG antibodies against Schu S4 LPS as E. coli strain JC8031. This strain was chosen for its ability to well as vaginal and bronchoalveolar IgA antibodies. Importantly, hypervesiculate, due to genetic knockout of tolRA (40), and has been glycOMVs significantly extended time to death upon subsequent used extensively in OMV engineering applications (27, 28, 33). challenge with F. tularensis Schu S4 and provided complete OMVs were isolated from JC8031 cells expressing the Schu S4 O-PS protection against challenge with F. tularensis subsp. holarctica gene cluster from pGAB2 (17) and subjected to Western blot

E3610 | www.pnas.org/cgi/doi/10.1073/pnas.1518311113 Chen et al. Downloaded by guest on September 27, 2021

PNAS PLUS ++ ++ ++ analysis using an F. tularensis O-PS–specific antibody named “FB11” WaaL ++ pO-PS ++ ++ ++ ++ (37). We observed a classical ladder-like pattern typical of LPS (Fig. 2), which results from O-PS chain length variability generated by the Wzy polymerase (2, 16). F. tularensis O-PS was absent in OMVs derived from JC8031 cells carrying empty plasmid (Fig. 2). Likewise, when the Schu S4 O-PS antigen genes were expressed in strain 75 75 75 CE8032, which lacks the waaL gene encoding the ligase that trans- 75 fers O-PS to lipid A-core (Fig. 1) (2, 16), the resulting OMVs were 50 no longer detected with the FB11 antibody (Fig. 2). 50 50 50 To demonstrate the generality of the approach, an expanded 37 kDa 37 repertoire of pathogen-mimetic O-PS structures was expressed in 37 kDa kDa OMVs. Specifically, plasmids containing the O-PS gene clusters 37 kDa from a variety of gram-negative pathogenic bacteria, including uro-

O-PS F. tularensis E. coli O7 E. coli O78 E. coli O148

++ ++ ++ pathogenic E. coli (UPEC) strain VW187 (O7:K1), enterotoxigenic ++ WaaL E. coli (ETEC) strains O78 and O148, P. aeruginosa strain PA103, pO-PS ++ ++ ++ ++ Shigella dysenteriae 1 strain W30864, Shigella flexneri serotype 2a, and Yersinia enterocolitica strain 6471/76, were transformed in strain JC8031. OMVs prepared from these cells were all cross-reactive with antibodies specific for the respective O-PS structures (Fig. 75 75 75 2). In contrast, control OMVs prepared from either CE8032 cells 75 expressing the same O-PS pathway genes or JC8031 cells carry- 50 50 ing empty plasmids were not detected by the cognate O-PS– 50 50 37 specific antibodies (Fig. 2). In the case of OMVs displaying kDa 37 Y. enterocolitica O-PS, we observed a smear rather than a clear 37 kDa 37 kDa kDa ladder; however, this smear is typically produced after electro- phoresis of LPS preparations from these bacteria (41). Taken together, these results suggest that lipid A-core in OMVs was P. aeruginosa O11 S. dysenteriae S. flexneri Y. enterocolitica O3 glycosylated with heterologous, strain-specific O-PS structures. Fig. 2. Incorporation of pathogen-specific O-PS in OMVs. Western blot + The Outer Surface of Intact OMVs Is Remodeled with F. tularensis O-PS. analysis of OMV fractions isolated from E. coli JC8031 (WaaL, ) or CE8032 − − Given our interest in creating a vaccine candidate against (WaaL, ) cells carrying an empty plasmid (pO-PS, ) or a heterologous O-PS pathway plasmid (pO-PS, +) corresponding to the pathogenic strain in- F. tularensis, OMVs generated by strain JC8031 carrying pGAB2, “ ” dicated below each panel. The O-PS pathway plasmids and empty control termed Ft-glycOMVs, were further characterized. To confirm plasmids are provided in SI Appendix, Table S3. Antibodies specific to each that F. tularensis O-PS was on the outer surface of vesicles, dot blots O-PS (SI Appendix, Table S4) were used to detect heterologous glycan

were performed by spotting fractions containing Ft-glycOMVs di- structures displayed on the glycOMVs. Molecular mass markers are labeled SCIENCES rectly onto nitrocellulose membranes without any denaturation on the right. steps. Only the OMV fractions derived from JC8031 cells carrying APPLIED BIOLOGICAL plasmid pGAB2 were detected by the FB11 antibody (SI Appendix, Fig. S1A), suggesting that nondenatured, intact vesicles carried structurally characterized. Western blot analysis using FB11 revealed F. tularensis O-PS on their surface. As expected, nondenatured nearly identical laddering for Ft-glycOMVs compared with hybrid vesicles derived from CE8032 carrying pGAB2 did not give a strong E. coli LPS capped with the F. tularensis O-PS (Ft-glycLPS) signal using FB11 (SI Appendix,Fig.S1A).Thesizeandshapeof extracted directly from intact JC8031 cells carrying pGAB2 (Fig. Ft-glycOMVs appeared indistinguishable from control OMVs (SI 3A), indicating that the engineered LPS molecules in the outer Appendix,Fig.S1B), indicating that incorporation of foreign O-PS membrane of JC8031 are structurally similar to those loaded in into E. coli LPS structures had no visible effect on vesicle nano- OMVs. Compared with the native F. tularensis Schu S4 LPS structure. Next, we determined whether F. tularensis O-PS detected (FtLPS), the height of these ladders (i.e., the chain length of in the pelleted supernatant was associated with intact vesicles or O-PS) was notably shorter (Fig. 3A). with released outer membrane fragments. To this end, the OMV- The O-PS repeating unit in native FtLPS is the tetrasaccharide containing fraction isolated from JC8031 cells carrying pGAB2 was [2)-β-Qui4NFm-(1→4)-α-GalNAcAN-(1→4)-α-GalNAcAN-(1→3)- separated by density gradient ultracentrifugation. Western blotting β-QuiNAc-(1→] (43). To determine the structure of the carbohy- and Coomassie staining of the resulting fractions revealed that O-PS drate moiety in Ft-glycOMVs, we performed NMR analysis on LPS glycans and total proteins comigrated to denser fractions (SI Ap- derived from JC8031 carrying pGAB2. Ft-glycLPS extracted from pendix,Fig.S1C and D), reminiscent of the gradient profiles seen these cells was delipidated by mild acid hydrolysis and purified by previously for intact OMVs and OMV-associated proteins (27, 42). size-exclusion chromatography (SEC). SEC yielded carbohydrate After nondenaturing dot blotting, the FB11 antibody was observed fractions that were subjected to structural analysis by 1D and 2D to cross-react with these same, denser fractions, confirming that NMR. The 1D proton spectrum of the isolated product revealed O-PS glycans were on the exterior surface of intact vesicles (SI Ap- the presence of over 15 signals with different intensities in the pendix,Fig.S1C). It is particularly noteworthy that OMVs generated anomeric region (δ 5.5–4.4), and 2D NMR spectra revealed the from JC8031 cells carrying pGAB2 were observed to cross-react with presence of many spin systems. By study of the 2D COSY, heter- the mouse IgG2a antibody FB11. We conclude that the O-PS onuclear single-quantum coherence (HSQC), and heteronuclear structure generated on OMVs is immunologically relevant given that multiple bond correlation (HMBC) spectra (SI Appendix,Fig.S2), FB11 targets a unique terminal F. tularensis O-PS epitope, confers four residues belonging to 2-acetamido-2-deoxy-galacturonamide survival to BALB/c mice infected intranasally with the F. tularensis (GalNAcAN), 4,6-dideoxy-4-formamidoglucose (Qui4NFm), and type B LVS, and prolongs survival of BALB/c mice infected in- N-acetylglucosamine (GlcNAc), in a 2:1:1 ratio, could be discriminated tranasally with highly virulent F. tularensis type A strain Schu S4 (37). (SI Appendix,TableS1). In the 1D NMR spectrum, the signals at 5.41 and 5.03 ppm corresponded to the anomeric protons of two Structural Characterization of Heterologous F. tularensis O-PS. To shed GalNAcAN residues, designated as residues B and C. The HSQC light on the identity of the heterologous O-PS, Ft-glycOMVs were spectrum showed downfield signals for C-4 of residue B at 81.3 ppm

Chen et al. PNAS | Published online June 6, 2016 | E3611 Downloaded by guest on September 27, 2021 A

MW Ft-glycOMVsFt-glycLPSFtLPS [M+Na]+ FB11 B 250 100 100 849.2 75 90 50 80 37 70 + 60 [M+K] 25 50 20

40 865.2 15 30

Intensity (%) kDa 20 10 0 400 720 1040 1360 1680 Mass (m/z)

460.4 C Y C 676.5 2 3 Y 646.5 3 Qui4NFm GalNAcAN GalNAcAN HOH CGlcNAc 2 H C H NOC H NOC 3 2 2 O OH O O O O Precursor HN O O 849.2 [M+Na]+ OHC HO NHAc -CHO HO OH HO NHAc HO NHAc 821.8 -H O 100 2 ABC D 831.6 90 Z 80 2 442.4 -H O 70 2 Y Y 60 39.1 2 3 Loss of GalNAcAN Loss of Qui4NFm Fig. 3. Structural analysis of heterologous F. tular- 50 ensis O-PS. (A) Western blot analysis of Ft-glycOMVs, 40 Z 676.5 2 646.5 C Ft-glycLPS, and FtLPS. Blots were probed with the 30 3 FB11 antibody. Molecular mass (MW) markers are Intensity (%) Loss of GlcNAc labeled on the left. (B and C) MALDI-TOF MS spectra: 20 460.4 1 + 23.1 442.4 MS of isolated O-PS tetrasaccharide m/z 849.2 [M 10 + + Na] and 865.2 [M+K] (B); and product-ion MS/MS 0 + 9 186 364 542 720 898 of m/z 849.2 [M+Na] (C). The fragment ions were Mass ( / ) reported using the Domon and Castello nomencla- m z ture (62).

and of residue C at δ 78.0, in accordance with a 4-substituted peaks at m/z 849.2 and at m/z 865.2 correspond to the sodium and α-D-GalNAcAN residue (43). The anomeric signal at 4.50 ppm potassium adducts of the tetrasaccharide, respectively (Fig. 3B). To was attributed to terminal Qui4NFm (residue A). The methyl further characterize the topology of this oligosaccharide, the ion at + group of this residue resonates at 1.18 ppm (44). The assignment 849.2 [M+Na] was subjected to tandem MS. In the resulting MS2 of residues Dα and Dβ was complicated by extensive overlap of spectrum (Fig. 3C), the Y3 ion and Y2 ion at m/z 676.5 and at m/z signals stemming from other carbohydrate material. The broad 460.4, respectively, showed a loss of Qui4NFm and GalNAcAN signals at ∼5.15 ppm (Dα) and ∼4.57 ppm (Dβ) belong to re- from the nonreducing end. The presence of fragment ions at m/z ducing end N-acetylglucosamine (GlcNAc) (SI Appendix, Table 646.5 (C3)andm/z 442.4 (Z2) indicated a loss of GlcNAc and S1). The 13C chemical shifts, deduced from the HSQC spectrum, GalNAcAN from the reducing end. This result confirmed the showed the downfield position of C-3 of Dβ at 82.8 ppm, in- Qui4NFm-GalNAcAN-GalNAcAn-GlcNAc sequence. dicating a 3-substituted residue (45). The linkage sequence of the Ft- monosaccharide was determined by the HMBC spectrum. The Structural Diversification of Lipid A Yields Less Toxic glycOMVs. 4-substitution of residues B and C was supported by correlations LPS is a main contributing factor in triggering host immune response during infection through recognition of lipid A, also known as en- between H-1 of B and C-4 of C and between H-1 of A and C-4 of dotoxin, by toll-like receptor 4 (TLR4). Immune recognition of lipid B, respectively. Furthermore, the correlation between H-1 of C A results in production of proinflammatory cytokines that are crucial and C-3 of Dβ was clearly observed. On the basis of these 1H and to fight infection but may also contribute to lethal septic shock at 13C NMR data, it can be concluded that the isolated compound high levels (46). Thus, for glycOMVs to be a viable vaccine platform, is a tetrasaccharide of the following structure: it is necessary to reduce the toxicity of lipid A while also maintaining its adjuvanticity. One such LPS derivative, monophosphorylated lipid AB C DA(MPL)fromSalmonella minnesota R595, is an approved adjuvant with reduced toxicity (47). MPL is a mixture of monophosphorylated lipids, with the primary species being pentaacylated, mono- To confirm this conclusion from NMR, we analyzed the isolated phosphorylated lipid A. In contrast, native E. coli lipid A is charac- oligosaccharide using MALDI-TOF MS in positive ion mode. The terized by the presence of six acyl chains and two phosphate groups.

E3612 | www.pnas.org/cgi/doi/10.1073/pnas.1518311113 Chen et al. Downloaded by guest on September 27, 2021 Indeed, MS analysis of isolated lipid A from selected E. coli strains, 100 PNAS PLUS including JC8031 or JC8031 carrying plasmid pGAB2, revealed a prototypical hexaacylated, bis-phosphorylated lipid A (SI Appendix, 80 Fig. S3 A–C). To mimic the MPL structure in our glycOMVs, we adopted a lipid A remodeling strategy described by Trent and co- workers (34). Specifically, we deleted the acyltransferase-encoding 60 lpxM gene in JC8031, resulting in strain JH8033 that synthesized PBS pentaacylated lipid A in the absence or presence of pGAB2 (SI FtLPS 40 empty OMVs (JC8031) Appendix,Fig.S3D and E). Expression of the F. tularensis phos- empty OMVs (JH8033)

phatase LpxE from plasmid pE resulted in a strain that produced Percent survival nearly homogenous pentaacylated, monophosphorylated lipid A (SI 20 Ft-glycOMVs (JC8031 pGAB2) Appendix,Fig.S3F). When combinations of lipid A-modifying en- Ft-glycOMVs (JH8033 pGAB2) zymes (e.g., LpxE and E. coli lipid A palmitoyltransferase PagP 0 coexpressed from plasmid pEP; Salmonella typhimurium lipid A 3′-O- 0 14723 56 8 deacylases PagL and LpxR, and PagP coexpressed from plasmid Days post infection pLPR) were coexpressed, a more heterogeneous mixture was observed as a consequence of the substrate specificity and limited expression level of the transmembrane lipid A-modifying enzymes (SI 100 Appendix,Fig.S4A and B), as seen previously (34). Importantly, JH8033 cells carrying pGAB2, or pGAB2 along with any of the 80 plasmids encoding lipid A-modifying enzymes, produced F. tularensis O-PS on the cell surface on par with that produced by JC8031 60 carrying pGAB2 (SI Appendix, Fig. S3G). Likewise, glycOMVs harvested from these strains also displayed the F. tularensis O-PS at levels comparable with glycOMVs from JC8031 (SI Appendix, 40 Fig. S3G). Ft-glycOMVs (JH8033 pE pGAB2) Percent survival Next, toxicity of whole cells and OMVs was evaluated by 20 Ft-glycOMVs (JH8033 pEP pGAB2) measuring human TLR4 activation in HEK-Blue hTLR4 re- Ft-glycOMVs (JH8033 pLPR pGAB2) porter cells. These cells express hTLR4 and respond to activation of the receptor by the production of secreted embryonic 0 alkaline phosphatase (SEAP) (34). Upon incubating this reporter 0 14723 56 8 cell line with whole bacterial cells, significantly lower TLR4 acti- Days post infection vation (P < 0.01) was observed for all JH8033 strains compared – with their JC8031 counterparts (SI Appendix,Fig.S5A), with sig- Fig. 4. Ft-glycOMVs delay onset of lethal disease with Schu S4. Kaplan Meier survival analysis of nine groups of BALB/c mice, five mice per group, nals that were comparable with a previously detoxified strain: SCIENCES immunized i.p. with the following: PBS, FtLPS, empty OMVs derived from namely BN2 (as W3110 ΔlpxM) (34). Notably, the presence of the

JC8031 or JH8033, Ft-glycOMVs derived from JC8031 pGAB2 or JH8033 APPLIED BIOLOGICAL F. tularensis O-PS did not have any impact on TLR4 activation. pGAB2 (Upper); and Ft-glycOMVs derived from JH8033 pE pGAB2, JH8033 The TLR4 activation assay was also run by treating the HEK-Blue pEP pGAB2, or JH8033 pLPR pGAB2 (Lower). To ensure that an equivalent reporter cell line with purified OMVs. As with whole cells, OMVs amount of LPS was used in each case, the LPS content of OMVs and purified derived from all JH8033 strains showed significantly reduced ac- LPS was normalized based on reactivity to FB11 antibody. Mice were boosted tivation of TLR4 compared with OMVs derived from the corre- 28 d after the original immunization with the same antigen and amount as sponding JC8031 strain (SI Appendix,Fig.S5B). the original dose. At 56 d after the primary injection, all mice were challenged i.p. with 25 cfus of F. tularensis Schu S4. Survival of mice in the Ft-glycOMVs Protect Against Lethal F. tularensis Challenge. Ft-glycOMV groups compared with those in the PBS or empty OMV control An ef- < fective vaccine for tularemia will likely require multiple antigens, groups was found to be significant (P 0.05; log-rank test). but, as an initial step in determining whether Ft-glycOMVs might be a candidate vaccine component for a multivalent vaccine, we To confirm the reproducibility of these results, a second nearly evaluated their protective efficacy in mice infected with the identical challenge was performed, except with two additional highly virulent F. tularensis type A strain Schu S4, which has an control groups: Ft-glycLPS alone and “sham” glycOMVs from LD of <10 colony-forming units (cfus) in mice (48). BALB/c 50 JC8031 cells expressing S. dysenteriae O-PS genes (Sd-glycOMVs). mice were immunized by an i.p. route with Ft-glycOMVs derived At 56 d after the initial dose, immunized mice were challenged from either JC8031 cells, JH8033 cells, or JH8033 cells carrying a with 22 cfus of F. tularensis Schu S4 via i.p. injection, and survival plasmid with the lipid A-modifying enzymes, purified FtLPS, of the mice was monitored. As above, mice immunized with empty OMVs from JC8031 cells, empty OMVs from JH8033 < cells, or PBS. At 56 d after the initial dose, immunized mice were Ft-glycOMVs demonstrated a significantly (P 0.05) delayed time challenged with 25 cfus of F. tularensis Schu S4 via i.p. injection, to death (mean increase of 3 d) compared with PBS-treated and survival of the mice was monitored. All mice receiving one of control mice, with all mice in this group surviving until day 6 and the Ft-glycOMV preparations survived until day 6 or 7, which three of the mice surviving until day 7 (SI Appendix,Fig.S6and corresponded to a significantly (P < 0.05) delayed time to death Table S2). This increase in time to death was specific to the (mean increase of 2.0–2.4 d) compared with PBS-treated control F. tularensis O-PS on OMVs because mice immunized with Sd- mice (Fig. 4 and SI Appendix, Table S2). In contrast, mice im- glycOMVs experienced no such increase in protection (P > 0.1) munized with either purified FtLPS or either of the empty OMV (SI Appendix, Fig. S6 and Table S2). Immunization with polysac- preparations all succumbed to infection within 5 d (Fig. 4 and SI charides alone also afforded no protection against challenge be- Appendix, Table S2), which was the same time as the PBS control cause mice that received either FtLPS or Ft-glycLPS perished at group. Importantly, there was no significant difference in survival the same rate as control mice that had received PBS (P > 0.1), between any of the various Ft-glycOMV–treated groups, suggesting with all mice except one dying within 4 d (one mouse receiving that detoxified Ft-glycOMVs afforded the same level of protection Ft-glycLPS succumbed to infection on day 5) (SI Appendix,Fig.S6 against pathogen challenge as their unmodified counterpart. and Table S2).

Chen et al. PNAS | Published online June 6, 2016 | E3613 Downloaded by guest on September 27, 2021 To determine whether Ft-glycOMVs can provide cross-strain A 10000000 p < 0.01 protection against other bacteria having structurally similar O-PS structures, immunized mice were challenged with two different F. tularensis subsp. holarctica (type B) LVS isolates, both sig- 1000000 nificantly less virulent than Schu S4. Specifically, LVS Iowa, which has an intranasal LD50 of ∼3,000–4,000 cfus, and the significantly more virulent LVS RML (intranasal LD50 is ∼175 100000 cfus) were used. Mice were immunized identically as above with Ft-glycOMVs derived from JC8031 or JH8033 cells, or PBS. At 56 d after the initial dose, immunized mice were challenged with 10000 4–400 cfus of LVS RML or LVS Iowa. All of the PBS-treated control mice when infected i.p. with only 4 cfus of either LVS isolate died within a week. The PBS-treated control mice in- Ft LPS-specific IgG titers 1000 fected with LVS RML all died on day 5 whereas those infected with LVS Iowa all died by day 7 (four mice died on day 6 and the last one on day 7) (SI Appendix, Fig. S7 A and B and Table S2). 100 PBS FtLPS JC8031 JH8033 JC8031 JH8033 JH8033 JH8033 JH8033 In contrast, mice immunized with Ft-glycOMVs were completely empty empty pE pEP pLPR protected against challenge by either strain up to 400 cfus, which OMVs OMVs Ft-glycOMVs was the highest dose tested (SI Appendix, Fig. S7 A and B and Table S2). Not only did we observe excellent protection against B p < 0.01 both of these strains, but none of the Ft-glycOMV–vaccinated 100000000 mice even seemed sick, suggesting that protection was probably day 56 IgG1 higher than we were able to see in this experiment. Taken to- day 56 IgG2a gether, our results demonstrate that Ft-glycOMVs offer pro- 10000000 tection against both type A and type B infection. 1000000 Ft-glycOMVs Induce a Mixed Th1/Th2 Response. To confirm that the

protective effects seen with Ft-glycOMVs correlated with in- 100000 creased antibody titers, the levels of FtLPS-specific IgGs were assessed in mice before challenge. Using ELISA with native FtLPS as the antigen, the total IgG titers for mice receiving Ft- 10000 glycOMVs were significantly increased (P < 0.01) compared with

all other groups as early as 14 d after immunization (SI Appendix, Ft LPS-specific IgG titers 1000 Fig. S8). At this time, the mean IgG titer was two orders of magnitude greater than the mean titer of PBS control group mice. This 100 differential became further amplified after the booster injection, PBS FtLPS JC8031 JH8033 JC8031 JH8033 JH8033 JH8033 JH8033 empty empty pE pEP pLPR reaching a maximum difference of three orders of magnitude at OMVs OMVs 56 d (Fig. 5A and SI Appendix,Fig.S9A). Likewise, immunization Ft-glycOMVs with Ft-glycOMVs derived from JH8033 cells carrying pGAB2, or Fig. 5. Ft-glycOMVs boost production of FtLPS-specific IgG antibodies. pGAB2 along with any of the plasmids encoding lipid A-modifying (A) FtLPS-specific IgG titers in endpoint (day 56) serum of individual mice (black enzymes, was significantly higher than the PBS and FtLPS control dots) and median titers of each group (red lines). Nine groups of BALB/c mice, groups (P < 0.01) (Fig. 5A). Importantly, there was no significant five mice per group, immunized i.p. with the following: PBS, FtLPS, empty OMVs difference in IgG titers after immunization with Ft-glycOMVs derived from JC8031 or JH8033, Ft-glycOMVs derived from JC8031 pGAB2 or harboring WT lipid A versus remodeled lipid A (P > 0.2) (Fig. 5A), JH8033 pGAB2, and Ft-glycOMVs derived from JH8033 pE pGAB2, JH8033 pEP pGAB2, or JH8033 pLPR pGAB2. To ensure that an equivalent amount of LPS was suggesting that detoxification of OMVs had no measurable effect used in each case, the LPS content of OMVs and purified LPS was normalized on their immunogenicity or adjuvanticity. IgG antibody titers were based on reactivity to FB11 antibody. Mice were boosted on day 28 with the further broken down by analysis of IgG1 and IgG2a titers, wherein same doses. (B) Median IgG subtype titers measured from endpoint serum with mean IgG1-to-IgG2a antibody ratios served as an indicator of a IgG1 titers in gray and IgG2a in black. An asterisk (*) indicates statistical signif- Th1- or Th2-biased immune response. Mice immunized with icance (P < 0.01; Tukey–Kramer HSD) of antibody titers against PBS control Ft-glycOMVs showed a significant (P < 0.01) increase in mean titers group. A double asterisk (**) indicates statistically significant difference (P < 0.05; of both FtLPS-specific IgG1 and IgG2a (Fig. 5B and SI Appendix, unpaired t test) in IgG1 and IgG2a titers within the group. Fig. S9B). The higher IgG1 versus IgG2a titers suggested a slight bias toward a Th2 response. The relative titers of IgG1 and IgG2a < subtypes from groups immunized with JH8033-derived Ft-glycOMVs protection against F. tularensis LVS challenge, significantly (P were comparable with the titers observed for JC8031-derived Ft- 0.01) enhanced FtLPS-specific IgA production in the bron- glycOMVs. Classically, a Th1-biased immune response is impor- choalveolar lavage (BAL) and vaginal lavage (VL) fluids, as well tant for intracellular pathogens such as F. tularensis;however, as the sera of immunized mice above that of empty OMVs, several groups have shown the importance of both Th1 and Th2 FtLPS, or PBS (shown for BAL and VL fluids in SI Appendix, immune responses for this particular pathogen (36). Fig. S10 A and B, respectively). The increased IgA titers correlated with a similarly significant (P < 0.01) increase in total Ft-glycOMVs Induce Mucosal IgA Production. Recent studies in- FtLPS-specific blood sera IgG titers (SI Appendix, Fig. S10C). dicate that IgA antibodies also play a significant role in protection against F. tularensis infection (49). As the predominant Discussion antibody found at mucosal sites, increased IgA production pro- In the present study, we describe a glycoconjugate vaccine plat- vides one possible means of enhancing protection against mu- form that leverages the immunological potential of recombinant cosal infection. Consistent with the latter, Ft-glycOMVs derived OMVs. This platform is founded in part on our previous finding from JH8033 cells, which significantly delayed time to death that remodeling the surface of OMVs with weakly immunogenic against F. tularensis Schu S4 challenge and generated 100% protein antigens yielded OMV-based vaccine candidates that

E3614 | www.pnas.org/cgi/doi/10.1073/pnas.1518311113 Chen et al. Downloaded by guest on September 27, 2021 boosted antigen-specific IgG levels (28). In fact, the response tibodies and thus dampens the protective effects of these host PNAS PLUS elicited by the engineered OMVs rivaled that obtained when the molecules (52). same protein antigen was adsorbed to the FDA-approved adjuvant One major route of F. tularensis infection is through inhalation alum (28), suggesting that OMVs function not only as nano- and other mucosal routes, and thus the presence of mucosal IgA particulate vaccine carriers but also as vaccine adjuvants (50). The antibodies is important in protection (49). To stimulate a protective basis for this adjuvanticity is likely due to the fact that OMVs (i)are mucosal immune response, vaccines must often be introduced readily phagocytosed by professional antigen-presenting cells; through mucosal routes, such as intranasal administration. Here, (ii) carry pathogen-associated molecular patterns (PAMPs) within we have shown that glycOMVs can generate an antigen-specific their structure that can stimulate both innate and adaptive immu- mucosal IgA response through s.c. administration, and this re- nity; and (iii) possess strong proinflammatory properties (21–23). sponse correlates with both high antigen-specific IgG titers in sera Because carbohydrates are also commonly known to be weak as well as protection against challenge. antigens (3–5), we hypothesized that delivery of specific poly- Antibodies alone do not provide hosts with protection against saccharide structures by engineered OMVs would enhance the tularemia. Indeed, studies have shown that adaptive immunity immune response to these weakly immunogenic epitopes due to against F. tularensis also requires a robust cell-mediated response the natural adjuvanticity of the OMV carriers. To test this hy- (53). Specifically, a T cell-dependent response is required to control pothesis, we created glycoengineered OMVs by combining the infection and is likely to hinge on the activation of macrophages. vesicle formation process with heterologous glycan biosynthesis Incidentally, F. tularensis is known to target macrophages and is able machinery in laboratory strains of E. coli. An attractive feature of to suppress the early inflammatory responses necessary in contain- γ our approach is the fact that different plasmid-encoded O-PS bio- ing the pathogen (54). Thus, early IFN activation of macrophages synthetic pathways can be readily transformed into E. coli,enabling is vital to control infection (55). Intracellular cytokine staining of “ ” splenocytes from mice vaccinated with Ft-glycOMVs revealed a a plug-and-play platform for the creation of glycOMVs that + surface display heterologous glycotopes from pathogenic bacteria. population of CD3 T cells that responded to restimulation with γ In the most notable example, hypervesiculating E. coli strain FtLPS in vitro with increased production of IFN (SI Appendix, F. tularensis Fig. S11), suggesting the generation of a small T cell-dependent JC8031 harboring the Schu S4 O-PS pathway genes – yielded OMVs that were glycosylated with a structural mimetic of response. However, this response was not limited to the Ft-glycOMV F. tularensis O-PS. Although glycan analysis revealed subtle chem- vaccinated group; similar shifts in T-cell population were seen in the FtLPS group as well as the PBS control group (SI Appendix, Fig. ical structural differences between the tetrasaccharide unit found in γ native FtLPS and the heterologous O-PS on Ft-glycOMVs, these S11). This finding, coupled with the fact that the shift in IFN - structural differences did not seem to significantly alter the prop- producing T cells was small, suggests that the observed T-cell erties of the O-PS. Indeed, the heterologous glycan exhibited a activation may be nonclassical because the antigen is not one classically associated with MHC presentation. Indeed, the small laddering pattern that was still recognized by FB11 antibodies shift in IFNγ-producing cells may be the result of stimulating γ/δ generated against the native FtLPS structure (37). T cells, a rare subset of T cells capable of MHC-independent The best evidence for authentic glycomimicry, however, was the activation (56). fact that vaccination with the resulting Ft-glycOMVs significantly One concern with the use of bacterial OMVs as a vaccination SCIENCES boosted the production of FtLPS-specific IgG antibodies as early as – platform is toxicity as a result of the presence of LPS on the 2 wk after the initial immunization and by as much as 2 3ordersof APPLIED BIOLOGICAL membrane surface. This concern may be addressed by chemically magnitude above all controls, including native FtLPS. This finding stripping away LPS from OMVs through the use of polymyxin B is particularly noteworthy in light of the generally observed phe- columns (23, 28). However, because our strategy involves as- nomenon that the immune response generated against purified sembling O-PS directly upon lipid A, stripping away LPS would LPS is T cell-independent and does not result in the production of remove the desired polysaccharide epitope as well. To circum- antigen-specific IgG antibodies (4), as was confirmed here with vent this issue, the lipid A structure of JC8031 E. coli was native FtLPS and engineered Ft-glycLPS. The high IgG titers and remodeled at the genetic level to yield a variant that is signifi- broad response produced as a result of vaccination with Ft- cantly less toxic, as measured by hTLR4 activation, while still glycOMVs suggests stimulation of immunological responses that retaining desirable immunomodulatory qualities. Previous com- are otherwise nonexistent against classical T cell-independent an- binatorial engineering of E. coli lipid A demonstrated that re- tigens. Indeed, T cells and the presence of toll-like receptor signals moval of an acyl chain by LpxM yields a pentaacylated lipid A on OMVs may serve secondary roles during immune responses structure with significantly reduced toxicity (34). Further de- (51). Importantly, the robust immune response elicited by glycOMVs toxification was achieved by removal of the 1-phosphate group by provided protection against lethal challenge by F. tularensis Schu S4, expression of F. tularensis LpxE (34). Here, we showed that strain as demonstrated by an increased time to death compared with JH8033, which was engineered to produce pentaacylated lipid A, vaccination with different controls, including FtLPS alone. The induced significantly lower TLR4 activation compared with the extended protection was attributed to the presence of the O-PS parental JC8031 strain producing a prototypical hexaacylated, bis- because no protection was afforded to mice vaccinated with phophorylated lipid A structure. In our hands, the further expres- sham OMVs containing a nonspecific O-PS structure (Sd-glycOMV). sion of LpxE (or any other lipid A-modifying enzymes including In addition to protection against this type A strain of F. tularensis, PagP, PagL, and LpxR) did not further reduce activation in either glycOMVs also provided complete protection against two dif- the JC8031 or JH8033 strain background, suggesting that LpxM- ferent F. tularensis type B strains that displayed similar O-PS mediated deacylation is responsible for the observed detoxification structures in the outer leaflet of their outer membranes. Spe- of E. coli lipid A. The resulting detoxified Ft-glycOMVs stimulated cifically, mice vaccinated with any of the different Ft-glycOMV FtLPS-specific IgG antibody titers that were nearly indistinguish- preparations were able to clear the infections caused by F. able from those elicited by Ft-glycOMVs bearing native E. coli tularensis subsp. holarctica LVS RML and LVS Iowa, and survive. lipid A, suggesting that there was no loss in adjuvant activity for At present, the underlying reason or reasons for why glycOMVs glycOMVs with remodeled lipid A, including pentaacylated, were more effective against the LVS isolates than the more vir- monophosphorylated structures resembling MPL. ulent Schu S4 remain unknown. However, one possibility might Overall, the results from this study represent a promising proof be related to the observation that highly virulent Schu S4, but of concept for the use of engineered OMVs as a platform for the not the closely related LVS, is able to bind the host serine delivery of carbohydrate-based vaccines. This system combines the protease plasmin, which allows evasion of opsonization by an- benefits of natural and synthetic vaccines into a singular platform

Chen et al. PNAS | Published online June 6, 2016 | E3615 Downloaded by guest on September 27, 2021 that overcomes many of the production and formulation hurdles glass plate to remove any residual organic phase and determine the mass of the that have plagued other glycoconjugate-based vaccines. Moreover, purified LPS. the vesicle architecture helps surface-exposed membrane antigens (e.g., proteins and polysaccharides) maintain their physico-chemical Fractionation of OMVs. Prepared OMVs were separated by density-gradient stability (24). Our results clearly show that glycOMVs are an all- ultracentrifugation as previously described (27). Briefly, OMVs were prepared as above but resuspended in a 50-mM Hepes, pH 6.8 solution. This in-one antigen, adjuvant, and delivery platform that is able to gen- solution was adjusted to 45% (vol/vol) Optiprep (Sigma) in 1.5 mL. All other erate a robust antibody response, induce a T-cell response, and confer Optiprep layers were prepared using the same 50-mM Hepes, pH 6.8 solu- protection against lethal challenge, whereas the polysaccharide anti- tion. Optiprep/Hepes gradient layers were added to a 12-mL ultracentrifuge gen alone failed in all three criteria. Compared with conventional tube as follows: 0.33 mL of 10%, 0.33 mL of 15%, 0.66 mL of 20%, 0.66 mL of approaches for producing glycoconjugate vaccines, glycOMV vaccine 25%, 0.9 mL of 30%, 0.9 mL of 35%, and 1.5 mL of 45% containing the production is significantly less complicated, less time consuming, less prepared OMVs, and enough 60% to nearly fill the tube. Gradients were expensive, and more scalable. It requires only one cultivation step to centrifuged (Beckman-Coulter; TiSW41 rotor; 180,000 × g; 3 h; 4 °C), and generate the final product, which can be easily and economically then a total of 10 fractions of 0.5 mL each were removed sequentially from isolated by a single ultracentrifugation step (28, 50). Another ad- the top of the gradient. These fractions were analyzed by Western blot and dot blot analyses as described below. vantage is that, by combining the polysaccharide biosynthesis and conjugation steps in a single, nonpathogenic strain of E. coli,final Western Blot Analysis. OMV and LPS samples were prepared for SDS/PAGE products are well-defined and can be flexibly tailored for specific analysis by boiling for 15 min and cooling to room temperature in the diseases simply by rewiring the polysaccharide biosynthesis pathway. presence of loading buffer containing β-mercaptoethanol. Samples were run Best of all, this result can be accomplished without ever having to on 12% (wt/vol) polyacrylamide gels (Mini-PROTEAN TGX; Bio-Rad) and handle or cultivate pathogenic bacteria. Finally, it should be pointed transferred to a PVDF membrane. After blocking with a 5% (wt/vol) milk outthatotherbiomolecularfeaturesofOMVs(e.g.,proteinsand solution, membranes were probed first with a primary antibody against the lipids) can also be engineered (33), in harmony with OMV vaccine specified O-PS and then with the corresponding HRP-conjugated secondary designs that contain a high density of specific carbohydrate structures, antibody (SI Appendix, Table S4). Signal was visualized using HRP substrate and either an X-ray film developer or a ChemiDoc Imaging System (Bio-Rad). making it possible in the future to create designer OMVs against a wide variety of targets with tunable immunomodulatory effects. Dot Blot Analysis. OMV samples were prepared by making the appropriate dilutions and spotting directly onto a nitrocellulose membrane, or by boiling Materials and Methods for 10 min and cooling to room temperature before spotting on the mem- Bacterial Strains and Plasmids. The bacterial strains and plasmids used in this study brane. After blocking with a 5% (wt/vol) milk solution, membranes were are described in SI Appendix,TableS3.Briefly,E. coli strain JC8031, a tolRA mutant probed first with the mouse mAb FB11 to F. tularensis LPS and then with HRP- strain that is known to hypervesiculate (40), was used for preparation of OMVs. conjugated anti-mouse IgG. Signal was visualized using HRP substrate and Strain CE8032, a waaL::Kan mutant derived from JC8031, was used as a control either an X-ray film developer or a ChemiDoc Imaging System (Bio-Rad). (57). Strain JH8033 was generated from JC8031 using P1 transduction of the lpxM::kan allele from the Keio collection as described in previous work (58). Electron Microscopy. Structural analysis of vesicles was performed via trans- Plasmids pE, pEP, and pLPR were constructed previously (34). F. tularensis Schu S4 mission electron microscopy as previously described (28). Briefly, vesicles were was provided by Jeannine Peterson (Centers for Disease Control and Prevention, negatively stained with 2% (wt/vol) uranyl acetate and deposited on 400- Fort Collins, CO). F. tularensis subsp. holarctica LVS Iowa is the original ATCC 29684 mesh Formvar carbon-coated copper grids. Imaging was performed using an strain that has been passaged for several years in the B.D.J. laboratory and was FEI Tecnai F20 transmission electron microscope. originally provided by Karen Elkins (US Food and Drug Administration, Rockville, MD). F. tularensis subsp. holarctica LVS RML was provided by Katy Bosio [Rocky Preparation of Ft-glycLPS for Structure Determination. An overnight culture of Mountain Laboratories (RML), NIAID, NIH, Hamilton, MT]. LVS RML was originally E. coli JC8031 carrying pGAB2 was subcultured in 4 L of LB medium. The acquiredbyFranNano(UniversityofVictoria, Victoria, BC, Canada). The strain culture was grown overnight, and the cell pellet was collected via centrifu- designations of both LVS isolates have been confirmed by the absence of pdpD, gation. The cell pellet was suspended in 2 mL of water, to which nine vol- absence of pilA, and deletion in the C terminus of FTT0918 (59). umes of ethanol were added, and agitated for 1 h at room temperature. After centrifugation at 3,000 × g for 15 min, the supernatant was removed, Cell Growth and Preparation of OMVs. OMVs were prepared as described pre- and the cells were resuspended in 10 mL of 90% (vol/vol) ethanol and viously (28). Briefly, a plasmid containing a specific O-PS pathway (SI Appendix, extracted again with nine volumes of ethanol for 15 min at room temper- Table S3) was transformed into the hypervesiculating E. coli strain JC8031, or ature with agitation. The cells were then pelleted via centrifugation and related strain, and selected on medium supplemented with the appropriate an- resuspended in 20 mM Tris·HCl, pH 8.0 containing 2 mM CaCl2, and digested tibiotic. An overnight culture of a single colony was subcultured into 100–200 mL overnight with 2–5 mg/mL Proteinase K at room temperature. Digestion was of Luria–Bertani (LB) medium. The culture was grown to mid-log phase, at which followed by ultracentrifugation at 100,000 × g for 16 h. The pellet was then time protein expression was induced with L-arabinose (0.2%) or isopropyl β-D-1- subjected to phenol/water extraction. All of the samples were transferred thiogalactopyranoside (IPTG) (0.1 mM), if necessary. Cell-free culture supernatants into glass tubes, freeze-dried, and resuspended in 5 mL of water. The cell were collected 16–20 h postinduction and filtered through a 0.2-μm filter. Vesicles suspension was heated to 65 °C with stirring and extracted with 5 mL of were isolated by ultracentrifugation (Beckman-Coulter; TiSW28 rotor; 141,000 × g; preheated 90% (wt/vol) phenol for 1 h. The suspension was cooled on ice, 3 h; 4 °C) and resuspended in PBS. OMVs were quantified by the bicinchoninic- and the mixture was centrifuged at 3,000 × g. The phenol phase was acid assay (BCA Protein Assay; Pierce) using BSA as the protein standard. reheated and reextracted with 5 mL of hot water. This process was repeated one more time. The combined aqueous phases were dialyzed (1000-Da Preparation of LPS. Purified F. tularensis subsp. holarctica LPS (FtLPS), whose O-PS MWCO), freeze-dried, and resuspended in 1.8 mL of 20 mM Tris·HCl, pH 8.0

repeats are identical in structure to the O-PS repeats in F. tularensis subsp. containing 2 mM MgCl2. A 100-μL aliquot of 7 mg/mL DNase I in 20 mM tularensis Schu S4 LPS (38), was obtained from BEI Resources. LPS derived from Tris·HCl, pH 8.0 and 2 mM MgCl2 was added to the sample. After incubation E. coli carrying pGAB2 (Ft-glycLPS) was prepared using a modification of a pre- for 3 h at 37 °C, 100 μL of 17 mg/mL RNase A was added, which was followed

viously published protocol (60). Briefly, an overnight culture of a single colony was by another 3 h incubation at 37 °C. Finally, CaCl2 was added to a final subcultured into 500 mL of LB medium. The culture was grown overnight (16–20 h), concentration of 2 mM, and the sample was digested with 400 μg of Pro- and the cell pellet was collected by centrifugation. The pellet was resuspended in teinase K overnight at room temperature. The Proteinase K was inactivated 10 mL of lysis buffer [2% (wt/vol) SDS, 4% (vol/vol) β-mercaptoethanol, and at 100 °C for 5 min, and the samples were ultracentrifuged at 100,000 × g 100 mM Tris·HCl, pH 7.5] and heated in a boiling water bath for 10 min. Pro- overnight. The pellets containing isolated LPS were lyophilized and sub- teinase K was added to a final concentration of 2 mg/mL and incubated at 50 °C jected to a chromatographic separation. The crude LPS was dissolved in overnight. The next morning, phenol was added, and the mixture was incubated 50 mM ammonium acetate buffer and injected into an Agilent 1200 HPLC at 70 °C for 15 min, with vortexing every 5 min. The mixture was cooled on ice equipped with a refractive index (RI) detector. The separation was per- and then centrifuged for 10 min at 13,000 × g. The aqueous phase was collected formed on a Superose 12 10/300 GL column, equilibrated, and eluted by and extracted with ether and then centrifuged for 5 min at 13,000 × g.The 50 mM ammonium acetate, pH 5.5 at a flow rate of 0.5 mL/min. The fraction aqueous phase was collected, containing the LPS. This solution was dried on a eluted at void volume was collected and freeze-dried. The dried fraction was

E3616 | www.pnas.org/cgi/doi/10.1073/pnas.1518311113 Chen et al. Downloaded by guest on September 27, 2021 resuspended into DOC buffer (200 mM NaCl, 10 mM Tris·HCl, 0.25% deoxy- significance was determined using a log-rank test compared with survival of the PNAS PLUS cholate sodium, 1 mM EDTA, pH 9.2) and injected into an Agilent 1200 PBS control group. The protocol number for the animal studies was no. 1305086 HPLC equipped with RI detector and a Superdex 75 10/300 GL column approved by the University of Iowa Animal Care and Use Committee. equilibrated previously with DOC buffer. DOC buffer was used as eluent with a flow of 0.5 mL/min. The major fractions were collected and dialyzed Mucosal Response Immunizations. Groups of 10 BALB/c female mice aged 6 to using a 2,000 molecular weight cut-off (MWCO) membrane against three 8 wk old (The Jackson Laboratory) were each immunized s.c. with either PBS changes of buffer containing 9% EtOH, 4 mM Tris, and 40 mM NaCl, fol- (pH 7.4) alone (control) or 100 μL of PBS (pH 7.4) containing 10 μgofFt- lowed by three changes of deionized (DI) water. The retentate containing glycOMVs from JH8033, 10 μg of empty OMVs from JH8033, or 2 μgofFtLPS. the LPS was lyophilized. Next, the isolated LPS was dissolved in 500 μLof1% Each group of mice was boosted 28 d after the initial immunization with the acetic acid and incubated at 100 °C overnight. The supernatant was taken same dosage. Blood was collected from each mouse from the mandibular and freeze-dried after centrifugation at 6,000 × g for 30 min and subjected to sinus immediately before the initial and boost immunizations. Forty-two further analysis. days after the initial immunization, the mice were killed, and blood was collected via cardiac puncture. Mucosal samples were also collected via NMR Spectroscopy and MALDI-TOF MS. For NMR spectroscopy, the sample was bronchoalveolar lavage and vaginal lavage. The protocol number for the dissolved in D2O (99.8% D; Aldrich), freeze-dried, and again dissolved in animal studies (2009-0096) was approved by the Institutional Animal Care 280 μLofD2O (99.96% D; Cambridge Isotope Laboratories) containing 0.5 μLof and Use Committee at Cornell University. acetone as internal reference. The sample was placed into a 5-mm Shigemi NMR tube with magnetic susceptibility plugs matched to D2O; 1D proton, 2D gradient Enzyme-Linked Immunosorbent Assay. FtLPS-specific antibodies produced in correlation spectroscopy (gCOSY), zero quantum-filtered total correlation spec- immunized mice were measured via indirect ELISA using a modification of a troscopy (zTOCSY), adiabatic rotating frame nuclear Overhauser effect spec- previously described protocol (28). Briefly, sera were isolated from the col- troscopy (ROESYad), and multiplicity-edited gradient HSQC (gHSQC) spectra lected blood draws after centrifugation at 2,200 × g for 10 min. Ninety-six– were acquired on a Varian 600-MHz instrument at 30 °C. The mixing times for well plates (Maxisorp; Nunc Nalgene) were coated with FtLPS (5 μg/mL in zTOCSY and ROESY were 80 and 200 ms, respectively. The spectra were refer- PBS, pH 7.4) and incubated overnight at 4 °C. All PBS used was at pH 7.4. The δ = δ = enced relative to the acetone signal ( H 2.218 ppm; C 33.0 ppm). For MALDI- next day, plates were washed three times with PBST (PBS, 0.05% Tween 20, TOF analysis, the experiments were performed in reflector-positive ion mode 0.3% BSA) and blocked overnight at 4 °C with 5% (wt/vol) nonfat dry milk using an AB SCIEX TOF/TOF 5800 (Applied Biosystems). The acquisition mass (Carnation) in PBS. Samples were serially diluted, in triplicate, between 1:100 range was 200–6,000 Da. Samples were prepared by mixing on the target 1-μL and 1:12,800,000 in blocking buffer and added to the plate for 2 h at 37 °C. sample solutions with 1 μL of 2,5-dihydroxybenzoic acid in 50% (vol/vol) meth- Plates were washed three times with PBST and incubated for 1 h at 37 °C in anol as matrix solution. the presence of one of the following horseradish peroxidase-conjugated antibodies: goat anti-mouse IgG (1:25,000; Abcam), anti-mouse IgG1 Mouse Immunizations and F. tularensis Challenge. Six groups of 10 BALB/c (1:25,000; Abcam), anti-mouse IgG2a (1:25,000; Abcam), or anti-mouse IgA female mice aged 6 to 8 wk old [National Cancer Institute (NCI)] were each (1:5,000; Abcam). After three additional washes with PBST, 3,3′-5,5′-tetra- immunized i.p. with 100 μL of PBS containing LPS or OMVs, prepared as methylbenzidine substrate (1-Step Ultra TMB-ELISA; Thermo Scientific) was described. All PBS used was at pH 7.4. The six groups were immunized with added, and the plate was incubated at room temperature for 30 min. The μ μ either PBS alone (control), 2 g of native F. tularensis LPS (FtLPS), 10 gof reaction was halted with 2 M H2SO4. Absorbance was quantified via micro- OMVs from JC8031 or JH8033 cells carrying no plasmid (empty OMVs), 10 μg plate spectrophotometer (Molecular Devices) at a wavelength of 450 nm. of OMVs from JC8031 cells harboring pGAB2 (Ft-glycOMVs), or 10 μgof Serum antibody titers were determined by measuring the lowest dilution that OMVs from JC8033 cells harboring pGAB2 and plasmids encoding for lipid A resulted in signal three SDs above background. Statistical significance was SCIENCES modifying enzymes. Before immunization, the LPS content of OMVs and determined using Tukey’s post hoc honest significant difference (HSD) test purified LPS was quantified based on reactivity to FB11 antibody in ELISA and compared against the PBS control case. APPLIED BIOLOGICAL format and by a standard colorimetric assay to detect 2-keto-3-deoxy- octonate (KDO), a core sugar component of LPS, as described previously (28). Intracellular Cytokine Staining. Splenocytes were seeded in 96-well plates at a The amount of LPS in each preparation was then normalized to ensure that density of 1 × 106 cells per well in complete RPMI 1640 and supplemented an equivalent amount of LPS was administered in each case. Each group of with 10% (vol/vol) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 U/mL mice was boosted with an identical dosage of antigen 28 d after the priming IL-2 (eBioscience). To each well, 100 μg/mL FtLPS was added and incubated at dose. Blood was collected from five mice of each group from the mandibular 37 °C for 24 h. Brefeldin A (eBioscience) was added 4 h before harvesting. sinus immediately before and 14 d after the first immunization, immediately Cells were then harvested, blocked with anti-CD16/32, and stained with before the boosting dose, and at 14 and 28 d after the boosting dose. Alexa 488-conjugated anti-CD3e. Cells were washed and fixed using 2% = Terminal splenectomies were performed on one-half (n 5) of all six groups (wt/vol) paraformaldehyde (eBioscience). Cells were then permeabilized at 56 d after the priming dose. The remaining five mice in each of the six with 0.1% saponin (eBioscience) and incubated with anti-INFγ, anti-TNFα,or groups were challenged i.p. with 25 cfus of F. tularensis Schu S4, and the anti–IL-4, all PE-Cy7.5–conjugated. Data were collected on a FACScalibur health of the mice was examined daily for signs of disease. A separate flow cytometer (Becton Dickinson) and analyzed using FlowJo (Treestar). All challenge was performed as described above with the following changes. antibodies used in this section were sourced from eBioscience unless μ Mice groups were immunized with either PBS alone (control), 2 g of native noted otherwise. F. tularensis LPS (FtLPS), 2 μg of LPS derived from JC8031 cells producing heterologous F. tularensis O-PS from pGAB2 (Ft-glycLPS), 10 μg of OMVs Characterization of Mutant Lipid A. Lipid A was prepared from 15-mL cultures from JC8031 cells harboring pGAB2 (Ft-glycOMVs), or 10 μg of sham OMVs and analyzed using a MALDI-TOF/TOF (ABI 4700 Proteomics Analyzer) mass from JC8031 cells producing heterologous S. dysenteriae O-PS from pSS37 spectrometer in the negative ion linear mode as previously described (61). (Sd-glycOMVs). The five mice in each of the groups were challenged i.p. with 22 cfus of F. tularensis Schu S4, and the health of the mice was examined TLR4 Activation Assay. HEK-Blue hTLR4 cell lines were purchased from Invi- daily for signs of disease. vogen and maintained according to the manufacturer’s specifications. Cells Challenge against F. tularensis subsp. holarctica LVS was performed as were plated into 96-well plates at a density of 1.4 × 105 cells per mL in HEK- follows. Groups of five 6- to 8-wk-old BALB/c female mice (NCI) were each Blue detection media (Invivogen). Antigens were added at the following immunized i.p. with 100 μL of PBS containing LPS or OMVs, prepared as concentrations: 104 cells per mL for whole cells; and 10 ng/mL for OMVs. described. All PBS used was at pH 7.4. The groups were immunized with Purified E. coli O55:B5 LPS (Sigma-Aldrich) and detoxified E. coli O55:B5 either PBS alone (control) or with 10 μg of OMVs from JC8031 or JH8033 cells (Sigma-Aldrich) were added at 100 ng/mL and served as positive and nega- harboring pGAB2 (Ft-glycOMVs). Mice were boosted with the same dosages tive controls, respectively. Plates were incubated at 37 °C, 5% CO for 10–16 h, 28 d after the initial immunization. At 56 d after the initial immunization, 2 after which time the plates were analyzed using a microplate reader at each group was challenged i.p. with either F. tularensis LVS RML or 620 nm. Statistical significance was determined via unpaired t test. F. tularensis LVS Iowa at 4, 40, or 400 cfus. The PBS control groups were challenged with 4 cfus of either strain. The health of the mice was examined ACKNOWLEDGMENTS. We thank Cynthia Leifer and Brian Rudd for help with daily for signs of disease. experiments, as well as for helpful discussions of the manuscript; Joanna Goldberg, For all of the above experiments, when an animal became moribund, it was Markus Aebi, Mikael Skurnik, Rebecca Thomas, Renato Morona, and Roland killed according to the procedure in the approved protocol. Mice were moni- Lloubes for strains, plasmids, and antiserum used in this work; and Dana Ries for tored until 14 d, at which time a Kaplan–Meier plot was generated. Statistical expert assistance. We are grateful for use of the Cornell Center for Materials

Chen et al. PNAS | Published online June 6, 2016 | E3617 Downloaded by guest on September 27, 2021 Research Shared Facilities, which is supported through NSF MRSEC Program Grant (toP.A.).WealsoacknowledgetheNewYorkStateOfficeofScience,Technology DMR-1120296; and for use of the University of Iowa Carver College of Medicine and Academic Research (NYSTAR) Distinguished Faculty Award (to M.P.D.); Chem- Biosafety Level 3 Core Facility. This work was supported by NSF Grants CBET ical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, 1159581 and CBET 1264701 (both to M.P.D.); an NSF GK-12 “Grass Roots” Fellow- US Department of Energy Grant DE-FG02-93ER20097 (to P.A.); Army Research ship (to L.C.); and NIH Grants GM088905-01 (to M.P.D.), EB005669-01 (to D.P. and Office Grant W911NF-12-1-0390 (to M.S.T.); and Project 14 of the Midwest Re- M.P.D.), AI044642 (to B.D.J.), AI057160 (to B.D.J.), GM008629 Training Grant in gional Center of Excellence (MRCE) for Biodefense and Emerging Infectious Disease Genetics (to J.R.F.), AI064184 (to M.S.T.), AI076322 (to M.S.T.), and GM10349010 Research (B.D.J.).

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