Bacterial strains, cell lines and plasmids used in this study are listed in Table 1; oligonucleotides are listed in Table 2. AC53, a spontaneous streptomycin (Sm)-resistant mutant of the clinical isolate E7946 (O1 El Tor Ogawa), or ETEC H10407-S, a Sm-resistant mutant of the clinical isolate H10407, were used as wild-type strains (V-WT and E-WT). E. coli strain DH5αλpir and SM10λpir were used for genetic manipulations. Unless stated otherwise, strains were cultivated in Lysogeny broth (LB) or on LB agar plates with aeration at 37°C. If required, antibiotics and other supplements were used in the following final concentrations: streptomycin (Sm), 100 μg/ml; ampicillin (Ap), 100 μg/ml or in combination with other antibiotics 50 μg/ml; kanamycin (Km), 50 μg/ml; IPTG, 0.1 mM; glucose (Gluc), 0.2%; and sucrose (Suc), 10%.

The isolation of chromosomal DNA, PCR reactions, the purification of plasmids or PCR products, the construction of suicide and expression plasmids as well as the subsequent generation of deletion mutants were carried out as described previously using derivatives of pCVD442 or pMMB67EH (Seper et al., 2011; Leitner et al., 2015; Pressler et al., 2016). Qiagen plasmid kits were used for isolation of plasmid DNA; QIAquick^® Gel extraction and QIAquick^® PCR Purification kits (Qiagen) were used for purifying DNA fragments. PCR reactions for subcloning were carried out using the Q5^® High-Fidelity DNA Polymerase (NEB), while Taq DNA Polymerase (NEB) was used for all other PCRs. Construction of ompA in-frame deletion mutants in V. cholerae and ETEC were carried out as described by Donnenberg and Kaper (1991) using derivatives of pCVD442, i.e., pΔompA-V or pΔompA-E. The suicide vector pΔompA-V was already available from a previous study (Song et al., 2008). For construction of pΔompA-E, ∼800 bp PCR fragments located up- and downstream of the ompA were amplified using the oligonucleotide pairs ompA_E_SacI_1 and ompA_E_EcoRI_2 as well as ompA_E_EcoRI_3 and ompA_E_XbaI_4 with chromosomal DNA from E-WT as template (Table 2). After digestion of the PCR fragments with the appropriate restriction enzyme (NEB) indicated by the name of the oligonucleotide, they were ligated into pCVD442, which was digested with the appropriate restriction enzymes. Unless noted otherwise, ligation products were transformed into DH5αλpir and ApR colonies were characterized for the correct constructs by PCR. To obtain deletion strains, generated derivatives of pCVD442 were transformed into E. coli Sm10λpir and conjugated into V. cholerae or ETEC. Exconjugants were purified by Sm^R/Ap^R selection. Sucrose selection was used to obtain Ap^S colonies and chromosomal deletions were confirmed by PCR, respectively.

The Lpp-OmpA-RBD^Vch (LOR^Vch) and Lpp-OmpA-RBD^ETEC (LOR^ETEC) fusion proteins were designed in silico and the corresponding sequences including a 5′ untranslated region harboring a unique KpnI restriction site and an optimal Shine-Dalgarno sequence (Supplementary Figure 2) were synthesized and subcloned into the standard vector system pMK by the GeneArt Gene Synthesis platform (Thermo Fisher Scientific). Thus, constructs were provided as pMK-V-LOR and pMK-E-LOR. Finally, the expression plasmid pLOR-V and pLOR-E were constructed using the oligonucleotides LOR_V_1 and LOR_V_BamHI_2 as well as LOR_E_1 and LOR_E_BamHI_2 for amplifying the fusion construct using the ordered plasmids as template. This added a C-terminal FLAG-tag epitope to the constructs. The resulting PCR fragments were purified, digested with KpnI and BamHI, and ligated into pMMB67EH, which has been digested with the same enzymes. Ligation products were transformed into DH5αλpir and ApR colonies were characterized for the correct constructs by PCR.

Vibrio cholerae as well as ETEC OMVs were isolated as previously described (Schild et al., 2008; Leitner et al., 2013, 2015). Briefly, overnight cultures grown in LB medium of the respective strains were diluted (1:100) in fresh LB and cultivated overnight at 24°C and 180 rpm. The cells were then removed from the supernatant by centrifugation (9,500 × g, 20 min, 4°C) and subsequent sterile filtration (0.22 μm). The OMVs present in the supernatant were pelleted through subsequent ultracentrifugation (150,000 × g, 4°C, 4 h) and resuspended in saline to generate a 1000-fold concentrated OMV suspension compared to the original filter-sterilized supernatant. Protein concentration was determined using Bradford assay (Bio-Rad Laboratories, Protein Assay Dye Reagent) according to the manufacturer’s manual. To quantify the LPS content of OMVs, purpald assays were performed as described previously using 3-deoxy-D-mannooctulosonic acid (Kdo) (Sigma-Aldrich) as a standard (Roier et al., 2016).

To estimate the size distributions of the isolated OMVs, dynamic light scattering (DLS) was carried out using Zetasizer Nano ZS90 (Malvern, United Kingdom) as previously described (Zingl et al., 2021b). Samples were diluted 1:1000 in saline and processed at 25°C under standard settings [Dispersant Refractive Index = 1.331, viscosity (cP) = 0.89]. Three measurements were performed using a measurement angle of 173° (backscatter), auto measurement duration, and “seek for optimal position” as positioning setting.

Female BALB/c mice (Charles River Laboratories) were used in all experiments in accordance with the rules of the ethics committee at the University of Graz and the corresponding animal protocol, which has been approved by the federal ministry BMBWF (protocol: 39/12/75ex2017/18). Mice were housed with food and water ad libitum and monitored under the care of full-time staff. All animals were acclimated for 1 week before any procedures were carried out and were approximately 8 weeks old at the start of the immunization.

Eight-week-old female mice were intranasally immunized with OMVs (25 μg) in saline at days 0, 14, and 28 as described previously (Schild et al., 2008; Leitner et al., 2013). A group of sham (saline)-immunized control mice were housed in parallel with the immunized mice for the duration of each experiment. Overall two independent immunization rounds for each immunization group were performed with at least three mice per group. Comparison of the results from the independent immunization rounds revealed no differences in the induction of a humoral immune response in the respective immunization group.

Blood samples as well as fecal pellets were collected from immunized and sham-immunized adult mice throughout the immunization study and processed as previously described to monitor the induced immune response (Schild et al., 2008, 2009). Briefly, blood samples were collected from immunized and sham immunized mice on days 0, 14, 28, 42, and 60. The collected blood was allowed to clot at room temperature (RT) for 30 min after which serum was isolated by removing the blood clot using a sterile toothpick followed by centrifugation (15 min, 1,000 × g). The supernatant representing the serum of each sample was removed, diluted threefold in PBS/sodium azide (0.05%), and subsequently stored at −80°C. In addition, aliquots of the sera collected on day 60 were stored at −80°C without any dilution or addition of sodium azide of to be used in cell culture neutralization assays. In case of stool samples, freshly voided fecal pellets collected on days 42 and 60 were vacuum-dried for 10 min before their weight was recorded. Immunoglobulins were extracted by adding 1 ml of extraction buffer [PBS, 0.01% sodium azide, 5% fetal calf serum, 1 tablet complete EDTA-free protease inhibitor cocktail (Roche) per ml] per 100 mg dry-weight feces. After vortexing the samples for 15 min at 4°C, solid material was separated by centrifugation (2 min, 13,000 × g) and the supernatants were stored at −80°C.

For immunoprecipitation 9 μl of SARS-CoV-2 Spike Protein (RBD) Antibody (PA5-114451, Thermo Fisher Scientific) were mixed with 100 μg OMVs (protein equivalent determined by Bradford) and adjusted to final volume of 200 μl using saline. Antibodies were allowed to bind to the OMVs for 30 min at RT under gentle rocking before the OMVs were pelleted by centrifugation (75,600 × g, 4 h, 4°C). The OMV pellet was washed once with 1 ml saline and centrifuged again (75,600 × g, 2 h, 4°C) before the sample was finally resuspended in 40 μl saline and subjected to immunoblot analyses.

To analyze the protein content of OMVs, the standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure in combination with 12% gels and the PageRuler Prestained Protein Ladder (Thermo Fisher Scientific) as a molecular mass standard were used. Approximately 7.5 μg of each sample was loaded and either stained according to the procedure of Kang et al. (2002) or transferred to a nitrocellulose membrane (Amersham) for immunoblot analysis, which was essentially performed as described previously (Roier et al., 2012). Either the anti-FLAG^® M2-HRP monoclonal antibody (A8592, Sigma Aldrich) was used as sole antibody or SARS-CoV-2 Spike Protein (RBD) Antibody (PA5-114451, Thermo Fisher Scientific) was used in combination with the HRP-linked anti-rabbit IgG (111-035-003, Jackson ImmunoResearch) were used as primary and secondary antibody, respectively.

Temporal immune responses of different Igs, half-maximum total Ig titers and mucosal immune responses to VΔΔΔ or EΔΔΔ OMVs (5 μg/ml in PBS, pH 7.4) as well as the determination of the half-maximum total Ig titers to SARS-CoV-2 Spike Protein S1 (5 μg/ml in PBS, pH 7.4, RP-87679 Thermo Fisher Scientific) were carried out essentially as described previously (Roier et al., 2012; Leitner et al., 2013, 2015) using appropriate purified mouse Ig isotype standard (IgA, 553476; IgG1, 557273; or IgM, 550963, BD Biosciences) as well as horseradish peroxidase-conjugated goat anti-mouse antibodies (IgA, 62-6720; IgG1, A10551; IgM, 62-6820; IgG, IgM, IgA, PA1-84388, Invitrogen) in combination with the TMB peroxidase substrate reagent set (BioLegend). Optical densities were monitored at 450 nm with a FLUOstar Omega microplate reader (BMG Labtech). Starting dilutions of the mouse sera were 1:10 for quantification of the temporal immune responses and half-maximum total Ig titers against the Spike protein, 1:100 for quantification of the half-maximum total Ig titers against OMVs, or 1:400 for quantification of the temporal immune responses against OMVs. Starting dilutions of the fecal pellet extracts for quantification of the mucosal immune responses against OMVs were 1:10. If the highest sample concentration (sera or fecal pellet extracts) used did not yield any detectable absorption the value was set to the respective limit of detection of the assay stated in each figure legend.

Plasmids pHAGE2-CMV-luciferase-IRES-ZsGreen, pHDM-Spike, pHDM-Hgpm2, pHDM-tat1b, and pRC-CMV-rev1b were transfected into HEK 293T cells as described (Crawford et al., 2020). The plasmids and HEK 239T-ACE2 cell line were obtained from BEI Resources (Manassas, VA, United States). Transfection was confirmed by scoring green fluorescence of the cells 24 h later. Viral particles were collected from cell culture supernatants after 48 h as described (Crawford et al., 2020) and were stored in single-use aliquots at −80°C. The titer of thawed virus stock was 1.5 × 10⁵ infectious units/ml.

Neutralization assays were performed essentially as recently described (Crawford et al., 2020). Briefly, serial dilutions of each mouse serum were made in duplicate in 96-well plates (12565501, Fisher Scientific) in Dulbecco’s Modified Eagle Medium (DMEM) (MT10013CV, Corning) supplemented with 10% heat-inactivated fetal bovine serum (16140063, Gibco), and penicillin (100 U/ml) and streptomycin (100 μg/ml) (15140148, Gibco). A volume of 150 μl DMEM containing 3.1 × 10³ infectious units of pseudotype virus was added to each well and incubated for 1.2 h at 37°C in a 5% CO₂ incubator to allow antibodies to bind. This amount of virus gave a multiplicity of infection of approximately 0.08. Polybrene (TR1003E, Sigma) was added to each well to increase the efficiency of viral infection just before transferring the contents of each well to semi-confluent monolayers of HEK 293T-ACE2 cells in solid white 96-well plates (07-200-591, Fisher Scientific). The plates were incubated for 48 h at 37°C in a 5% CO₂ incubator to allow for viral infection. Infection by the Spike-pseudotyped lentivirus expressing Luciferase-IRES-ZsGreen was quantified by measuring light production after removing spent medium and adding Bright-Glo Luciferase reagent (E2610, Promega). After mixing for 20 s by swirling, each plate was incubated in the dark for 2 min, followed by measuring relative light units (RLU) on a Synergy^TM HT Microplate Reader using Gen5 data analysis software (BioTek). Background was initially assayed from uninfected cells, virus in the absence of cells, or DMEM medium alone, and all were equivalent. Therefore, in subsequent experiments, each plate contained at least two DMEM alone wells. The maximum infection on each plate was measured by infecting at least four wells of HEK 293T-ACE2 cells with pseudotype virus that had not been neutralized with serum. The fraction infectivity was calculated by averaging technical replicates and dividing by the average of the maximum infectivity on that plate. The inhibitory concentration 50% (IC50) was calculated graphically.

The data is generally presented as median with interquartile range as some data sets were not normally distributed. Consequently, data were analyzed using the Mann–Whitney U test or a Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons. Differences were considered significant at p-values of < 0.05. GraphPad Prism version 8 was used for all statistical analyses.

We recently demonstrated that OMVs derived from ETEC or V. cholerae lacking one functional secondary lipid A acyltransferase, MsbB (also referred to as LpxN) retain their potential to induce a high-titer, protective immune response but show a significantly reduced pro-inflammatory cytokine induction suggesting lower endotoxicity (Leitner et al., 2013, 2015). Moreover, cholera toxin subunits A and B in V. cholerae, and the heat-labile enterotoxin catalytic A subunit in E. coli were deleted. Thus, presence of these secreted toxins in OMVs is precluded (Leitner et al., 2015). In addition to these modifications resulting in detoxified OMVs, we also deleted ompA, encoding the outer membrane protein OmpA, which crosslinks the periplasmic peptidoglycan and the outer membrane. Deletion of ompA has been reported to result in elevated OMV release in various gram-negative bacteria (Song et al., 2008; Schwechheimer and Kuehn, 2015). Concordantly, the engineered triple mutants VΔΔΔ and EΔΔΔ showed a significant three- to six-fold increase in OMV production compared to the parental WT strains (V-WT and E-WT), respectively (Supplementary Figure 1). Thus, the engineered triple mutants VΔΔΔ and EΔΔΔ are optimized V. cholerae and ETEC platform strains for production of OMVs to be used for vaccination.

To ensure efficient translocation to the outer membrane and external exposure of the SARS-CoV-2 RBD domain, we adapted the Lpp-OmpA’ fusion strategy, which has been used previously to display a variety of heterologous proteins on the bacterial surface of E. coli (Francisco et al., 1992; Stathopoulos et al., 1996; Daugherty et al., 1998; Earhart, 2000). The general design of the LOR fusion constructs was similar for V. cholerae and ETEC (Figure 1A). The LOR fusion constructs consist of a N-terminal signal sequence and next nine amino acids of the major outer membrane lipoprotein (Lpp), followed by a two-amino-acid linker, and then approximately 110 amino acids of OmpA, i.e., amino acids 46–146 of ETEC and amino acids 51–158 of V. cholerae OmpA. The Lpp fragment directs the fusion protein to the outer membrane, with the acylated N-terminal end being anchored in the inner leaflet of the outer membrane. The OmpA fragment contains five membrane-spanning segments with the C-terminus residing externally (Francisco et al., 1992). Via an additional four-amino-acid linker the heterologous peptide consisting of the entire RBM and the majority of the RBD of SARS-CoV-2 is fused to the C-terminal end. As Lpp and OmpA of V. cholerae and ETEC share only 38% identity, the amino acid sequence as well as the SARS-CoV-2 Spike protein is neither optimal for E. coli nor V. cholerae; we have used species-specific fusion constructs using the individual lpp and ompA sequences as well as codon optimized sequences (obtained via the IDT codon optimization tool) of the SARS-CoV-2 RBD for each species, namely, LOR-V and LOR-E, respectively (Supplementary Figure 2). Along the design, a unique KpnI restriction site for subsequent cloning into expression vectors and an optimized Shine Dalgarno sequence ensuring high translation efficiency were included in the 5′ untranslated region. Synthesized fragments were PCR-amplified, which added a C-terminal FLAG-tag epitope to the fusion proteins, and subcloned into the IPTG-inducible broad range vector pMMB67EH (see Supplementary Figure 2 and “Materials and Methods” section for details) resulting in the LOR fusion expression constructs pLOR-V and pLOR-E.

Expression of the LOR fusion proteins in VΔΔΔ and EΔΔΔ should result in their localization on the bacterial cell surface and therefore also on the surface of OMVs, which bud from the outer membrane. Successful localization of LOR in OMVs was confirmed by immunoblot analyses (Figure 1B and Supplementary Figure 3). Detection of LOR fusion proteins with anti-FLAG and anti-RBD antibodies showed specific bands with the expected molecular weight of approximately 36 kDa for V. cholerae and 38 kDa for ETEC for the OMVs derived from VΔΔΔ pLOR-V and EΔΔΔ pLOR-E, but not for OMVs isolated from strains harboring the empty vector (VΔΔΔ p and EΔΔΔ p). Thus, OMVs of both species can be decorated with significant amounts of the LOR. Furthermore, outward exposure of RBD part on OMVs was confirmed via an immunoprecipitation assay using OMVs derived from VΔΔΔ pLOR-V, VΔΔΔ p, EΔΔΔ pLOR-E, and EΔΔΔ p as antigen in combination with anti-RBD antibodies. After incubation of OMVs and anti-RBD antibodies, samples were subjected to centrifugation and washing steps to purify OMVs. Finally, immunoblot analyses with the immunoprecipitated samples were performed to detect anti-RBD antibody (raised in rabbits) bound to the OMV samples using an HRP-conjugated anti-rabbit antisera (Figure 1C). A Kang-stained gel executed in parallel served as a loading control (Supplementary Figure 4). Heavy chains of rabbit IgG, indicating bound anti-RBD antibodies to OMVs, were detected at the expected molecular weight of approximately 55 kDa in immunoprecipitations using OMVs derived from VΔΔΔ pLOR-V and EΔΔΔ pLOR-E, but not for OMVs isolated from strains harboring the empty vector (VΔΔΔ p and EΔΔΔ p). The accessibility of the LOR fusion proteins on OMVs by the anti-RBD antibody strongly suggests that the RBD portion is residing externally on the surface of the OMVs.

Finally, we determined the size distribution and quantified the biomass of OMVs liberated by the parental WT strains (V-WT and E-WT), the triple mutants (VΔΔΔ and EΔΔΔ) as well as the donor strains for the OMV-vaccine candidates used herein (i.e., VΔΔΔ p, EΔΔΔ p, VΔΔΔ pLOR-V, and EΔΔΔ pLOR-E) (Supplementary Figure 5). These analyses confirmed the increased OMV biomass amounts liberated by the triple mutants in both species.

In order to investigate the immune response upon immunization with LOR-decorated OMVs (LOR-OMVs), we intranasally immunized mice with OMVs derived from VΔΔΔ pLOR-V, VΔΔΔ p, EΔΔΔ pLOR-E, and EΔΔΔ p according to our standard immunization protocol (Schild et al., 2008; Figure 2A). Briefly, mice were intranasally immunized on days 0, 14, and 28 receiving 25 μg OMVs (protein equivalent) per immunization. In total, seven different immunization groups were tested (Figure 2B). Four groups received the same OMVs at days 0, 14, and 28 and were therefore only immunized with OMVs from either VΔΔΔ pLOR-V, VΔΔΔ p, EΔΔΔ pLOR-E, or EΔΔΔ p. Two groups (EVE pLOR-E/V and EVE p) were immunized with alternating regimen and received OMVs from EΔΔΔ pLOR-E on days 0 and 28 as well as OMVs from VΔΔΔ pLOR-V on day 14 (EVE pLOR-E/V) or EΔΔΔ p on days 0 and 28 as well as VΔΔΔ p on day 14 (EVE p). A change of the OMV donor species between the immunizations was thought to specifically boost the immune response against the RBD of SARS-CoV-2 being the common antigen on OMVs derived from VΔΔΔ pLOR-V and EΔΔΔ pLOR-E. Finally, a sham-immunized group receiving saline at days 0, 14, and 28 served as control.

To monitor the immune responses in sera of immunized and sham-immunized control mice by enzyme-linked immunosorbent assay (ELISA), OMVs derived from VΔΔΔ pLOR-V or EΔΔΔ pLOR-E were used as coating material allowing the detection of temporal IgM, IgG1, and IgA responses to the respective OMVs (Figure 3). Herein we focused on IgG1 as previous studies revealed IgG1 to be the dominant IgG subclass induced after intranasal immunization with OMVs (Schild et al., 2008, 2009; Bishop et al., 2010; Leitner et al., 2013, 2015). As reported earlier, immunization with V. cholerae or ETEC OMVs induces a species-specific immune response without any significant cross-reactivity (Leitner et al., 2015). Thus, groups immunized with OMVs solely derived from one species were only tested for immune responses against OMVs derived from the same species, while immunization groups with alternating regimen and the sham-immunized control group were tested for immune responses against both, OMVs derived from V. cholerae and ETEC. The antibody titers of the sham-immunized control group were determined for days 0 and 60 and remained below the limit of detection or at very low levels for both time points against OMVs derived from V. cholerae and ETEC, respectively. In general, the induction of an immune response was characterized by an Ig titer increase during the vaccination period. Thus, IgM titers peaked at days 28 or 42, while IgG and IgA were slightly delayed and peaked on days 42 or 60. Not surprisingly, Ig titers of the immunization groups receiving the same OMVs at all three immunization were overall higher and reached the Ig peak earlier than the immunization groups with alternating regimen. Importantly, IgG1 and IgA titers remained stable or even increased between days 42 and 60, which indicates the induction of a robust immune response until day 60, the designated endpoint of this study.

Next, we evaluated the induction of a humoral immune response against the SARS-CoV-2 Spike protein. Based on the high IgG1 titers in serum samples we focused on this isotype to analyze the temporal immune response against the Spike protein (Figure 4). IgG1 titers of the sham-immunized control group as well as the immunization groups VΔΔΔ p, EΔΔΔ p, and EVE p remained below the limit of detection or at very low levels at all time points, respectively. In contrast, all three immunization groups receiving OMVs decorated with LOR, i.e., VΔΔΔ pLOR-V, EΔΔΔ pLOR-E, and EVE pLOR-E/V showed detectable IgG1 titers against the Spike protein from days 28 onward, which peaked on days 42 or 60. We also tried to determine serum IgA responses against the Spike protein, but IgA titers were overall lower compared to the IgG1 titers and remained below the limit of detection (0.04 μg/μl) even for several serum samples of the immunization groups receiving OMVs decorated with LOR. Unfortunately, this analysis was hampered by observable cross-reactivity of the secondary antibody with serum components or the Spike protein combined with relatively low IgA titers in the samples.

To further characterize the induced humoral immune response, sera from days 42 and 60 were used to determine the half-maximum total Ig titers against OMVs derived from VΔΔΔ pLOR-V or EΔΔΔ pLOR-E as well as against SARS-CoV-2 Spike protein (Figure 5). In comparison to the sham-immunized control group the immunization groups receiving OMVs showed a marked increase in total Ig titers for both time points against the respective OMVs derived from VΔΔΔ pLOR-V or EΔΔΔ pLOR-E (Figures 5A,B,D,E). Similar to the temporal immune response patterns, the OMV-specific responses were slightly lower in the immunization groups with alternating regimen (EVE pLOR-E/V and EVE p) compared to the immunization groups receiving the same OMVs at all three immunizations. In comparison to the sham-immunized control group, a significant increase of the total Ig titers against the Spike protein was observed at day 42 for the VΔΔΔ pLOR-V and EVE pLOR-E/V immunization group as well as for all immunization groups receiving OMVs decorated with LOR (i.e., VΔΔΔ pLOR-V, EΔΔΔ pLOR-E and EVE pLOR-E/V) at day 60 (Figures 5C,F). In contrast, to the OMV-specific immune response, the EVE pLOR-E/V immunization group showed slightly, but not significantly higher Ig titers against the Spike protein than the VΔΔΔ pLOR-V and EΔΔΔ pLOR-E groups. For both time points tested, the immune response against the Spike protein remained undetectable or at low levels for immunization groups receiving OMVs from strains harboring the empty vector (VΔΔΔ p, EΔΔΔ p and EVE p). In general, isotype-specific and total Ig titers did not massively drop between days 42 and 60 in the respective immunization groups suggesting the induction of a stable immune response (Figures 3–5).

In addition to the humoral immune responses, we also analyzed the induced mucosal immune responses by measuring the secretory IgA titers in fecal pellet extracts collected at days 42 and 60 against OMVs derived from VΔΔΔ pLOR-V or EΔΔΔ pLOR-E (Figures 6A–D). Fecal pellet collection was preferred over other methods (e.g., saliva collection or lung lavage) as it seems less stressful and allowed us to keep the mice alive for later time points. Furthermore, it has been shown in earlier studies that secretory IgA antibodies reflecting the mucosal immune response can also be found in feces upon OMV immunization and that IgA titers in feces correlate by trend with those found in the respiratory tract, e.g., saliva (Vetvik et al., 1998; Hirano et al., 2006; Schild et al., 2009).

Generally, the mucosal immune response on days 42 and 60 showed a similar pattern as the half-maximum total Ig titers. All immunization groups receiving OMVs showed a significant increase in secretory IgA titers against the respective OMVs compared to the sham-immunized control group (Figures 6A–D). In majority of the cases, the secretory IgA titers against OMVs were slightly lower in immunization groups with alternating regimen (EVE pLOR-E/V and EVE p) compared to the immunization groups receiving the same OMVs at all three immunizations. We also tried to determine secretory IgA levels against the Spike protein in fecal pellet extracts, but for the majority of samples the titers remained below or close to the limit of detection (1 μg/g). As described above, this analysis was hampered by observable cross-reactivity of the secondary antibody with components of the fecal pellet extract or the Spike protein combined with relatively low concentration of mucosal IgA in the samples.

To determine if the vaccines were capable of eliciting neutralizing antibodies, a Spike-pseudotyped lentivirus neutralization assay was used as previously described (Crawford et al., 2020). Functionality of the assay was confirmed by using commercially available anti-RBD antisera as positive control and PBS as solvent (negative) control, which revealed a robust neutralizing activity of the anti-RBD antisera, but not for the solvent control (Supplementary Figure 6). Briefly, a pseudotype lentivirus expressing the SARS-CoV-2 Spike protein on its envelope and, upon infection, luciferase in the host cell cytoplasm, was incubated with each mouse serum to allow antibody binding, followed by infection of HEK 293T cells expressing the ACE2 receptor. The fraction infectivity was determined by measuring RLU of the monolayers. An amount of virus was used to achieve a signal >1000 above background. Sera from the sham-immunized control group and immunization groups receiving OMVs from strains harboring the empty vector (VΔΔΔ p, EΔΔΔ p, and EVE p) showed no measurable neutralization activity. Using the highest sera concentration, i.e., 1:4 dilution, a significant neutralizing activity was observed for all three immunization groups receiving OMVs decorated with LOR, i.e., VΔΔΔ pLOR-V, EΔΔΔ pLOR-E and EVE pLOR-E/V, compared to the sham-immunized control group (Figure 7). In case of sera from the EVE pLOR-E/V group a significant neutralizing activity was also detectable for following two dilutions (1:14 and 1:49). The IC50 for the serum from the immunization group with alternating regimen (EVE pLOR-E/V) was approximately 1:142, which was about fourfold higher than for the repeating regimens (VΔΔΔ pLOR-V and EΔΔΔ pLOR-E).