Structural dynamics and immunogenicity of the recombinant and outer membrane vesicle-embedded Meningococcal antigen NadA

Domain: biology
Published: 2026-03-11
Editorial outcome: major_revision

Abstract
Structural knowledge of antigens in their native state can drive the design of optimized vaccine antigens that mimic the native epitope exposure and conformation. Here, by hydrogen-deuterium exchange mass spectrometry, we assessed the structural features of Neisseria Adhesin A (NadA), a meningococcal trimeric outer membrane protein, included as soluble recombinant antigen in the 4CMenB vaccine. We propose a structural annotation of the recombinant NadA and compare its structural dynamics with NadA in situ, as embedded in meningococcal outer membrane vesicles (OMVs). The observed conformational differences suggest that OMV-embedded NadA could be more susceptible to trimer opening and display a larger antigenic surface than the soluble antigen. Accordingly, mice immunized with OMV-embedded NadA elicited antibodies with superior bactericidal activity compared to the soluble antigen. Collectively, these data support the hypothesis that protein vaccine antigens presented in native-like environments can elicit a more potent immune response than recombinant forms.

Introduction
Subunit vaccines are often based on recombinant proteins, which can be rationally designed with excellent efficacy and safety profiles and produced with high purity1. Membrane proteins often represent suitable surface antigens of bacterial pathogens. However, membrane proteins are challenging to produce recombinantly in their ‘native’ conformation as extracting them from their membrane environment can compromise their native folding. The genome-based reverse vaccinology approach is a valuable technology to identify surface exposed bacterial membrane proteins suitable as vaccine candidates2–4 and their subsequent structural characterization (structural vaccinology) can enable a rational vaccine design5,6. In 2013, a reverse vaccinology-derived multicomponent subunit vaccine against Neisseria meningitidis serogroup B (MenB) was approved (4CMenB)7,8. One of the four protective components of 4CMenB is Neisseria adhesin A (NadA)3,8, a protein antigen able to induce high levels of bactericidal antibodies both in animal models3,9,10 and in humans11. NadA variant 3, the vaccine variant and hereinafter referred as NadA, is a flexible and elongated outer membrane protein that mediates adhesion to and invasion of host cells during bacterial infection and colonization of the human respiratory tract12. NadA is a member of trimeric autotransporter adhesins and shares with them the typical modular organization13. After removal of the signal peptide (aa 1-23), NadA presents an N-terminal head region (aa 24-73) projected away from the bacterial surface that is involved in host recognition and binding, an elongated central stalk (aa 73-344), and a beta-barrel C-terminal anchor that embeds into the bacterial outer membrane (aa 345-405). NadA still lacks a complete structural determination, although the X-ray structure of a short yet trimeric construct (NadA 24-170) has been solved14. The stalk of NadA 24-170 adopts a coiled-coil organization and is made of canonical heptad repeats and two uncanonical undecad repeats. Traditionally, in coiled coils, heptad repeats, denoted ‘abcdefg’, are characterized by hydrophobic residues at the first ‘a’ and fourth ‘d’ positions, which are packed inside the coiled coil15. In the uncanonical undecads ‘abcdefghijk’, hydrophobic residues are present also at the eighth position ‘h’15. NadA head domain is a small globule resulting from the partial opening of the trimer, and is shaped in a tripartite wing-like structure flanking an N-terminal heptad coiled-coil region14. The head and stalk domains of NadA, referred together as passenger domain, spontaneously assemble into a stable trimer16, allowing it to be included as recombinant soluble antigen in the current 4CMenB vaccine (NadA 24-350), without the C-terminal beta-barrel anchor. The recombinant soluble NadA form has already proven to elicit an effective immune response, with anti-NadA antibodies promoting bacterial killing and preventing the initial bacterial colonization11. However, it has been shown that not all anti-NadA antibodies isolated from vaccinated individuals are able to recognize the native antigen exposed on the bacterial membrane11, which may suggest that the protein conformation and epitope exposure of the soluble vaccine NadA and the bacterial NadA could differ or that not all epitopes are accessible in the native membrane bound form.

In this work, we thus aimed at comparing the conformational dynamics of the soluble antigen NadA, included in the current vaccine, and NadA in situ, in its native meningococcal membrane, as well as evaluating the immune response elicited by both NadA forms, in order to identify its optimal immunogenic state. To study NadA in situ, we leveraged the capability of outer membrane vesicles (OMVs) to faithfully reproduce the bacterial membrane environment, carrying antigens in an identical state as that adopted onto the bacterial surface. OMVs are spherical buds of outer membrane naturally released by Gram-negative bacteria, therefore their lipid, outer membrane protein and lipopolysaccharide (LPS) composition are the native ones17,18. For interrogating the conformational dynamics of NadA, we applied hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), a technology that has proved suitable for the analysis of membrane proteins in complex lipid environments19–24, including OMVs25. In HDX-MS, a protein is exposed to a deuterated buffer and the exchange of its back-bone amide hydrogens with deuterium is measured as a function of time using mass spectrometry. The HDX rate of these labile hydrogens is a highly sensitive reporter of the local structure and conformational dynamics26, such that HDX data can be of use to fill in missing structural data from electron microscopy (EM) or X-ray crystallography, or combined with them, to predict protein structure27.

By using HDX-MS data, here we propose a structural annotation of the NadA 24-350, gaining information on its entire structure, and unveiled that NadA can adopt an open trimer conformation. While the overall structures of the soluble vaccine antigen and the OMV-embedded form appear similar, we reveal differences at their conformational level, and that the open trimer conformation is favored in OMVs. Finally, we show that in mice, compared to the recombinant antigen, OMV-embedded NadA is capable of a better elicitation of antibodies, which also present higher functional quality, i.e. superior bactericidal activity compared those elicited by the recombinant antigen. As a whole, our data offer a structural basis for a potential optimization of the MenB antigen NadA and, in a broader perspective, showcase that antigen functional immunogenicity can be improved by displaying antigens onto OMVs.

Results
Structural annotation of NadA

The recombinant vaccine antigen NadA (NadA 24-350), hereinafter referred as rNadA, fails to crystalize, likely due to its flexibility14,28. We thus lack a complete determination of its structure, which prompted us to use HDX-MS to infer local dynamic information linkable to its secondary structural elements29,30. Our HDX-MS analysis yielded a map of 181 peptides, with 100% sequence coverage and high spatial resolution (10.24 of redundancy) (Supplementary Fig. 1). Deuterium labelling was performed for several time intervals and under different temperature conditions (Supplementary Table 1) to expand the dynamic and time window studied, thus sampling the HDX with high temporal resolution31. The results showed that rNadA contains regions that undergo rapid, medium, and slow HDX kinetics both in the head and stalk domains (Fig. 1a and Supplementary Fig. 2, uptake plots in Supplementary Dataset 1), and interestingly, that specific regions manifested bimodal HDX behavior, hence a low- and high-mass envelope distribution (Fig. 1b) (see paragraph: Identification of an open trimer conformation in NadA).

By using HDsite, we deconvolved peptide-level HDX data to residue-resolved kHX values of individual or groups of few back-bone amides32. The kHX provides information on the degree of engagement of back-bone amide hydrogens in intramolecular hydrogen-bonding, hence on the presence of stable secondary/tertiary structure33, which we combined with the structural information available for NadA 24-170 (PDB: 6EUN) to model the part of the stalk that still lacks structural determination. kHX values were extracted at single residue level for 19% of the sequence and with an average resolution of 3.92 amino acids for the rest (Supplementary Fig. 3, kHX plots in Supplementary Dataset 2). From the derived kHX values, we calculated residue-level protection factors (Supplementary Table 2), i.e. the unitless ratio between the rate of exchange for a completely unstructured state (kch) and the observed rate kHX, which are therefore intrinsically linked to dynamic properties33. In NadA 24-170, protection factors >250 (ranging from 103 to 106) were extracted for residues within the coiled coil head and stalk, whereas protection factors ≤250 were calculated for residues located in non-coiled coil regions, namely the wings (aa 65-74) and the N-terminal part of the head domain (residues 24-28), which in part lacks electron density in the X-ray structure previously determined14. For the part of the stalk of unknown structure (residues 168-350), most backbone amides displayed protection factors >250, indicative of coiled coil elements, except for selected amides within the regions spanning residues 252-254, 264-267 and 288-293 (protection factors <100), which suggests that the coiled coil stalk of NadA is interrupted by flexible structural elements. Based on the HDX data and the analysis of NadA primary sequence and coiled coil periodicities, from L168 the most likely assignment of NadA stalk resulted in fourteen heptad repeats and two uncanonical undecad repeats, one spanning residues 210-220 and the other one spanning residues 270-280; with two flexible regions of non-coiled coil structure assigned to residues 252-265 and 284-297 (Fig. 1c, Supplementary Table 2). The very low protection factors (<10) of the amides comprised in the C-terminus (aa 329-350) indicate a highly flexible and unstructured region, which is corroborated by the presence of numerous disruptive and polar amino acids, typical of intrinsically disordered segments34. We could thus infer that this part, which we denominated NadA tail, lacks a stable coiled coil structure (Fig. 1c). A low-resolution Cryo-EM structure confirmed that rNadA predominantly adopts an elongated and flexible structure (30 nm in length), with a clearly resolved rod-like organization. A globular density with a diameter of 5 nm, fitting the N-terminal head domain, was visible at one end, whereas a hook-like bend with a diameter of 3.7 nm was observable at the opposite end. The latter corresponds to the NadA tail, for which also Cryo-EM data supports the lack of a coiled coil organization (Fig. 1d, Supplementary Figs. 4 and 5).

Conformational differences between rNadA and OMV-embedded NadA

Successively, we aimed at investigating NadA structure in situ, as exposed on the meningococcal membrane surface. To do so, we analyzed meningococcal OMVs overexpressing NadA (NadA 24-405). In this native form, the sequence signal is cleaved off and the passenger domain of the protein extends outwards from the beta-barrel translocator domain, which is anchored and inserted into the outer membrane12. By label-free proteomic analysis, 296 proteins were identified in the OMVs, with predicted cytoplasmatic proteins representing 5.5% of the total protein content, revealing the high quality of the OMVs. NadA monomer was measured at 8.7% of the total protein content, corresponding to 2.9% of trimeric NadA (Supplementary Table 3). In negative staining transmission electron microscopy (NS-TEM), the OMVs appeared as spherical and intact particles with a diameter ranging from 30 to 100 nm, surrounded by a thick membrane carrying several appendages, corresponding to NadA, which are not visible in OMVs released by the bacteria that do not express NadA35,36 (Fig. 2a). We compared the conformational dynamics of rNadA and OMV-embedded NadA by HDX-MS (Figs. 2b and 3a). We carried out an HDX-MS experiment with integrated size-exclusion chromatography (SEC-HDX-MS), which enabled efficient lipid removal and allowed monitoring the HDX of 36 peptides spanning 72.8% of sequence37 (Supplementary Table 4, uptake plots in Supplementary Dataset 3). We observed that peptides of OMV-embedded NadA encompassing the insertion within residues 284-297 and the wings did not reveal significant differences in HDX compared to rNadA (Fig. 2b), indicating that these elements of flexibility are present both in the recombinant and native state. They could thus be pivotal structural elements. The tail of NadA, located upstream the beta-barrel anchor, showed characteristics of a structural reorganization in situ. In rNadA, peptide 327-340, spanning the tail, was found to be highly dynamic (fully exchanged at early time points), whereas in OMV-embedded NadA, we clearly observed that the peptide isotopic envelope was characterized by HDX bimodal behavior and overall reduced HDX (Fig. 3b). This phenomenon can be reasonably attributed to a structural rigidification of the NadA tail due to the proximity of the membrane anchor. Compared to rNadA, OMV-embedded NadA manifested reduced HDX in several segments of the stalk (within residues 78-93, 155-163, 172-211, 270-278) and in the head domain (residues 38-59) (Figs. 2b and 3a), indicative of a long-range propagation of the membrane stabilizing effect. Like rNadA, also OMV-embedded NadA displayed bimodal HDX behavior in its head domain (see paragraph Identification of an open trimer conformation in NadA), and the observed reduced HDX was due to the low-mass distribution (Fig. 3b). Considering the observed long-range propagation of the membrane stabilizing effects, we investigated whether there could be in NadA a bidirectional allosteric crosstalk between the head and stalk domains. We thus performed HDX-MS analysis of rNadA in complex to a known interactor hypothesized to bind to the head domain, Hsp9038–40 (Supplementary Table 5, uptake plots in Supplementary Dataset 4). We confirmed that Hsp90 directly associates with the NadA head domain, specifically at the level of residues 40-51, which showed decreased HDX when rNadA is bound to Hsp90 compared to the unbound state (Supplementary Fig. 6). Furthermore, we observed that its binding induced significantly increased HDX, meaning structural destabilization, in the NadA stalk (from residue 94) (Supplementary Fig. 6). This destabilization was clearly observable until the predicted coiled-coil interruption at residues 252-265, whilst appearing highly diminished downstream. Taken together, these data corroborate that an allosteric crosstalk exists in NadA and suggest two mechanistic features of this adhesin: 1) the coiled coil stalk presumably bends, twists or rigidifies when both termini are subjected to stimuli, like the membrane constrain at the C-terminus or interactors to the N-terminal head domain, giving rise to a bidirectional head-anchor crosstalk; and 2) the predicted coiled-coil interruptions likely play a role in releasing the torsional stress of the trimeric stalk when this is perturbed.

Identification of an open trimer conformation in NadA

As aforementioned, several regions of the coiled coil head and the stalk of rNadA exhibited bimodal isotopic envelopes (Supplementary Figs. 7 and 8), which we clearly observed also in OMV-embedded NadA (Fig. 3b and Supplementary Fig. 9). Bimodal HDX envelopes are characterized by a low- and a high-mass distribution distinguishable on the m/z scale, indicative of the presence of two different populations in solution: one in which the protein backbone amides are more prone to exchange compared to the other one41. In a pulse labelling HDX experiment on rNadA, both isotopic distributions displayed unvaried centroid mass and intensity, disproving irreversible unfolding or dissociation of the trimer in solution over time (Supplementary Fig. 10). For rNadA, the two populations are not observed to interconvert during the HDX time course studied, indicating them as conformationally distinct, with a very slow interconversion rate. For OMV-embedded NadA, the two populations do show a minor interconversion during the HDX time course thus indicating a somewhat faster interconversion rate42 (Supplementary Figs. 7 and 8). The alternative more accessible population mainly manifested in coiled coil regions flanking flexible elements, i.e. in proximity of the discontinuity between 252-265 of the stalk (peptides 206-249, 254-274, Supplementary Figs. 2 and 8) and of the wings in the head domain (peptides 36-58, 38-59, 40-59, 40-50, 74-93, Supplementary Figs. 2 and 7). These data denote that these flexible elements give a certain degree of conformational freedom to the NadA trimer, yielding the coiled coil to occupy co-existing more closed (tight) and more open (relaxed) trimeric states. The analysis of a shorter form of NadA (NadA 24-149), containing approximately 70% of dissociated monomeric form14, suggested that the open trimer conformation in rNadA does not adopt a fully monomeric state, as the high-mass distributions of rNadA display lower centroid masses compared to NadA 24-149 at early time points (Supplementary Fig. 7). This experiment also rules out the presence of dissociated monomers in the rNadA sample, given that NadA 24-149 manifested a clear HDX bimodality in the coiled coil segment downstream of residue 87, unlike rNadA. In rNadA, the closed and open trimer conformations (low-/high-mass distributions) appear to be distributed in approximately 9/1 ratio respectively (Supplementary Figs. 7 and 8). Intriguingly, in OMV-embedded NadA, the HDX bimodal behavior of the head domain appeared to favor the high-mass distribution compared to rNadA (Fig. 3b and Supplementary Fig. 9) and bimodal HDX was also observed in the extreme N-terminus (peptide 24-39), a phenomenon absent in rNadA. The initial abundance of the low- and high-mass distributions in OMV-embedded NadA cannot be accurately quantified due to a faster closed-to-open conformational transition than for rNadA; however, in the earliest time frames where the least interconversion has occurred (time points 11 sec and 1 min), peptides 40-58 and 40-59 show a ratio of 3/1 between the low- and high-mass distributions (Fig. 3b and Supplementary Fig. 9). We could not confirm the presence of bimodal HDX in the stalk of OMV-embedded NadA because of the lack of sequence coverage, but we clearly show that the head domain of NadA in situ appears to manifest a more populated open trimer conformation compared to rNadA. Considering that the head domain is primarily responsible for binding as well as adhesion function and harbors the main epitopes11, it is reasonable to speculate that the open trimer conformation could influence the adhesive and immunogenic properties of NadA through expansion of its exposed surface.

Evaluation of the immune response elicited by rNadA and OMV-embedded NadA

Owing to the conformational differences highlighted by the HDX-MS analysis -both in NadA head domain and stalk- we compared the immunogenicity of the 4CMenB vaccine recombinant form of NadA (NadA 24-350) and OMV-embedded NadA (NadA 24-405). CD-1 mice were immunized with 20 μg of rNadA (a saturating dose in mice3,10) or 5 μg (total protein content) of MenB or E. coli OMVs overexpressing NadA. Very recently, Croia et al.43 reported that this amount of OMVs was sufficient to elicit saturating levels of antibodies against OMV proteins expressed at concentrations ≥1.5-2%, which is in the range of the percentage of NadA in the OMVs used in our study. MenB OMVs from a NadA knock-out strain (NadA KO) were used as negative internal control. The elicited NadA-specific antibody titers were evaluated by ELISA using post-III immune sera of individual mice. rNadA and both NadA-OMV-based formulations elicited comparable antibody titers that result significantly higher than the negative control (Fig. 4a).

To estimate the serum bactericidal activity (SBA) of the elicited antibodies, mice sera were tested against the BZ83 meningococcal natural strain. The BZ83 strain was selected as test strain from a panel of strains as it is mismatched for the PorA variant and also for the other MenB major antigens, such as fHbp and NHBA, therefore all bactericidal responses elicited by the 5/99 meningococcal OMVs-derived sera against the BZ83 strain are expected to be NadA-specific. Expectedly, no killing was achieved with the serum derived from mice immunized with MenB OMVs from the NadA KO (Fig. 4b), demonstrating that, while responses to PorA or other antigens could be elicited, no functional contributions to the SBA derive from antibodies to other OMV components. Conversely, a highly functional response was achieved with sera derived from mice immunized with NadA-OMVs (SBA titers exceeding 65,000) compared to the response raised by rNadA (Fig. 4b). These data indicate that, even though the recombinant and OMV-based formulations elicit similar NadA-specific IgG titers, the OMV formulations show improved functional immune response and at lower dose of antigen administrated.

Methods
Ethics statements. Animal husbandry and experiments were ethically reviewed and carried out in accordance with European Directive 2010/63/EU, in compliance with relevant guidelines (Italian Legislative Decree N. 26/2014) and the GSK’s policy and guidelines on the care, welfare and treatment of animals, in GSK animal facilities located in Siena, Italy (AAALAC accredited).

Recombinant NadA proteins and Hsp90. rNadA (NadA 24-350) is the soluble recombinant form of NadA present in the 4CMenB vaccine, for which purification and characterization have been previously reported3,10,39,68. rNadA 24-149 was from a previous study14. Recombinant Hsp90 was from a previous study40.

Preparation of OMVs. The meningococcal OMVs were prepared from 5/99 strain (PorA subtype P1.5,2 and ΔlpxL1, ΔSynx,) deleted of NadA repressor (ΔNadR) to overexpress NadA69. The N. meningitidis strain was first pre-inoculated into 7 mL of Meningitidis Chemical Define Medium I- medium (MCDMI) at an OD600 ranging from 0.15-0.2 and incubated at 37 °C at 180 rpm until mid-exponential phase (=0.8-0.9 OD). The mid-exponential pre-cultures were inoculated into 50 mL medium in 250 mL baffled flasks and grown overnight (16-18 h) until late stationary phase at 37°C and 180 rpm. Successively, the bacterial cells were pelleted by centrifugation for 30 min at 3,400 × g and 4°C, and the culture supernatants filtered (pore size 0.22 µm). For the generation of the E. coli OMVs, vaccine strains pET21 plasmid expressing the full-length nadA was transformed into E. coli BL21(DE3) ΔTolR strain. BL21(DE3) ΔTolR strains were pre-cultured overday in 7 ml of HTMC medium supplemented with 100 µg/ml of ampicillin. The cultures were diluted 1:100 in 50 ml of HTMC liquid medium and incubated overnight at 30°C, 5% CO2, 160 rpm. Bacteria were centrifuged at 3400 × g for 15 min, resuspended into 60 ml of fresh HTMC broth supplemented with 1mM IPTG and incubated for 3 h at 37°C, 5% CO2, 160 rpm to mid-exponential phase. The bacterial cells were pelleted by centrifugation for 30 min at 3400 × g and 4°C and the culture supernatants filtered (pore size 0.22 µm). OMVs were collected from the filtered supernatants of meningococcal and E. coli cultures by ultracentrifugation for 2 h at 9,6000 × g and 4 °C, and an additional washing step with PBS was performed. The OMV pellets were re-suspended in PBS at a concentration of 1 µg/µL.

Identification of rNadA peptides. Peptide identification was performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of peptides generated upon on-line pepsin digestion, setting buffer conditions and chromatographic gradient as for deuterated samples. Peptide ions were fragmented by collision-induced dissociation using argon as collision gas and MS/MS spectra were acquired in data independent acquisition mode (MSE). MS/MS data were processed with ProteinLynx Global Server (PLGS) version 3.0 (Waters). DynamX version 3.0 (Waters) was used to filter peptides by selecting those fragmented at least 2 times, presenting 0.2 fragments per amino acid, and found at least in 4 out of 5 acquired MS/MS files, to ensure confident peptide identifications. Additionally, the MS trace of the identified peptides was visually inspected and peptides with insufficient signal-to-noise ratio were discarded. The identification of the peptides for OMV-embedded NadA was not performed due to the lack of sufficient material.

Continuous labelling of rNadA. rNadA was diluted in PBS at room temperature or on ice and the temperature let equilibrate for 30 min prior labelling. HDX was then initiated by 4.17-fold dilution with deuterated PBS buffer (at 95% D2O), yielding to 72.2% of final deuterium content in the reaction mixture. The labelling was performed at room temperature (for 1 min, 10 min, 30 min, 100 min, and 1,000 min) and on ice (for 4 s, 15 s, 30 s, 1 min). At selected time intervals, an aliquot was withdrawn and quenched 1:1 with ice-cold 300 mM phosphate buffer at 4 M Urea (final pH/D 2.5). After quenching, samples were immediately frozen in liquid nitrogen and stored at -80 °C until LC-MS analysis. The labelling for 30 min at room temperature and 1 min on ice was performed two additional times, producing triplicates. A maximally labelled sample was associated to this experiment.

Pulse labelling of rNadA. rNadA was diluted in PBS and let in a thermomixer set at 23°C. At various incubation times (15 s, 1 min, 10 min, 100 min, and 1,000 min), an aliquot was withdrawn and HDX was initiated by 4.17-fold dilution with deuterated PBS buffer (at 95% D2O), yielding to 72.2% of final deuterium content in the reaction mixture. After 15 s of deuterium labelling, the reaction was quenched 1:1 with ice-cold 300 mM phosphate buffer at 4 M Urea (final pH/D 2.5). After quenching, samples were immediately frozen in liquid nitrogen and stored at -80 °C until LC-MS analysis. A maximally labelled sample was associated to this experiment.

Continuous labelling of rNadA and rNadA 24-149. rNadA and rNadA 24-149 were diluted alone in PBS and let in a thermomixer set at 23°C for 30 min. HDX was then initiated by 4.17-fold dilution with deuterated PBS buffer (at 95% D2O), yielding to 72.2% of final deuterium content in the reaction mixture. At various time intervals (15 s, 1 min, 10 min, 100 min, and 1,000 min), an aliquot was withdrawn and quenched 1:1 with ice-cold 300 mM phosphate buffer at 4 M Urea (final pH/D 2.5). After quenching, samples were immediately frozen in liquid nitrogen and stored at -80 °C until LC-MS analysis. A maximally labelled sample was included in the data set.

Continuous labelling of rNadA and rNadA-Hsp90. rNadA was incubated alone in PBS or with Hsp90 (ratio 1:3.8 NadA: Hsp90) for 30 min at room temperature or on ice, in order to allow the complex to form and the temperature to equilibrate prior labeling. HDX was then initiated by 4.17-fold dilution with deuterated PBS buffer (at 95% D2O), yielding to 72.2% of final deuterium content in the reaction mixture. At various time intervals (15 s, 1 min, 10 min, 30 min, 100 min, and 1,000 min at room temperature; 15 s, 30 s, 1 min on ice), an aliquot was withdrawn and quenched 1:1 with ice-cold 300 mM phosphate buffer at 4 M Urea (final pH/D 2.5). After quenching, samples were immediately frozen in liquid nitrogen and stored at -80 °C until LC-MS analysis. The labelling for 30 min was performed two additional times, producing triplicates for that time point. A maximally labelled sample was associated to this experiment.

Continuous labelling of rNadA and OMV-embedded NadA. rNadA and OMV-containing NadA were diluted in PBS and let in a thermomixer set at 23°C for 30 min prior labelling. HDX was then initiated by 2.33-fold dilution with deuterated PBS buffer (at 95% D2O), yielding to 66.5% of final deuterium content in the reaction mixture. At various time intervals (11 s, 1 min, 10 min and 100 min at 23°C), an aliquot was withdrawn and quenched 1:1 with ice-cold 300 mM phosphate buffer at 4 M Urea (final pH/D 2.5). After quenching, samples were immediately frozen in liquid nitrogen and stored at -80 °C until LC-MS analysis. The labelling for rNadA was performed in triplicates for each time point.

Maximally labeled control. rNadA was diluted in Solvent A (0.23% formic acid), injected onto the LC system and digested on-line. The solvent eluting from the pepsin column, containing peptides generated upon enzymatic cleavage, was collected and evaporated to dryness. Peptides were resuspended in deuterated PBS buffer at equal deuterium content as in the protein labelling mixture and the exchange was allowed for 2 h. The reaction was quenched 1:1 with ice-cold 300 mM phosphate buffer at 4 M Urea (final pH/D 2.5). Samples were immediately frozen in liquid nitrogen and stored at -80 °C until LC-MS analysis.

Liquid chromatography method for soluble recombinant proteins. Frozen quenched samples were rapidly thawed with a bench top centrifuge and injected onto a refrigerated UPLC system (NanoAcquity, Waters) with all chromatographic elements held at 0 °C. Except for the maximally labelled peptides, all protein samples passed through an in-house packed column containing pepsin immobilized on agarose resin beads at 20 °C. The generated peptides were trapped on a Vanguard column (BEH C18, 130 Å, 1.7 µm, 2.1 mm × 5 mm; Waters) and desalted for 2.5 min in solvent A (0.23% formic acid, pH 2.5) at a flow rate of 200 µl/min. Subsequently, the peptides were separated over an Acquity UPLC column (C18, 130 Å, 1.7 µm, 1.0 mm × 100 mm; Waters), with a 9 min-linear gradient rising from 8 to 40% of solvent B (0.23% formic acid in acetonitrile, pH 2.5) at 40 µl/min.

Size-exclusion chromatography coupled to liquid chromatography method for rNadA and OMV-embedded NadA. Frozen quenched samples were rapidly thawed and injected onto the refrigerated UPLC system with all chromatographic elements held at 0 °C. Protein and protein-lipid samples were analyzed by modifying the LC apparatus with the integration of a short size-exclusion chromatography (SEC) column in the HDX refrigerated chamber, placed after the pepsin column37. Samples were digested through a pepsin column held at 20 °C, then filtered through the SEC column (BEH SEC guard column, 125 Å, 1.7 µm, 4.6 x 30 mm, 1-80K; Waters). OMV lipids, when present, were retained in the SEC, whereas peptides passed and were trapped and desalted on a C18 Vanguard column for 4 min in solvent A, at a flow rate of 150 µl/min. Subsequently, peptides were separated over a C18 analytical column as described in the previous session. The SEC was cleaned on-line during the chromatographic gradient with four subsequent injections of 100 µL of 6M Urea in phosphate buffer (pH 2.5), whereas the pepsin column was removed, and a metal union mounted in its place.

Mass spectrometry method. Following the chromatographic separation, peptides were analyzed using a hybrid ESI-Q-TOF mass spectrometer (Synapt G2-Si, Waters). The instrument was set in positive ionization mode with spray voltage of 3 kV and the ions were further separated by ion mobility for enhancing peak capacity. The MS spectra were acquired in range from 200 to 2000 m/z, performing a scan every 0.5 s. Human Glu-fibrinopeptide (Sigma-Aldrich, St. Louis, MO) served as an internal standard and the peptide signal was recorded throughout all the analysis.

Data analysis. DynamX version 3.0 was used to calculate deuterium incorporation at peptide level. kHX values were calculated with HDsite software32, by applying the centroid fitting and normalizing the data for the maximally labelled control. Time points of the labelling on ice (≌0°C) were normalized to in-exchange time intervals at room temperature as elucidated in70.

Statistical approach. The statistical significance of a difference in HDX between two states was determined by calculating a confidence interval (CI) based on the pooled standard deviation of the peptide deuterium uptake (SD) in time points performed in triplicates following Weis’ recommendations71. For each state, the SDs were pooled using the root-mean-square, according to equation 1:

(1) SD = √ (∑SDi^2 / N)

where N is the number of peptides considered. A pooled SD for the states performed in triplicates, was subsequently calculated using equation 2 for the labelling rNadA-Hsp90:

(2) SD = √ (SD^2_pool1_rNadA_alone + SD^2_rNadA+Hsp90)

The restricted volume of OMV sample allowed to produce only singlets, while triplicates were carried out for the state rNadA at every time point. The propagated SD (SD_pool2) was thus calculated as for equation 3:

(3) SD = √ (SD^2_pool2_rNadA * 2)

The pooled SD calculated by eq. 2 and 3 were used to calculate the CI at significance level of 98% and 99% respectively, with a zero-centered average difference in deuterium uptake considering a two-tailed distribution with two degrees of freedom (n=3), by using equation 4 and 5, respectively:

(4) CI(98%) = ±6.965 * (SD_pool1 / √n)

(5) CI(98%) = ±9.925 * (SD_pool2 / √n)

where n is the number of replicates performed, equal to 3. The higher CI was set for the comparison of rNadA/OMV-embedded NadA to compensate for the lack of triplicates in one state. Additionally, for peptides showing significantly different deuterium content between rNadA and OMV-embedded NadA, we checked that the ∆HDX was at least >5xSD calculated for rNadA in that time point (all peptides pass this threshold, and, in most instances, their ∆HDX is highly above this threshold).

Label free proteomics analysis of the OMVs. Five micrograms of OMV proteins were digested with trypsin using the iST kit (Preomics) according to the manufacturer’s protocol. Data acquisition was performed on a Q Exactive Plus (Thermo Scientific) mass spectrometer equipped with an electrospray interface (ESI) and coupled to an Acquity UPLC I-Class (Waters). Peptides were injected and separated on an Acquity UPLC CSH Peptide C18 130 Å column (1 mm X 150 mm, 1.7 μm, Waters) set at 50 °C using a flow rate of 0.05 mL/min in gradient mode (mobile phase A consisted of 3% (v/v) acetonitrile 0.1% (v/v) formic acid in water and a mobile phase B of 0.1% (v/v) formic acid in acetonitrile). The following gradient was used: 0–28% B in 40 min, 28–85% B in 5 min, holding at 85% B for 5 min and re-equilibration at 0% B for 10 min. Acquisition was performed in Data Dependent Acquisition (DDA) mode (Top5). Data base search was performed using Peaks software by Bioinformatics solutions, version X, using the public data base containing the 1950 annotated proteins of the reference strain NZ-05/33 of Neisseria meningitidis. We also searched for the presence of PTMs (312, common and uncommon PTMs) using the Peaks PTM module of the Peaks Studio software. LFQE was performed using the “quantification Peaks module”. The identified proteins were first classified as lipoproteins using Signal P72 and the cellular localization of the other proteins was assigned by PsortB 3.073. For quantification, the measured proteins were normalized to the total amount of identifiable proteins and expressed as percentage of the total protein amount. No PTM was identified for NadA in OMVs, except for few peptides carrying deamidated asparagine and glutamine residues at too low abundance for the PTMs to be considered as confident identification.

Negative staining electron microscopy. OMVs diluted at 80 ng/μL in PBS were loaded onto a copper 200-square mesh grid of carbon/formvar rendered hydrophilic by glow discharge (Quorum Q150R S). The excess solution was blotted off after 30 s using Whatman filter Paper No.1 and then the grids were negatively stained with NanoW (Nanoprobes) for 30 s and then blotted using Whatman filter Paper No.1 and let air dry. Micrographs were acquired using a Tecnai G2 Spirit Transmission Electron Microscope (final magnification of 120000x), equipped with a CCD 2kx4k camera.

Cryo-electron microscopy. Data collection was performed at the FEI Company (Eindhoven, The Netherlands). Purified recombinant NadA variant 3 concentrated 1 mg/ml was diluted 1/15 times to a final concentration of 0.067 mg/ml with buffer composed of 20 mM Tris and 150 mM NaCl, pH 7. Quantifoil R2/2 grid (400 mesh Cu, Electron Microscopy Sciences) was rendered hydrophilic with 15 mA current for 90 second by glow discharge in a EmiTech K100X. 2.5 µl of the specimens were deposited onto the grid and vitrified using a Mark IV Vitrobot (FEI Company) with a blotting time of 4 second and humidity of 100% at 4°C. The plunge- frozen samples were imaged with a Titan Krios electron microscope operating at 300kV in focus with a Volta Phase plate with focus determined manually at a single offset position. A prototype Falcon 3 detector was used at 47,000 X nominal magnification with a pixel size of 1.75 A/pixel. A total dose of 120 electrons/Å2 was applied to each image space (details of Cryo-EM data collection, refinement and validation statistics in Supplementary Table 6). The image processing was performed within Scipion Software platform (http://scipion.cnb.csic.es)74 which provides several 3DEM software packages unified into an interface. Particles in the images presented a clear preferred orientation with mainly lateral views, due probably to the length of the molecule that exceeds the ice thickness. Moreover, due to the probable difficult classification of “heads” and “tails”, the particle picking was performed on half of the particles and 15273 “heads-tails” were extracted. The particles were classified several times to prune the set in 2D using Relion 2.075 and the best classes obtained were used to generate an initial model within EMAN276. This map was subjected to 3D classification in Relion resulting in two different models with distinct features: one with a globular extremity and a upright stalk while the other presented a straight part ended with a broaden bent part, presumably corresponding to the head and the tail, respectively. Particles were cleaned again using reference free 2D classification in Relion and the best 6270 particles were re-extracted from the original micrographs with a bigger box size of 320 pixels in order to obtain the full-length protein. Since the recombinant NadA full-length was known, an initial model of the correct length was created using Chimera program77 joining the initial volume of the head and the tail and extending the central part and low filtered in order not to generate bias. This raw initial model was 3d classified in Relion against the latest set of 4439 particles generating a structure of around 25 Å calculated using FSC=0.143 criteria78. The FSC has been obtained by using Projection Matching program by Xmipp79. To improve the resolution of this model, 7327 particles were manually selected with a smaller box size of 240 pixels to avoid an overlapping of particles in the same box. This latest dataset was pre-processed and 3D classified using Relion to generate two maps and a new set of 5735 particles formed by the best ones that were later re-extracted with improved centering to meliorate the alignment of the particles. The best 3D volume obtained from classification was processed with Xmipp3 tools to be filtered map and to create a tight soft mask. Finally, this masked density was used as an initial template for the 3D refinement in projection matching with the 5735 particles. The final resolution reached 14 Å measured with FSC=0.143 criteria. Local resolution was calculated using MonoRes80 while the angular distribution was tested with Relion package. To confirm the overall structure, a fitting with the crystal structure of NadAV3 (PDB: 6EUN) was performed using ChimeraX software.

Mouse immunizations. Female CD1 mice, 6-8-week-old, were purchased from Charles River Laboratories and kept in a controlled environment (individually ventilated cages; 22 ± 3 °C; 12 h/12 h light/dark cycle). Our mice were commonly group-housed, so we only used females as they exhibit generally a reduced level of aggressiveness in medium to long housing conditions, which can affect scientific outcome81. Five mice per group across 4 groups were used to detect the expected effect size and ensure reliable statistical power, considering the typical variability in assays59. This sample size adheres to ethical standards of animal welfare by minimizing the number of animals used, in line with the principle of reduction from the 3Rs framework. Animals have been randomized before allocation to groups using an Excel file. To guarantee a more heterogenous and representative immune response, CD1 mice, which are outbred mice, were used in this study, instead of the more classical Balb/c or C57BL/6 inbred mice. Animals and their housing and husbandry were checked daily, and their well-being and health status were recorded in a dedicated logbook according to the local standard operating procedures. Final bleeding was performed under general anesthesia and animals were euthanized by cervical dislocation before recovery from anesthesia. Mice were immunized intra-peritoneally (IP). For each injection, the mice received a total dose of 5 μg of OMV vaccines or 20 μg of NadA recombinant protein3,10. The OMVs or the recombinant protein vaccines were absorbed with aluminum hydroxide (Alum, 3 mg/ml) and administered in three doses at day 1, 21 and 35. Blood was taken at day 0, 20, 34 for further analysis and bleed out was performed on day 49.

IgG antibody titers elicited against NadA. Serum antibody titers against NadA were measured by ELISA, which was performed in a similar manner as previously described81. Microtiter plates were coated overnight at 4°C with 0.015 μM of recombinant purified NadA (variant 3). After overnight incubation, plates were washed three times with washing buffer (0.05% Tween 20 in PBS) and saturated with saturation buffer (2.7% polyvinylpyrrolidone in water) for 2 h at 37 °C. Then, plates were washed three times. Sera samples were then serially diluted in the dilution buffer (1% Bovine Serum Albumin, and 0.05% Tween 20 in PBS) and incubated for 2 h at 37 °C. After incubation, plates were washed three times with the washing buffer and incubated for 90 min at 37 °C with alkaline phosphatase-conjugated anti-mouse antibodies (Sigma-Aldrich), diluted 1:2000 in dilution buffer. After incubation, plates were washed and then incubated at room temperature for 30 min with a ready-to-use solution of alkaline phosphatase yellow liquid substrate system for ELISA (Sigma-Aldrich). After the incubation, 100 μL/well of 4 N NaOH solution was added to stop the enzymatic reaction, and the optical density was analyzed using a plate reader at a dual wavelength of OD 405/620–650 nm. The antibody titers of the single samples are expressed in Units ELISA per milliliter (UE/ml) and are calculated from a standard curve as part of the same assay.

Serum Bactericidal Assay (SBA). Serum bactericidal activity against N. meningitidis strain BZ83 (PorA subtype P1.5,10) was evaluated as previously described82 with pooled baby rabbit serum (Cederlane) used as the complement source (rSBA). Bacteria were grown in Mueller Hinton broth (MH), plus 0.25% (w/v) glucose, at 37°C with shaking until early exponential phase (OD600 of ∼0.25) and then diluted in Dulbecco’s saline phosphate buffer (Sigma) with 0.1% (w/v) glucose and 1% (w/v) BSA (Bovine Serum Albumin) to approximately 10^5 CFU/ml. The incubation with 25% of baby rabbit complement with or without polyclonal mouse sera at different dilutions was performed at 37°C (or 30°C as specified for respective experiments) with shaking, for 60 min. Serum bactericidal titers were defined as the serum dilution resulting in a at least 50% decrease in the CFU/ml after 60 min of incubation of bacteria with the reaction mixture, compared to the control CFU/ml at time zero.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study addresses the important question relevant for vaccine design - whether natively presented antigen is immunologically superior to recombinant protein antigen. Noteworthy results: The HDX-MS analysis of NadA displayed natively on OMVs shows conformational differences to recombinant NadA that may affect exposure of immunological epitopes. This is addressed next by murine immunogenicity studies but there are some limitations in how the assays have been conducted. A more direct approach is needed to support the hypothesis that protein vaccine antigens presented in native-like environments elicit a more potent immune response than recombinant forms.\nGeneral comments: To compare the immunogenicity of the above 2 forms, a more direct comparison would be OMVs from MenB which natively displays NadA versus OMVs from MenB NadA KO mixed with recombinant NadA such that they have the same background activity from OMVs per se but possess the 2 different forms of NadA. It is known from the proteomics data in this paper what percentage NadA constitutes of the total protein content of the former and thus an appropriate amount of recombinant NadA could be mixed with OMVs of the latter to directly compare the two. Instead, this study compares OMVs from MenB naturally displaying NadA with recombinant NadA protein using 2 different concentrations of protein for each. The impact of other antigens such as PorA will affect the potency of the OMV vaccine which means it is difficult to compare OMVs in vivo with a recombinant protein on its own. The authors do recognise this as it is mentioned (lines 333 and 334) “We cannot exclude that the increased bactericidal activity in the OMV formulation may be due to a shift in specific NadA IgG subclasses due to the adjuvant effect of LPS…”. However, in this study, the OMVs are derived from a meningococcal strain detoxified in its LPS due to a deletion in lpxL1 (described in the Methods). OMVs from a MenB NadA KO on its own has been included as a separate negative internal control, likewise E. coli OMVs displaying NadA but the relevance of their use is not made clear. The method for isolation of E. coli OMVs is missing.\nSpecific comments: All in vivo data should ideally include a placebo control group to establish the baseline for each response measured from experimental groups. More clarity is needed for some of the in vivo figures. For example, the y axis on Figure 4A showing ELISA data states GMT EU/mL. GMT is not defined in the manuscript. This should be IgG followed by units for concentration since a standard curve was used to convert absorbance values to concentration. It is unclear from the Methods whether the standard curve was conducted as part of the same assay (thus giving reliable data) or not as the use of a “reference” standard curve is described. The method and results are missing details such as the concentration of secondary antibody and substrate and suppliers. Figure 4A legend states antibody but should be more specific i.e., ELISA is to measure NadA-specific IgG.\nFigure 4B: it is surprising that MenB OMVs from NadA KO showed very little SBA response given the presence of other antigens such as PorA, particularly since the strain from which the OMVs were derived and the strain used in the SBA have the same PorA subtype (P1.5.2). A comment on this should be included.\nFigure 4C For the inhibition of bacterial adhesion assay, in the Introduction it states that OMV-embedded NadA elicits in mice antibodies of higher functional quality, capable of better preventing meningococcal adhesion to epithelial cells. A meningococcal adherence blocking assay would be the relevant assay to perform in this study. However, the Methods and Results sections describe that the bacteria used were E. coli harbouring the empty pET21 vector or E. coli expressing NadA and it is unclear why E. coli has been used. Whilst 4 vaccines are compared in Figure 4A and 4B, one of these 4 is omitted from Figure 4C (MenB OMVs from the NadA KO). The results for this (for one dilution) appear in the supplementary section (Figure S10) whereby OMVs from the MenB NadA KO are compared with OMVs from E. coli harbouring the empty vector ie 2 NadA negative controls. The data presented in this figure show that the former elicits no blocking activity whilst the latter 90-95% blocking which is not surprising given that E. coli was used as the organism for adherence. More interpretation linking the relevance of the data to the strains used for different experiments should be provided.", "reviewer_2": "This article by Calvaresi et al. utilises a novel structural technique to advance knowledge of the structure of a complex outer membrane protein and major bacterial vaccine antigen. The most compelling aspects of this article are the utilisation of the HDX technique to firstly determine the structural elements of the NadA protein that were missing from the crystallography-derived structure and secondly to identify regions of flexibility that allow for structural changes during interactions with the membrane or binding partners. These aspects of the manuscript will be of interest to a wide readership as they indicate how the HDX approach can be used in understanding flexible regions of proteins and how protein structure impacts on function.\nThe other major finding of this article is that embedding the NadA protein into a membrane (in outer membrane vesicles) results in elicitation of higher levels of bactericidal antibodies. This aspect is an important finding with precedent elsewhere in the literature that underpins the authors conclusion that the more open structure of the membrane-embedded protein allows the immune system to generate antibodies against structural elements that are hidden in the recombinant protein. These novel epitopes mimic epitopes presented on the surface of meningococcal cells resulting in a higher bactericidal effect. This is an important finding that will resonate with vaccinologists but is less compelling as no evidence is presented that the increased in bactericidal titre is due to the presence of such antibodies. The authors also only test against a strain where this vaccine antigen is over-expressed and hence not representative of strains found in human populations. Nevertheless the extrapolation of the observed data is compelling and likely to be proven as correct in the near future.\nThis article is well written and presented and hence there are only a few minor suggestions and comments.\nLine 181-182. I am unclear about how the Hsp90 binding is affecting the HDX values (as shown in Fig S5). The graph indicates that values for 40-51 are increased but this is described as decreased HDX in the text (I am not sure if a 1 sentence explanation in the legend may prevention confusion). I was also specifically unsure about the 40-51 region as shown in Figure S5 as the values seem to go up at 10 minutes but not other times. Does binding only elicit a transitory effect? This may be deserving of comment in the text.\nLine 231. The 5 ug of total protein for the OMVs implies 0.5 ug of NadA as line 150 indicates that ~10% of total protein is NadA. There is therefore a 40-fold difference in amount of injected NadA protein for the OMVs versus rNadA. Is this likely to have influenced results? Will more protein increase the likelihood of eliciting non-bactericidal antibodies? Conversely, is it possible that the high levels of recombinant NadA from aggregates that mask certain epitopes?\nLine 241-244. Is the clear difference in SBA titre due to masking of the tail region in OMV formulations? Antibodies binding to this region would be less likely to elicit a bactericidal effect but may be highly immunogenic and hence mask generation of antibodies to other regions. Could a similar effect be elicited by a rNadA lacking the tail region?\nFigure 4 should indicate that panel C utilises E. coli-NadA in the adhesion assay as readers are likely to conclude that this is inhibition of meningococcal adherence. This is particularly likely as panel B utlises the BZ83 meningococcal strain.\nLine 352. Missing word or rephrase. ‘similarly to what previously observed’", "reviewer_3": "The manuscript by Calvaresi and colleagues describes the comparison of recombinantly produced soluble NadA from meningococcus and membrane-embedded NadA. The recombinant NadA is the antigen used in the 4CMenB vaccine containing residues 24 to 350. The membrane-embedded NadA appears to comprise residues 1-405 (this is not clear in the text). The authors characterise the structural dynamics using hydrogen-deuterium exchange mass spectrometry (HDX) and electron microscopy (EM). They find specific differences in the dynamics of the NadA N-terminal head region, which is more mobile in the membrane-embedded protein, and the C-terminal region adjacent to the outer membrane, which is more flexible in the recombinant protein version. The authors use the data from EM and HDX to generate a more complete quasi-atomic model of the NadA trimer. They hypothesise that the membrane-embedded NadA antigen resembles more the native version, with different epitopes displayed and therefore will elicit a superior antibody response. Immunisation of mice using recombinant NadA or NadA embedded in outer membrane vesicles (OMVs) from E.coli and Meningococcus confirms that all antigens elicit an IgG antibody response. But the OMV-embedded NadA is superior in preventing meningococcal adhesion to Chang liver cells.\nThe manuscript is clearly written and easy to follow. I am not in a position to comment on the HDX and immunisation procedures and will focus mostly on the EM part of the work which requires revision.\nCryo-EM work:\n-Figure 1D: the fit of the crystal structure into the EM map is not visible. The angular relationship between the two views (90 degrees?) should be indicated, and a third view should be included.\n-Figure S4: A representative cryo-EM micrograph is missing showing the quality of the ice and the particles. Furthermore, the 2D class averages should have a scale bar, be enlarged and of higher resolution.\n-A supplementary figure should include a gold-standard FSC curve, and show the local resolution as indicated in the methods part.\n-The authors should show a FSC map-model to indicate the quality of the fit of the crystal structure into the cryo-EM map, or a correlation coefficient from Chimera.\n-The top view depicted is too small – it should be enlarged. A zoom into the fit would be really helpful. Again, the angular relationship between the views shown should be indicated.\n-The methods part does not include which version of Relion (line 553) was used for image processing.\n-The workflow for cryo-EM is unclear. I suggest including a workflow as a supplementary figure. Did the authors apply three-fold symmetry to the particle? The workflow figure should show the 2D class averages that went into the reconstruction and the initial 3D model the authors generated in Chimera (line 561)\n- The cryo-EM map has to be deposited into the EM data bank and the model fitted into the map needs to be deposited into the PDB.\nNegative stain EM of OMVs with NadA:\n-How could the authors be sure that the particles depicted in Fig. 2A are NadA? Is NadA the only protein with this shape and size in the OMVs?\n-There is a discrepancy between the ‘representative’ micrograph (Fig. 2A) and the ‘representative’ 2D class average (Fig. 2B). The particles highlighted on the surface of the OMVs appear all bent – in fact, a zoom in depicting several of these particles would be helpful. In contrast, the ‘representative’ 2D class average is straight. How can this be explained ? More 2D class averages should be shown, and not just the best one (the methods part describes this class average as the best one, thus it is not representative , line 536).\n-The 2D class average shown in Figure 2B should be larger and have a scale bar.\n-The details visible in the 2D class average in Figure 2B are remarkable. It would be good to show a collection of individual particles aligning to this class to show that the individual particles share these features and details.\n-The methods part indicates that 20 micrographs were collected and 800 particles picked. This indicates that 40 particles could be picked per micrograph. This is not possible for the ‘representative‘ micrograph shown in Figure 2A – I see max 15 particles in the micrograph. How do the authors explain the discrepancy?\nOther points:\n-The authors should clarify if the NadA versions displayed on E.coli and MenB OMVs are full-size/ native or truncated versions e.g. lacking the first 24 residues.\n-the changes in conformation upon Hsp90 binding detected by HDX MS are interesting. Would it be possible to show this with adhesin domains (Lox-1 receptor domain)?\n-Line 301: The percentage of open conformation is indicated for recombinant NadA; it should also be indicated for the NadA-OMV.\n-The recombinant NadA and the NadA-OMVs are not identical. How can the authors be sure that the ‘closed’ and ‘open’ conformations they identify by HDX are identical between the two constructs?\n-Regarding the neutralisation experiments (Fig. 4): The observed effect likely is only partially due to the membrane-associated NadA representing the native NadA better than the recombinant NadA. The authors should also discuss that the display of several copies of NadA on a vesicle elicits a stronger immune response – similar to VLPs displaying many copies of an epitope being more immunogenic than a peptide epitope alone.\nMinor comments:\nFigure 1A: the X-axis should have units.\nLine 184f: the sentence is incomplete and does not make sense.\nLine 273: NadA 24-350 is not full-length. Please correct this throughout the text clarifying which type of construct was used by indicating boundaries.\nLine 346: I am not sure if a statement like ‘our work represents a first attempt to’ is allowed, and I am also not convinced that it is indeed the first attempt to explain the immunogenic difference observed between native and recombinant antigen. I suggest adding ‘to the best of our knowledge’\nLine 538: correct to FEI Company, Eindhoven, The Netherlands\nLine 679: the journal is not indicated in this reference.", "reviewer_4": "The paper by Calvaresi et al. explores the structural dynamics of NadA in both its recombinant form and in a native-like OMV formulation. The techniques used are HDX-MS and electron microscopy. These OMVs have a history of use in a range of biophysical methods and are a nice intermediate between recombinant and native state. It is a great vehicle for HDX-MS experiments, and on that basis alone I’m excited about the paper. It should open the door to more HDX experiments of (bacterial) membrane proteins, and the results themselves are interesting (reminiscent of similar structural transitions in the RBD of Sars CoV2).\nThe work is well conducted, but I do have some concerns over the interpretation of findings and a set of more minor matters.\n1. My main concern is attributing the differential functional response to the enhanced conformational dynamics of the head domain. I agree that its dynamics are different than the rNadA, but there may be other reasons for the differential response. A more nuanced interpretation may be necessary, or some additional information provided. Consider:\na. Is there evidence of equivalence between the recombinant NadA and the OMV-NadA? For example, are the authors sure that there are no post-translational modifications that could explain the effect? I can’t tell from the description of the MS analyses.\nb. Similarly, it’s clear that the stalk stands well above the vesicles, but because EM images are always selectively filtered, are the authors sure that there are no other interactions with NadA in the OMV?\nc. And if the interactions are unique to the primary structure of NadA, notice that Figure 2c shows unique conformations across the length of the protein (from head to tail), in the form of stabilizations. Stabilization should not be equated with “hiding” antigenic regions. They can also expose new regions (or increase their population).\n2. HDX results. They look great, but I have a few questions.\na. The HDX summary tables show a much lower sequence coverage than described in lines 105ff. Is a table missing?\nb. I like HDsite but it makes assumptions that should be stated somewhere in the text (i.e., equivalency of back-exchange for all peptides). It can’t (or shouldn’t) be universally applied.\nc. It would be better if the descriptions of stability we simply referred to in terms of protection factors. These are well understood by the community. I find the combined use of both log(kHX) and protection factors a bit awkward.\nd. The mixed populations are interesting and an important element of the conclusions. Does figure S7 really show bimodality? It is hard to see this in the figure. Also I think S7 isn’t an OMV-embedded sample as stated in the text (or the caption is wrong?)\ne. I’m a little concerned that the OMV samples were not analyzed in replicate. Can the authors comment on the implications of this?\nGeneral:\n1. Did the authors mean figure 1C on line 136?\n2. Line 140; the resolution of the EM data is a bit low to conclude something ISN’T there (coiled coil).\n3. Define GMT in the caption of Figure 4a.\n4. The axes in the figures (spectral inserts) are really small and difficult to see.\n5. Line 86: should be \"combined with…\"\n6. Line 184 \"resulted pronounced…\" Not sure what is meant.\n7. Line 860 should be “adhesion with the…”"}, "author_response": "Our response: We gratefully thank the reviewer for these comments. The design of an appropriate strategy to compare the functionality of the antibodies raised by NadA recombinant antigen and its membrane-embedded counterpart was indeed challenging, due to the lack of unambiguous literature data on how to compare recombinant and native antigens. Indeed, in Fantappiè et al. (https://doi.org/10.3402/jev.v3.24015) and Bartolini et al. (https://doi.org/10.3402/jev.v2i0.20181), recombinant antigens were compared both to their OMV-embedded form and when mixed with empty OMVs, with apparent slightly different results; while in Croia et al. (https://doi.org/10.3390/membranes13110882) the antigens were compared between their OMV-embedded form and in their recombinant form mixed with empty OMVs.\nWe take the opportunity to clarify our approach.\n• A combination of ‘empty’ OMVs supplemented with the recombinant NadA was not used in our setup because we believe that it could result in a ‘non-specific’ binding of recombinant NadA on the OMV surface, due its adhesive nature, with implications in masking potentially relevant epitopes of the recombinant form. Our hypothesis is also in line to what recently reported by Croia et al. (https://doi.org/10.3390/membranes13110882), who evoked a probable capacity of OMVs to “absorb” soluble antigens by establishing stable interaction antigen-OMVs. In light of this, we have decided to proceed by comparing the response of the soluble recombinant NadA and OMV-embedded NadA in a similar manner as carried out by Huang et al. (doi: 10.1038/srep37242), and acknowledged the strong adjuvanticity of the OMVs per se, which is well documented.\n• Regarding the doses for our study, we chose specifically 20 ug of recombinant NadA since it is routinely used as a saturating dose in mice in several publications related to our 4CMenB vaccine, attaining maximal antigen-specific antibody responses (10.1084/jem.20020407, https://doi.org/10.1073/pnas.0603940103, 10.1371/journal.pone.0110047). Concerning the 5 μg of OMVs, several publications report that antigen-specific immune responses could be obtained with relatively low doses of OMVs in animal models (doi: 10.1038/srep37242., 10.3402/jev.v3.24015). In addition, very recently Croia et al. (https://doi.org/10.3390/membranes13110882) provided an extensively study on how the amount of recombinant antigens presented in E. coli OMVs influence the antigen-specific antibody response in mice and concluded that a 5 𝜇g-dose of OMVs is generally sufficient to elicit plateau levels of antibodies against OMV proteins expressed at concentrations ≥1.5-2%, which is in the range of concentration of NadA in OMVs. If anything, our selected doses put the OMV-derived NadA at a significantly lower dose with respect to the recombinant NadA, which would rather bias the data against it rather than for it. We added these clarifications and references in the main text and modified the text, which now reads:\nLines 229-233: “CD-1 mice were immunized with 20 μg of rNadA (a saturating dose in mice3,10) or 5 μg (total protein content) of MenB or E. coli OMVs overexpressing NadA. Very recently, Croia et al.43 reported that this amount of OMVs was sufficient to elicit saturating levels of antibodies against OMV proteins expressed at concentrations ≥1.5-2%, which is in the range of the percentage of NadA in the OMVs used in our study.”\n• With respect to the specificity of the functional responses of anti-NadA IgGs, we immunized mice with OMVs derived from MenB strain 5/99 and, among a panel tested, selected the strain BZ83 for the SBA assay, as the latter is mismatched for PorA variant and also for the other major antigens, such as fHbp and NHBA, therefore all bactericidal responses elicited by the 5/99 meningococcal OMVs-derived sera against the BZ83 strain are expected to be NadA-specific. While responses to PorA or other antigens might be elicited, ultimately no contribution to the SBA is expected to derive from any other OMV components, as also demonstrated by the negative SBA titers of the MenB OMV devoid of NadA (fig. 4b). This also underlines the significance of our negative control. We have now clarified this aspect in the text.\nLine 238-245: To estimate the serum bactericidal activity (SBA) of the elicited antibodies, mice sera were tested against the BZ83 meningococcal natural strain. The BZ83 strain was selected as test strain from a panel of strains as it is mismatched for the PorA variant and also for the other MenB major antigens, such as fHbp and NHBA, therefore all bactericidal responses elicited by the 5/99 meningococcal OMVs-derived sera against the BZ83 strain are expected to be NadA-specific. Expectedly, no killing was achieved with the serum derived from mice immunized with MenB OMVs from the NadA KO (Fig. 4b), demonstrating that while responses to PorA or other antigens could be elicited, no functional contributions to the SBA derive from antibodies to other OMV components.”\n• Furthermore, we acknowledge that the adjuvant nature of the LPS in an OMV-based formulation may generate more bactericidal responses, even with similar IgG levels, if more IgG2a subclass is present. However, as the reviewer remarks, the MenB OMVs are detoxified in LPS due to the lpxL1 mutation, whilst the E. coli OMVs are not and contain LPS with full endotoxin activity and adjuvant potential. We clarified this concept in the text. Lines 351-356 now read:\n“We cannot exclude that the increased bactericidal activity in the OMV formulations may be due to a shift in specific NadA IgG subclasses due to the adjuvant effect of LPS60,61 and particularly by an increase of IgG2a, which in mice has higher complement fixing effector activity62. However, although our MenB OMVs contained genetically detoxified LPS whereas the E. coli OMVs had LPS with full endotoxin activity, no significant differences in functionality were observed between the two OMV formulations.”\n• We take the opportunity to highlight that another factor playing a potential role in the enhanced functional immune response of the OMV-based formulations is their high-density display of NadA, as demonstrated also for Virus-like Particles and Nanoparticles, such as NadA displayed on ferritin (10.1038/nri2868, 10.1016/j.csbj.2015.11.001, 10.1038/s41423-022-00897-8, and 10.3390/ijms24076183). This aspect has been mentioned by reviewer 3 and is now clarified in the text.\nLines 343-350: “By comparing the immune response elicited by recombinant and OMV-embedded native NadA, we first observed that, although the dose of NadA administrated as recombinant antigen was approximately 40 times higher than the antigen in the OMVs, all formulations elicited comparable NadA-specific antibody titers. This could be attributable to a higher antigen density presentation on the OMV surfaces that facilitates B cell activation by cross-linking B cell receptors56,57, as reported for antigens displayed on Virus-Like Particle58 or Nanoparticles57,59, or to the effects of OMV proteins carrying a plethora of MHC II epitopes that raise a large pool of effector helper CD4+ T cells, as highlighted by Croia et al.43”\n• We thank the reviewer for highlighting that the method for the isolation of the E. coli OMVs in the “Materials and Methods” section was missing. We have now inserted it in the paragraph “Preparation of OMVs”, which now reads:\nLines 412-421. “For the generation of the E. coli OMV vaccine strains pET21 plasmid expressing the full-length nadA was transformed into E. coli BL21(DE3) ΔTolR strain. BL21(DE3) ΔTolR strains were pre-cultured overday in 7 ml of HTMC medium supplemented with 100 µg/ml of ampicillin. The cultures were diluted 1:100 in 50 ml of HTMC liquid medium and incubated overnight at 30°C, 5% CO , 160 rpm. Bacteria 2 were centrifuged at 3400 × g for 15 min, resuspended into 60 ml of fresh HTMC broth supplemented with 1 mM IPTG and incubated for 3 h at 37°C, 5% CO , 160 rpm to mid-exponential phase. The bacterial cells 2 were pelleted by centrifugation for 30 min at 3400 × g and 4°C and the culture supernatants filtered (pore size 0.22 µm). OMVs were collected from the filtered supernatants of meningococcal and E. coli cultures by ultracentrifugation for 2 h at 9,6000 × g and 4 °C, and an additional washing step with PBS was performed. The OMV pellets were re-suspended in PBS at a concentration of 1 µg/µL.”\nSpecific comments: All in vivo data should ideally include a placebo control group to establish the baseline for each response measured from experimental groups. More clarity is needed for some of the in vivo figures. For example, the y axis on Figure 4A showing ELISA data states GMT EU/mL. GMT is not defined in the manuscript. This should be IgG followed by units for concentration since a standard curve was used to convert absorbance values to concentration. It is unclear from the Methods whether the standard curve was conducted as part of the same assay (thus giving reliable data) or not as the use of a “reference” standard curve is described. The method and results are missing details such as the concentration of secondary antibody and substrate and suppliers. Figure 4A legend states antibody but should be more specific i.e., ELISA is to measure NadA-specific IgG.\nOur response: We thank the reviewer for giving us the opportunity to better clarify how we conducted our ELISA experiment.\n• The GMT has been substituted by IgG titers that, as mentioned by the reviewer, are more appropriate.\n• In our study, we used a method validated for the release of the GMP lots of NadA. Each plate contains positive and negative controls to validate the assay as well as the standard curve. We have expanded the ELISA paragraph in the Material and Methods (lines 623-637), which now reads:\n“Serum antibody IgG titers against NadA were measured by ELISA, which was performed in a similar manner as previously80. Microtiter plates were coated overnight at 4°C with 0.015 μM of recombinant purified NadA (variant 3). After overnight incubation, plates were washed three times with washing buffer (0.05% Tween 20 in PBS) and saturated with saturation buffer (2.7% polyvinylpyrrolidone in water) for 2 h at 37 °C. Then, plates were washed three times. Sera samples were then serially diluted in the dilution buffer (1% Bovine Serum Albumin, and 0.05% Tween 20 in PBS) and incubated for 2 h at 37 °C. After incubation, plates were washed three times with the washing buffer and incubated for 90 min at 37 °C with alkaline phosphatase-conjugated anti-mouse antibodies (Sigma-Aldrich), diluted 1:2000 in dilution buffer. After incubation, plates were washed and then incubated at room temperature for 30 min with a ready-to-use solution of alkaline phosphatase yellow liquid substrate system for ELISA (Sigma-Aldrich). After the incubation, 100 μL/well of 4 N NaOH solution was added to stop the enzymatic reaction, and the optical density was analyzed using a plate reader at a dual wavelength of OD 405/620–650 nm. The antibody titers of the single samples are expressed in Units ELISA per milliliter (UE/ml) and are calculated from a standard curve as part of the same assay.”\n• The legend of Fig. 4A has been revised to increase its clarity and now reads:\n‘A) NadA-specific IgG titers of sera from mice immunized with NadA-OMVs (5 µg) or with rNadA (20 µg) as measured by ELISA. Each dot represents an individual mouse serum while lines indicate the median value within each immunization group.\nFigure 4B: it is surprising that MenB OMVs from NadA KO showed very little SBA response given the presence of other antigens such as PorA, particularly since the strain from which the OMVs were derived and the strain used in the SBA have the same PorA subtype (P1.5.2). A comment on this should be included.\nOur response: We thank the reviewer for this comment as it spotted an oversight in reporting the PorA subtypes of the N. meningitidis strains used in this study; we addressed this point also in one of the previous responses. Mice were immunized with OMVs from MenB strain 5/99, which bears PorA of subtype P1.5.2, while the strain BZ83, which bears a PorA subtype P1.5,10, was used for the SBA assay. The diverse sequence of the immunogenic loop VR2 explains the very low SBA response of MenB OMVs from NadA KO. This mistake is now corrected in the main text.\nLines 638-648 now read: “Serum bactericidal activity against N. meningitidis strain BZ83 (PorA subtype P1.5,10) was evaluated as previously described80…”. In this sentence, the name of the strain was also corrected from BZ883 into BZ83.\nFigure 4C: For the inhibition of bacterial adhesion assay, in the Introduction it states that OMV-embedded NadA elicits in mice antibodies of higher functional quality, capable of better preventing meningococcal adhesion to epithelial cells. A meningococcal adherence blocking assay would be the relevant assay to perform in this study. However, the Methods and Results sections describe that the bacteria used were E. coli harbouring the empty pET21 vector or E. coli expressing NadA and it is unclear why E. coli has been used. Whilst 4 vaccines are compared in Figure 4A and 4B, one of these 4 is omitted from Figure 4C (MenB OMVs from the NadA KO). The results for this (for one dilution) appear in the supplementary section (Figure S10) whereby OMVs from the MenB NadA KO are compared with OMVs from E. coli harbouring the empty vector ie 2 NadA negative controls. The data presented in this figure show that the former elicits no blocking activity whilst the latter 90-95% blocking which is not surprising given that E. coli was used as the organism for adherence. More interpretation linking the relevance of the data to the strains used for different experiments should be provided.\nOur response: We thank the reviewer for this comment and welcome the opportunity to clarify.\n• For the binding inhibition assay, we replicated an optimized assay where NadA is expressed in E. coli, which has already been extensively used to probe the NadA-mediated adhesion function in a specific way (please see some examples: https://doi.org/10.1111/j.1365-2958.2004.04423.x, https://pubmed.ncbi.nlm.nih.gov/22066472/ and https://doi.org/10.1128/jb.00430-10). As N. meningitidis has diverse, and likely redundant, virulence factors, determining the precise role of NadA is difficult. However, E. coli expressing NadA helps to isolate its specific contribution, as reported here https://pubmed.ncbi.nlm.nih.gov/22066472/. We have now clarified this aspect in the result session (lines 253-257), which now reads:\n“As asserted in a previous work40, the use of the E. coli heterologous system is preferred to assess the NadA-specific contribution to epithelial cell adhesion due to the potential presence of redundant adhesion factors on the meningococcus surface. Importantly, our negative control E. coli without NadA (E. coli-pET21) showed no adhesive properties to these cells (Fig. S11).”\n• In addition, we have revised the text to give clarity regarding the use and description of the controls in Fig. S10 (now become S11). On the left side, we report that no inhibition of bacterial adhesion (100% adhesion) is seen upon incubation with sera from MenB NadA KO (1:20 dilution), as expected. The least diluted condition (1:20) was evaluated as one would therefore expect analogous results under the more diluted conditions. On the right side of the same figure, we reported the use of E. coli transformed with empty pET21 vector (E. coli with no expression of NadA), as a negative control. The significance of such control has been previously described in other works (https://doi.org/10.1111/j.1365-2958.2004.04423.x and https://doi.org/10.1128/jb.00430-10). We have better clarified the use of these controls in the caption of Fig. S11, that now reads:\n“Percentage of adherent bacteria (E. coli expressing NadA) preincubated with sera raised against MenB OMVs NadA KO (1:20 dilution) in duplicates (R1 and R2) and percentage of adhesion of E. coli cells not expressing NadA (transformed with empty plasmid pET21) in duplicates (R1 and R2)…”\nReviewer #2 (Remarks to the Author):\n- Line 181-182. I am unclear about how the Hsp90 binding is affecting the HDX values (as shown in Fig S5). The graph indicates that values for 40-51 are increased but this is described as decreased HDX in the text (I am not sure if a 1 sentence explanation in the legend may prevention confusion). I was also specifically unsure about the 40-51 region as shown in Figure S5 as the values seem to go up at 10 minutes but not other times. Does binding only elicit a transitory effect? This may be deserving of comment in the text.\nOur response: We welcome the opportunity to explain. In Fig. S5 (now Fig. S6), the y-axis indicates the difference in HDX (ΔHDX) between rNadA – rNadA bound to Hsp90. Positive values indicate that rNadA alone undergoes faster HDX than bound rNadA, hence a decreased HDX in the bound state. We better specified this in the text and in the fig. S6 caption.\nLines 177-180 “We confirmed that Hsp90 directly associates with the NadA head domain, specifically at the level of residues 40-51, which showed decreased HDX when rNadA is bound to Hsp90 compared to the unbound state (Fig. S6)”.\nFig. S6 caption: “Conformational impact of Hsp90 on rNadA. Difference in HDX between rNadA alone and in complex with Hsp90 labelled at room temperature (A) and on ice (B). Positive values indicate that rNadA undergoes faster HDX than bound rNadA, hence a decreased HDX in the bound state…”.\nWe added a similar sentence also in the caption of Fig. 2B, which now reads “Difference in HDX between rNadA and OMV-embedded NadA. Positive values indicate that rNadA undergoes faster HDX than OMV-embedded NadA, hence a rigidification of OMV-embedded NadA compared to rNadA”.\nWe note that the fact that the stabilizing effect (reduced HDX) upon Hsp90 binding is only apparent at two time points (1 and 10 min) does not indicate that binding elicits a transitory effect but rather that Hsp90 binding impacts the conformational environment around backbone amides in NadA that exchange on a fast to intermediate timescale.\n- Line 231. The 5 ug of total protein for the OMVs implies 0.5 ug of NadA as line 150 indicates that ~10% of total protein is NadA. There is therefore a 40-fold difference in amount of injected NadA protein for the OMVs versus rNadA. Is this likely to have influenced results? Will more protein increase the likelihood of eliciting non-bactericidal antibodies? Conversely, is it possible that the high levels of recombinant NadA from aggregates that mask certain epitopes?\nOur response: We thank the reviewer for highlighting this point. We addressed a specific comment about the doses used in a previous answer to reviewer 1. Concerning the reviewer’s point about the likelihood of increased amount of antigen to elicit non-bactericidal antibodies, we here provide a Serum Bactericidal Assay (SBA) performed from sera of mice immunized by doses of rNadA ranging from 1,5 to 20 ug (pool of seven animals per group, tested on MenB strain BZ83). The titers increased with higher rNadA amount used to immunize the animals indicating that increased doses of antigen do not appear to cause the generation of non-bactericidal antibodies.\nrNadA amount SBA\n20 ug 65536\n5 ug 32768\n1.25 ug 16384\nWe do not aim to insert these data in the manuscript since we believe that are out of the paper scope.\nRegarding the reviewer’s comment about possible aggregates at 20 ug of rNadA that could mask certain epitopes, we do not have any evidence of NadA aggregation at this amount and concentration, as demonstrated in the paper by Magagnoli et al. (doi: https://doi.org/10.1016/j.vaccine.2009.01.099), which reports the physicochemical characterization of the recombinant NadA. We would like also to mention another previous study reporting rNadA as a still monodisperse species after a heating period of 4 h at 37°C (Bozza G et al., https://doi.org/10.1371/journal.pone.0110047). Here, the authors showed a SEC profile of rNadA, where high-molecular weight aggregates (typically eluting in the first SEC fractions) are absent: SEC-HPLC profile of rNadA (from Bozza et al.)\nUpon preparing this response, we also realized that in the “Material and Methods” section, a paragraph reporting the provenience of rNadA and Hsp90 was missing and now added it, (Recombinant NadA proteins and Hsp90, lines 400-403) and included these two references.\n- Line 241-244. Is the clear difference in SBA titre due to masking of the tail region in OMV formulations? Antibodies binding to this region would be less likely to elicit a bactericidal effect but may be highly immunogenic and hence mask generation of antibodies to other regions. Could a similar effect be elicited by a rNadA lacking the tail region?\nOur response: We thank the reviewer for the pertinent consideration. Considering our repeated immunization data with the recombinant NadA of different length, we think that such possibility is unlikely.\nCharacterization of monoclonal antibodies (mAbs) elicited by mice immunization with full-length recombinant NadA (Giuliani M. et al., doi: 10.1038/s41598-018-22057-7) indicated only one mAb out of 18 are directed to the tail region (327-350), while all the others were reported to target the stalk and, mainly, the head. It is true that this single mAb was not bactericidal, but still its identification remains a single event. However, we cannot completely rule out such possibility and we believe that additional studies with monoclonal and polyclonal antibodies will help to clarify on this point. This is something we aim to investigate in the future. To emphasize this point, we have expanded the sentence in lines 377-381, which now reads:\n‘Additionally, epitope mapping studies with monoclonal and polyclonal antibodies elicited after immunization with rNadA and OMV-embedded NadA will clarify the plasticity of the humoral response and assess the relationship between the transient opening of NadA trimer on the cell surface and immunogenicity, as well as a potential immune refocusing effect resulting from differences in antigen accessibility in the two forms.’\n- Figure 4 should indicate that panel C utilises E. coli-NadA in the adhesion assay as readers are likely to conclude that this is inhibition of meningococcal adherence. This is particularly likely as panel B utlises the BZ83 meningococcal strain.\nOur response: The caption of Fig. 4C now reads “Inhibition of bacterial adhesion of E. coli expressing NadA exerted by sera raised against rNadA and NadA-OMVs formulations in two biological duplicates”. We note that we commented on the use of E. coli to probe NadA-mediated adhesion in an answer to reviewer 1.\n- Line 352. Missing word or rephrase. ‘similarly to what previously observed’\nOur response: The sentence now reads “as previously observed in the context of viral infections” (line 377).\nReviewer #3 (Remarks to the Author):\nCryo-EM work:\n- Figure 1D: the fit of the crystal structure into the EM map is not visible. The angular relationship between the two views (90 degrees?) should be indicated, and a third view should be included.\nOur response: The figure has been modified according to the request of the reviewer. The fit of the crystal structure (PDB 6EUN) into the EM map is now more visible. The angular relationship between the different views is now indicated, and a third view (a top view) is included. We thank the reviewer for these suggestions, which improve the quality of the figure.\n-Figure S4: A representative cryo-EM micrograph is missing showing the quality of the ice and the particles. Furthermore, the 2D class averages should have a scale bar, be enlarged and of higher resolution.\nOur response: A representative cryo-EM micrograph is now shown in a new figure (Fig. S5), which includes also the Cryo-EM workflow. The 2D class averages have been modified according to the reviewer’s suggestions and now include the scale bar. In addition, they have been enlarged and are of higher resolution.\n-A supplementary figure should include a gold-standard FSC curve, and show the local resolution as indicated in the methods part.\n-The authors should show a FSC map-model to indicate the quality of the fit of the crystal structure into the cryo-EM map, or a correlation coefficient from Chimera.\nOur response: A new figure (Fig S5) has been inserted with the FSC plot and related legend.\n-The top view depicted is too small – it should be enlarged. A zoom into the fit would be really helpful. Again, the angular relationship between the views shown should be indicated.\nOur response: These figures have been included in the Fig. 1d of the main text, according to the reviewer’s suggestions.\n-The methods part does not include which version of Relion (line 553) was used for image processing.\nOur response: We used Relion 2.0, and now included this information in the Material and Methods (line 593).\n-The workflow for cryo-EM is unclear. I suggest including a workflow as a supplementary figure. Did the authors apply three-fold symmetry to the particle? The workflow figure should show the 2D class averages that went into the reconstruction and the initial 3D model the authors generated in Chimera (line 561)\nOur response: We thank the reviewer for the suggestion. The addition of the cryo-EM workflow figure allows an easier comprehension of the reconstruction process followed. The cryo-EM workflow has been added as Fig. S4 as kindly suggested by the reviewer. No, three-fold symmetry has not been applied to particles.\n- The cryo-EM map has to be deposited into the EM data bank and the model fitted into the map needs to be deposited into the PDB.\nOur response: The cryo-EM map has been deposited in the EMDB data bank with the EMDB-ID EMD-19474. The cryo-EM map has been rigid-body fitted, using ChimeraX, with the crystal structure of NadA24-170 (PDB 6EUN). The PDB ID 6EUN has been indicated in the EMDB upload as the fitted crystal structure. No pdb/mmcif structural information model has been generated starting from the cryo-EM volume due to the very low resolution of the map. We also modified the Material and Methods reporting the correct PDB code of the structure used for rigid body fitting (PDB ID 6EUN).\nNegative stain EM of OMVs with NadA:\n-How could the authors be sure that the particles depicted in Fig. 2A are NadA? Is NadA the only protein with this shape and size in the OMVs?\nOur response: We thank the reviewer for the pertinent question. The immunogold (below reported) performed with an anti-NadA monoclonal antibody recognizing those appendages confirms NadA identity. We prefer not to include this figure in the current paper since it will be included in another paper under preparation. To address the reviewer’s concern, we have decided instead to insert a sentence in the text in lines 152-156, which now read:\n‘In negative staining transmission electron microscopy (NS-TEM), the OMVs appeared as spherical and intact particles with a diameter ranging from 30 to 100 nm, surrounded by a thick membrane carrying several appendages corresponding to NadA (Fig. 2a), which are not visible in OMVs released by the bacteria that do not express NadA35,36 (DOI: 10.1128/IAI.72.4.1914-1919.2004; DOI: 10.1002/pmic.200500164).\nHomologous and heterologous expression of NadA on OMV. From left to right: Immunogold electron microscopy on OMVs produced by meningococcal MenB ΔlpxL1 ΔNadA (negative control), MenB ΔlpxL1 ΔNadR strains (MenB-OMVs expressing NadA) and E.coli BL21ΔTolR_pET21b_NadA strain (E. coli OMVs).\n-There is a discrepancy between the ‘representative’ micrograph (Fig. 2A) and the ‘representative’ 2D class average (Fig. 2B). The particles highlighted on the surface of the OMVs appear all bent – in fact, a zoom in depicting several of these particles would be helpful. In contrast, the ‘representative’ 2D class average is straight. How can this be explained? More 2D class averages should be shown, and not just the best one (the methods part describes this class average as the best one, thus it is not representative , line 536).\n-The 2D class average shown in Figure 2B should be larger and have a scale bar.\n-The details visible in the 2D class average in Figure 2B are remarkable. It would be good to show a collection of individual particles aligning to this class to show that the individual particles share these features and details.\n-The methods part indicates that 20 micrographs were collected and 800 particles picked. This indicates that 40 particles could be picked per micrograph. This is not possible for the ‘representative‘ micrograph shown in Figure 2A – I see max 15 particles in the micrograph. How do the authors explain the discrepancy?\nOur response. We gratefully thank the reviewer for the thorough review of our data and understand the reviewer’s concern. After careful consideration, we have determined that the best approach is excluding some of the NS-TEM data (panel B of fig. 2) yet keeping the representative micrograph of the OMV overexpressing NadA (fig. 2A) to show the integrity of the OMVs and the presence of NadA appendages on their surface.\nThe decision to remove the aforementioned data was made after careful consideration of their relevance and impact on the overall conclusions of our study. Importantly, the data in question does not significantly impact the interpretation of our results or the conclusions drawn therefrom. In the interest of maintaining the highest level of scientific clarity, we concluded that including this data could potentially introduce ambiguity and detract from the quality of our research. We highlight that the data presented in the current version of the manuscript are already fully sufficient and appropriate to support the conclusions drawn from our study. Indeed, the primary conclusions and structural significance of our manuscript are predominantly based on the interpretation of the HDX-MS data, which per se provide robust foundations to support the main arguments of our study.\nOther points:\n-The authors should clarify if the NadA versions displayed on E.coli and MenB OMVs are full-size/ native or truncated versions e.g. lacking the first 24 residues.\nOur response: We thank the reviewer for the comment and have now specified that residues 1-23 comprise the signal peptide, which is cleaved off.\nLine 52 now reads “After removal of the signal peptide (aa 1-23), NadA presents an N-terminal head region (aa 24-73)…”.\nFurthermore, line 146 now reads: “To do so, we analyzed meningococcal OMVs overexpressing NadA (NadA ). In this native form, the sequence signal is cleaved off and the passenger domain…”\n24-405\n-the changes in conformation upon Hsp90 binding detected by HDX MS are interesting. Would it be possible to show this with adhesin domains (Lox-1 receptor domain)?\nOur response: We are indeed very interested in analyzing changes in conformational dynamics upon binding of other NadA-interactors. Lox-1 is an example. It would be particularly interesting since the binding site for Hsp90 and Lox-1 overlaps in NadA head domain (Liguori et al. DOI: 10.1128/mBio.01914-18). We would like to make the reviewer aware that we mapped by HDX-MS the interaction of NadA with other partners, specifically human Siglec-5 and Siglec-14. The work has just been accepted for publication and will be very soon available to the scientific community (Benucci et al., mBio, 2024, accepted manuscript).\n-Line 301: The percentage of open conformation is indicated for recombinant NadA; it should also be indicated for the NadA-OMV.\nOur response: The reviewer points out an important aspect and we take the opportunity to better clarify. For OMV-embedded NadA, we have purposely refrained to indicate the percentage of open conformation, whose abundancy appears clearly higher that in rNadA yet difficult to quantitate due to the observed closed-to-open conformation transition (manifesting as EX-1 kinetics). In contrast, in rNadA, the open conformation appears more stably populated (no apparent interconversion is observable), allowing us to determine its abundance. We have described these aspects in lines 199-203 and now further commented in lines 310-313:\n“We observed that the open conformation is present to a low extent (10% of total population) in the recombinant NadA, whilst it is significantly more populated in the OMV state (although its abundance cannot be accurately quantified due to the observed closed-to-open conformational transition).”\n-The recombinant NadA and the NadA-OMVs are not identical. How can the authors be sure that the ‘closed’ and ‘open’ conformations they identify by HDX are identical between the two constructs?\nOur response: We agree that we cannot define the structural properties of the open conformation(s) in detail by HDX-MS. Hence, our focus was to describe the presence of and dynamic transitions between the closed and open states, which is what HDX-MS can do well. However, we note that the peptide centroid masses of the open conformation in the two forms appears very similar, suggesting that the two open conformations are structurally similar if not identical.\n-Regarding the neutralisation experiments (Fig. 4): The observed effect likely is only partially due to the membrane-associated NadA representing the native NadA better than the recombinant NadA. The authors should also discuss that the display of several copies of NadA on a vesicle elicits a stronger immune response – similar to VLPs displaying many copies of an epitope being more immunogenic than a peptide epitope alone.\nOur response: We thank the reviewer for highlighting this important point. Indeed, we cannot exclude that the display of multiple NadA copies on the OMVs may be beneficial for eliciting functional antibodies. To the best of our knowledge, B-cell activation is facilitated not only by the repetition of epitopes on the OMV surface but also by a correct spacing of the antigen and by the size of the OMVs, which we have not investigated in the context of this paper. Anyway, we agree that it is very relevant to elaborate on this, therefore we inserted a comment in lines 343-350, which now reads:\n“By comparing the immune response elicited by recombinant and OMV-embedded native NadA, we first observed that, although the dose of NadA administrated as recombinant antigen was approximately 40 times higher than the antigen in the OMVs, all formulations elicited comparable NadA-specific antibody titers. This could be attributable to a higher antigen density presentation on the OMV surfaces that facilitates B cell activation by cross-linking B cell receptors56,57, as reported for antigens displayed on Virus-Like Particle58 or Nanoparticles57,59, or to the effects of OMV proteins carrying a plethora of MHC II epitopes that raise a large poll of effector helper CD4+ T cells, as highlighted by Croia et al.43”\nMinor comments:\nFigure 1A: the X-axis should have units.\nOur response: The name of the x-axis is indicated in the bottom graph (deuteration time) and the deuterium labelling times are indicated in the figure caption. No unit was specifically indicated for the x-axis to avoid cluttering and overcomplicating the graphs, as the time points followed have three different time units.\nLine 184: the sentence is incomplete and does not make sense.\nOur response: We have now rephrased as follow: “This destabilization was clearly observable until the predicted coiled-coil interruption at residues 252-265…”\nLine 273: NadA 24-350 is not full-length. Please correct this throughout the text clarifying which type of construct was used by indicating boundaries.\nOur response: We apologize for the confusion. This has been corrected in\nline 88: full-length NadA → NadA ,\n24-350\nline 146: full-length NadA → NadA. In detail: “….we analyzed meningococcal OMVs overexpressing NadA (NadA ). In this native form, the signal peptide has been cleaved off and the passenger domain of the\n24-405\nprotein extends outwards from the beta-barrel translocator domain, which is anchored and inserted into the outer membrane12”\nin line 277 “recombinant soluble meningococcal NadA” →soluble meningococcal vaccine antigen NadA\n24-350\nin line 283 full-length NadA (NadA ) →NadA\n24-350 24-350\nin line 297 full-length rNadA →NadA .\n24-350\nIn lines 276-278, we now highlighted that the recombinant protein does not comprise the beta-barrel anchor. The sentence now reads: “In this study, we applied HDX-MS to propose a structural model of NadA\n24-350\n(recombinant 4CMenB antigen without the beta barrel anchor), and analyzed the conformational dynamics of NadA in situ (NadA ), as natively exposed into OMVs….”.\n24-405\nFurthermore, in line 340-341, we have highlighted that we compared the immune response of the NadA form contained in the current vaccine, which is NadA . The sentence now reads: “Therefore, we evaluated the\n24-350\nimmune response elicited by NadA , the soluble recombinant vaccine form of 4CMenB, and OMV-embedded NadA (NadA ),….”\n24-350\n24-405\nWe also stressed this in lines 228-229: “…we compared the immunogenicity of the 4CMenB vaccine recombinant form of NadA (NadA ) and OMV-embedded NadA (NadA ) …”.\n24-350 24-405\nLine 346: I am not sure if a statement like ‘our work represents a first attempt to’ is allowed, and I am also not convinced that it is indeed the first attempt to explain the immunogenic difference observed between native and recombinant antigen. I suggest adding ‘to the best of our knowledge’.\nOur response: We thank the reviewer for this note, we have now rephrased the sentence by adding “To the best of our knowledge”, and “one of the first attempts”. By this sentence, we intended to convey the message that our work represents the first, or one of the first attempts, to explain those differences in immunogenicity by structural dynamic differences.\nLine 538: correct to FEI Company, Eindhoven, The Netherlands.\nOur response: This has been corrected.\nLine 679: the journal is not indicated in this reference.\nOur response: We thank the reviewer for pointing this out and have now corrected this reference, as well many others that were not formatted correctly.\nReviewer #4 (Remarks to the Author):\n1. My main concern is attributing the differential functional response to the enhanced conformational dynamics of the head domain. I agree that its dynamics are different than the rNadA, but there may be other reasons for the differential response. A more nuanced interpretation may be necessary, or some additional information provided. Consider:\na. Is there evidence of equivalence between the recombinant NadA and the OMV-NadA? For example, are the authors sure that there are no post-translational modifications that could explain the effect? I can’t tell from the description of the MS analyses.\nb. Similarly, it’s clear that the stalk stands well above the vesicles, but because EM images are always selectively filtered, are the authors sure that there are no other interactions with NadA in the OMV?\nc. And if the interactions are unique to the primary structure of NadA, notice that Figure 2c shows unique conformations across the length of the protein (from head to tail), in the form of stabilizations. Stabilization should not be equated with “hiding” antigenic regions. They can also expose new regions (or increase their population).\nOur response: We agree that these are very important aspects that could be discussed further. Below we touch upon all the reviewer’s points in turn.\na) As vaccine antigen, NadA has been extensively studied by us and other groups. To the best of our knowledge, neither rNadA nor native NadA have ever been reported covalently modified with PTMs by us or other groups. The classification of bacterial PTMs is a field on its infancy, making the selection of PTMs to be searched challenging. We re-analyzed the OMV proteomics raw data using the Peaks PTM module of the Peaks Studio software, which searches for the presence of 312 common and uncommon PTMs. None of them were identified in NadA OMV-embedded, except for few peptides with deamidated asparagine or glutamine residues at too low abundance for the PTMs to be confidently identified. We have modified the “Material and Methods” section “Label free proteomics analysis of the OMVs” to report on the PTM search and its result. The recombinant antigen does not present any PTM, as previously reported by Magagnoli et al., (https://doi.org/10.1016/j.vaccine.2009.01.099). We added in the section “Material and Methods”, references related to the physicochemical characterization of the recombinant proteins used in this study. Lines 400-402 now read: “rNadA (NadA ) is the soluble\n24-350\nrecombinant form of NadA present in the 4CMenB vaccine, for which purification and characterization have previously been reported”.\nb) The reviewer is definitely right. While NadA protrudes away from the bacterial outer membrane surface, it can bend and adopt different orientations (as reported for other TAAs), including orientations where the head domain or other parts of its structure could transiently interact with other proteins or components of the bacterial surface. However, we believe that if NadA was engaged in interactions with other membrane components, its capability of eliciting antibodies would have been reduced in OMVs. Conversely, we observed an increase of functional anti-NadA antibodies in OMVs, suggesting that the protein is accessible to the immune system. Anyway, we think it will be valuable to monitor differences and epitope accessibility in the two NadA forms by performing epitope mapping studies with monoclonal and polyclonal in future works, as mentioned also in response to reviewer 2.\nc) The reviewer’s point is very valid. Indeed, every conformational difference manifesting both in the form of increased or decreased HDX could contribute into a different epitope presentation between the two NadA forms. While we have mainly focused on relating the differences in immunogenicity to the different abundance of the open and closed conformation between the two forms, we agree that the stabilization highlighted in NadA-OMV stalk can also have a contribution, and this should be better highlighted. We have now edited the text accordingly.\nLines 227-229: “Owing to the conformational differences highlighted by the HDX-MS analysis -both in NadA head domain and stalk- we compared the immunogenicity of the 4CMenB vaccine recombinant form of NadA (NadA ).”\n24-350\nLines 365-370: “Moreover, the conformational asset of the stalk of NadA in OMVs, which appears conformationally stabilized in respect of rNadA as observed by our HDX-MS analysis, could induce the elicitation of antibodies able to better recognize the stalk of NadA on the bacterial surface thanks to a more correct epitope presentation, further contributing to the higher functionality of the immune response elicited by the OMV-based formulations.”\n2. HDX results. They look great, but I have a few questions.\na. The HDX summary tables show a much lower sequence coverage than described in lines 105ff. Is a table missing?\nOur response: In the HDX summary table, we initially reported only the data of our comparative HDX-MS experiments, i.e. NadA bound to Hsp90 vs unbound, and rNadA vs OMV-embedded NadA. We have now added the HDX-MS data used for the structural model, for which the HDX of 181 peptides was followed (as reported in lines 104-105; peptides reported in table 1 and fig. S1).\nb. I like HDsite but it makes assumptions that should be stated somewhere in the text (i.e., equivalency of back-exchange for all peptides). It can’t (or shouldn’t) be universally applied.\nOur response: HDsite (ref. 32) has been co-developed by Z. Kan, who is co-author of this work. The algorithm doesn't assume that all peptides have equivalent back-exchange. Instead, it uses a MaxD control to correct for back-exchange on a per amino acid basis by acknowledging that a given residue that appears in different peptides may have different back-exchange due to different LC retention times and calculating an effective back-exchange time for each peptide by using previously measured free reference peptide exchange rates under quench conditions (pH 2.5, 0 °C). As specified in lines 522-523, we used our MaxD sample to perform the back-exchange correction prior deconvolution of peptide-level data into residue-level data via HDsite.\nc. It would be better if the descriptions of stability we simply referred to in terms of protection factors. These are well understood by the community. I find the combined use of both log(kHX) and protection factors a bit awkward.\nOur response: We agree with the reviewer and have addressed this point in the whole paragraph “Structural model of NadA” of the result session. In Table 2, we have substituted the log(k ) values with k\nHX HX\nvalues, as those are better understood by the community. K values are also readable in Supplementary\nHX\ndataset 2. The protection factors were already available in Table 2.\nd. The mixed populations are interesting and an important element of the conclusions. Does figure S7 really show bimodality? It is hard to see this in the figure. Also I think S7 isn’t an OMV-embedded sample as stated in the text (or the caption is wrong?)\nOur response: We thank the reviewer for the comments and apologize for the lack of clarity. Both figs. S6 and S7 show spectra of representative peptides spanning the entire NadA sequence, including those that manifest bimodality. In fig. S6, the spectra of NadA are juxtaposed to those of NadA , highlighting the\n25-350 24-149\ndifference in bimodality between the two forms. As the reviewer noticed, in fig. S7, only few peptides (206-249 and 250-274) show bimodality, as this manifests only in proximity of flexible structural elements of the stalk (described in lines 203-206). We have now corrected the figure captions of figs. S6 and S7, which now read “HDX behavior of NadA at the resolution of peptide segments”. We also realized that the order of few spectra in fig. S7 was incorrect and corrected the figure.\ne. I’m a little concerned that the OMV samples were not analyzed in replicate. Can the authors comment on the implications of this?\nOur response: We welcome the opportunity to clarify. We were very limited in sample availability for the OMV-embedded NadA, also due to the large volume of OMV sample (15 µL) to submit for each injection. We therefore needed to choose whether performing a single time point in triplicates or 4 time points in singlet; for the scope of an HDX-MS experiment, we deemed more appropriate to choose the second approach. However, we mitigated the issue of the lack of replicates for OMV-embedded NadA by applying a very stringent statistics (lines 544-548), and still aligning to the recommendation for performing HDX-MS experiments (Masson, G. R. et al., doi: 10.1038/s41592-019-0459-y). Our threshold of significance has been then set at 0.62 Da, a threshold much higher than that we would normally set for assigning significant HDX differences by using the most common statistical approaches for HDX-MS data, given the obtained average standard deviations. We believe this threshold ensures great confidence in our data, which is also underscored by the consistency of the effects throughout the protein sequence and time points (Fig. 2c).\nGeneral:\n1. Did the authors mean figure 1C on line 136?\nOur response: Thank you for spotting this mistake; yes, we meant 1C, we have now corrected.\n2. Line 140; the resolution of the EM data is a bit low to conclude something ISN’T there (coiled coil).\nOur response: The reviewer is right; indeed, we do not make use of the Cryo-EM structure to conclude anything about secondary or tertiary structural elements of NadA. Our structural model is based on HDX-MS data. The Cryo-EM structure confirmed that the protein is flexible, while the globular density of the C-terminus, together with the primary sequence analysis, confirmed that this region is very likely not coiled coil (see paragraph: structural model of NadA).\n3. Define GMT in the caption of Figure 4a.\nOur response: We have now substituted GMT with IgG titres, as suggested by reviewer 1.\n4. The axes in the figures (spectral inserts) are really small and difficult to see.\nOur response: We have now made them bigger.\n5. Line 86: should be \"combined with…\"\nOur response: This has been corrected.\n6. Line 184 \"resulted pronounced…\" Not sure what is meant.\nOur response: We rephrased “This destabilization was clearly observable until the predicted coiled-coil interruption at residues 252-265…”\n7. Line 860 should be “adhesion with the…”\nOur response: The caption has been modified according to the request done by reviewer 2 and now reads ‘Inhibition of bacterial adhesion of E. coli expressing NadA exerted by sera raised against rNadA and NadA-OMVs formulations in two biological duplicates (R1 and R2)’."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Comments have been addressed satisfactorily.", "reviewer_2": "The revisions of the manuscript and reply to reviewer comments fully meets all my concerns about the original manuscript. I have no further comments.", "reviewer_3": "The manuscript by Calvaresi and colleagues was extensively revised addressing most of my comments.\nI have the following comments regarding the revised version of the manuscript:\n-The FSC curve (Figure S5) decreases and then increases to a correlation of >0.5 after ca 13 A. This indicates that the half maps have been filtered/masked (the relevant information is not provided). If this was not the case, it would mean that a higher correlation of the half maps is achieved at higher resolution than 10 A which is not possible (e.g. there is no sign of secondary structure elements in the 2D class averages). This needs to be addressed.\n-The local resolution of the map is not shown (not responding to my suggestion). I feel it would be important to see if regions of lower resolution correspond to the more flexible regions observed in HDX.\n- A FSC of map versus model is not shown. The authors explain in their response that they performed rigid body fitting of the crystal structure. However, they generate an atomic model based on HDX data in the present manuscript. I don’t understand why this model is not flexibly fitted into the EM density, also using the novel HDX information about structure and flexible regions of the NadA trimer.\n-The negative stain EM of OMV-NadA has been taken out of the revised manuscript. I don’t quite understand why the authors did not try to repeat the reconstruction with more data. Rather the authors state: ‘After careful consideration, we have determined that the best approach is excluding some of the NS-TEM data (panel B of fig. 2) yet keeping the representative micrograph of the OMV overexpressing NadA (fig. 2A) to show the integrity of the OMVs and the presence of NadA appendages on their surface.’\n-to show the presence of NadA on the OMVs (see above), immunogold data were included in the response to the reviewers which the authors intend to use for another manuscript.\nI feel that this is problematic for two reasons: (i) data were removed without an attempt to improve the data. (ii) The readers of the paper, if published in its present form, will have no way of knowing if the arrows in Figure 2A highlight NadA. Figure 2A is weak without additional supporting data. I would suggest including at least a side-by-side comparison of OMVs with and without NadA overexpression.\n-The percentage of open NadA trimer could not be determined for OMV-NadA based on HDX data. I feel this is a key point of the manuscript. High-resolution EM (focussing on the head domain) could allow to computationally sort particles into open and closed conformations for the recombinant NadA (where the ratio was estimated to be 1:9 for open and closed) and possibly also for OMV-NadA. In the current manuscript, the data supporting more open conformations in OMV-NadA are exclusively based on HDX data which could not be quantified.\n-The authors postulate allosteric communication between head and stalk domains of NadA based on HDX data. However, the data provided are not quantifiable in the case of wildtype NadA (OMV-NadA). To strengthen their case, the allosteric links could be corroborated using molecular modelling and molecular dynamics simulations.\n-Regarding the response to adjuvant point raised by another reviewer, I think that the comparison between OMV-NadA and recombinant NadA is not ideal. The authors ‘believe’ but did not show that rNadA could non-specifically bind to OMVs and mask important epitopes. I feel they need to provide this data for of rNadA with OMVs lacking NadA.", "reviewer_4": "The authors have most concerns. However, there is one point that need more direct attention. I do like reviewer one's advice to combine the recombinant with an NadA-null OMV. I believe this is important. The authors express worry over aggregation with the NadA-null OMV but (a) this is easy to test and (b) will not likely be an issue as they show data which demonstrates that NadA does not aggregate even at high concentrations. The potential for clarifying (and supporting) information is quite high and I would encourage them to make the attempt. All other concerns appear adequately addressed."}, "author_response": "We thank the reviewer for highlighting the inappropriate FSC. We now provide new FSC curve as Fig. S5 and added this sentence in the Materials and Methods ‘The FSC has been obtained by using Projection Matching program by Xmipp (XMIPP: A new generation of an open-source image processing package for electron microscopy. DOI: 10.1016/j.jsb.2004.06.006).\nWe have now provided the figure showing the local resolution as the reviewer suggested (panel in Fig. S4). However, we would like to point out that this figure may not be very informative towards the evaluation requested, as it is of low resolution.\nWe thank the reviewer for the insightful comment and understand that there may have been a misunderstanding regarding the description of our work. Indeed, we have not generated an atomic model of the full-length protein; rather, we have focused on collecting HDX data to highlight NadA dynamic properties and identify flexible motifs and potential interruptions in the coiled-coil organization. Based on these findings, we have proposed an annotation for the remaining portion, following established guidelines for coiled-coil annotation (Predicting Coiled Coils from Protein Sequences | Science). Based on the reviewer’s comment and to better clarify the scope of our work, we have now rephrased the sentence 'to generate a structural model' into 'to propose a structural annotation' in the abstract, in lines 89 and 105. We believe this sentence more accurately describes the scope of our work.\nAs such, we are unable to provide an FSC with a full-length model because we do not have a complete structural model for the entire NadA length. Instead, we have performed rigid body fitting of the crystal structure of NadA (PDB 6EUN), which covers the head domain and approximately half of its full-length stalk. A rigid body fitting was carried out using ChimeraX between the experimental Cryo-EM volume and a simulated ChimeraX map generated from the PDB 6EUN, both at same resolution, showing a correlation coefficient of 0.9.\nIn line to what the reviewer points out in a following comment, the Cryo-EM data are not very informative due to the low resolution and lack any structural details.\nWe appreciate the reviewer’s suggestion. However, despite our best efforts to enhance the reconstruction by incorporating more data, we were unsuccessful, likely due to the significant conformational heterogeneity of the sample. To accommodate the reviewer’s request, we have now included a side-by-side comparison of OMVs both with and without NadA in Fig. 2A and referenced in the main text two prior publications that presented negative stain EM data on bare OMVs (refs. 35 and 36: 10.1128/IAI.72.4.1914-1919.2004 and 10.1002/pmic.200500164). We hope that the additional data will address the reviewer's concerns satisfactorily.\nWe would like to clarify that our HDX data support that the NadA ‘open/close’ transitions in the solution-phase resemble transient localized “molecular breathing motions\" rather than stable globally distinct conformational states. As such, we believe such transient local solution-phase motions would be highly difficult to capture in our cryo-EM data, especially considering the low resolution. rNadA transitions to the open conformation more slowly, which allows a more accurate quantification of the initial populations, whereas in the OMV-embedded NadA, the transition between the close and open conformation happens faster. As such, our HDX-MS data do not allow an accurate quantification of the two conformations in OMV-embedded NadA. However, we note the spectra of peptides 40-58 and 40-59 at time points 11 sec and 1 min suggest that the open conformation in OMV-embedded NadA is three-fold more populated than in rNadA in that specific time window (Fig. 3 and S9) and we have now added a sentence in the text to highlight this observation. Lines 226-230 now reads:\n“The initial abundance of the low- and high-mass distributions in OMV-embedded NadA cannot be accurately quantified due to a faster closed-to-open conformational transition than for rNadA; however, in the earliest time frames where the least interconversion has occurred (time points 11 sec and 1 min), peptides 40-58 and 40-59 show a ratio of 3/1 between the low- and high-mass distributions (Figs. 3b and S9).”\nWe thank the reviewer for the suggestion; we have a somewhat different view of the significance of our data. HDX-MS is a well-established experimental method for detecting native solution-phase allosteric changes in proteins. On the basis of our HDX data, we clearly observe long-range allosteric effects, both in rNadA (head-stalk) and OMV-embedded NadA (anchor-stalk-head). We appreciate that, while we have detected an anchorhead communication in OMV-embedded NadA, the opposite effect has not been probed per se for OMV-embedded NadA, but we believe that showing it in rNadA (headstalk) completes the picture on the dynamic behavior of this protein, including the role of the predicted coiled-coil interruptions. While MD simulations can indeed be useful, we believe that our experimental data offer a more reliable, coarse-grained, view of native solution-phase allosteric effects in NadA.\nReviewer 1, who initially raised concerns regarding the immunogenicity strategy, has expressed satisfaction with the explanations and clarifications we provided. We feel that our responses have adequately addressed the key points raised, and the matter appears to be resolved to the satisfaction of the original reviewer.\nReviewer 1, who initially raised concerns regarding the immunogenicity strategy, has expressed satisfaction with the explanations and clarifications we provided. We feel that our responses have adequately addressed the key points raised, and the matter appears to be resolved to the satisfaction of the original reviewer."}, {"round": 3, "reviewer_comments": {"reviewer_1": "When using pooled biological fluid, ideally 3 independent experiments should be conducted and the individual technical replicate raw values shown. The revised Fig 4C is an improvement but as this is a single experiment, I suggest making it clear in the figure legend that this is representative of 3 independent experiments (with 3 technical triplicates) and refer to the original Fig 4C which can be placed in the Supplementary section. The negative control data should also be included in the new Fig 4C in the main body of the manuscript, rather than presented separately in the Supplementary section where it is currently.", "reviewer_2": "The changes to the manuscript have met my concerns and I have no further comments.", "reviewer_3": "• I noticed that the authors have changed Figure 2A. In the first revision, they presented two negative-stain micrographs side by side, comparing OMVs with and without NadA. In the current version, they have reverted to the low-quality micrograph from the original submission. On page 31 of the merged PDF, there is now only one micrograph, which differs from Figure 2A, and the ‘OMV-only’ control is missing. This figure panel should be reverted to the revised version, which included micrographs for OMVs with and without NadA.\n• I further note that the authors write in the legend of Figure 2A: “A) Representative raw of Negative Staining TEM image of meningococcal OMVs carrying on the surface several NadA copies (indicated with white arrows).” It is clear from the authors’ response that this is not a representative micrograph. In addition, “a raw” should be corrected to: “Negative-stain TEM micrograph of …”.\n• There is a typo in line 155: “…which are not visible in OMVs released by the bacteria that do not expressed…” This should be corrected to: “…which are not visible in OMVs released by the bacteria that do not express…”\n• Figures 4C and S11, which showed inhibition of bacterial adhesion of NadA-expressing E. coli by sera raised against rNadA and NadA-OMV formulations, have been deleted. This further reduces the scientific content of the manuscript.\n• I note that lines 251–265 describe the experiment corresponding to the now-deleted Figure 4C. This section should be removed.\nAs mentioned previously, I cannot comment on the novelty or technical difficulty of obtaining the HDX data. The insights provided by EM are very limited due to the low resolution of the 3D reconstruction. Image processing using XMIPP is not state-of-the-art; I wonder why the authors did not use a more commonly used image processing software such as CryoSPARC or Relion. Moreover, the authors did not provide a full-size atomic model based on the HDX data and AlphaFold 3 predictions. Given that Figure 2 presents an in-silico model of full-length NadA, this omission is disappointing. The authors could have integrated the HDX data with their EM results to highlight which regions of the structure are flexible. This could have been further complemented with molecular dynamics simulations.\nTaken together, I feel that the overall biological message of the manuscript that, compared to the recombinant shorter form of NadA, the OMV-embedded form of NadA adopts a more open conformation that enhances epitope exposure and elicits a stronger functional immune response is not surprising. The OMV-embedded NadA better mimics the protein’s natural state on the bacterial surface.", "reviewer_4": "I understand that some data availability issues required the retraction of data from the bacterial adhesion assay (figure 4C in particular). While unfortunate, as it was a nice part of the functional validation of their findings, there is sufficient supporting information to support their essential claims and the publication of the study."}, "author_response": "Our response. We fully agree with the reviewer’s recommendation that three independent experiments should be performed when using pooled biological fluid, and we confirm that this protocol was followed for Fig. 4C, which reports the inhibition of NadA-mediated adhesion by sera raised against rNadA and NadA-OMVs formulations. Unfortunately, while we recorded the averages of three technical replicates from three independent experiments in our laboratory notebook, the individual raw data points for two of the three experiments were not retained. In the interest of data integrity and transparency, we have decided to remove Fig. 4C, as well as the associated Supplementary Fig. 11 and their descriptions from the “Results” section. In consequence we also have revised the discussion in our manuscript to reflect these changes. Despite the absence of Fig. 4C, our findings still support the conclusion that OMV-embedded NadA elicits antibodies with stronger immune response of higher functional quality as demonstrated by superior bactericidal activity, compared to those elicited by the recombinant antigen. This enhanced activity can be correlated with the conformational differences identified through our structural mass spectrometry studies of rNadA and OMV-embedded NadA. We believe this supports our overall interpretation and maintains the integrity of our results.\nOur response: We thank the reviewer for pointing this out. The reviewer is correct: an error occurred during figure assembly, and the incorrect image and legend were inserted for Figure 2A in the last submission (revision C). Both the figure and its legend were already corrected in revision B and will be restored to that version in this new revision. We apologize for this oversight and appreciate the reviewer’s careful reading.\nOur response. We thank the reviewer; the typo will be corrected as suggested.\nOur response. We thank the reviewer for this comment. However, lines 251-262, which described the experiment in Fig. 4C, were present only in the rebuttal A, and not in the last rebuttal version C. Lines 251-258 of the current version describe the experiment in Fig. 4B (SBA).\nOur response. Thank you for these detailed and constructive points. We would like to clarify some of our choices and the scope of this work. We emphasize that gaining information on protein structure by HDX in its native state, especially within a complex OMV environment, is extremely challenging, and our achievement represents one of the few examples of such a study.\nWe agree with the reviewer that our EM data provide limited resolution for a full 3D reconstruction; for this reason, we did not integrate HDX data with EM results to highlight flexible regions.\nIn addition, we would like to clarify that RELION was indeed used in our workflow, in combination with Xmipp, all orchestrated within the Scipion framework. RELION (v2.0) was employed for particle picking, repeated 2D classification, and 3D classification. Xmipp was used for projection-matching refinement, mask generation, map filtering, and FSC calculation, which in our hands provided more stable results for this heterogeneous OMV dataset. Because our aim was not atomic-resolution or GPU-accelerated refinement, a purely RELION- or CryoSPARC-based pipeline would not have provided a clear advantage. Scipion allowed us to reproducibly combine the complementary strengths of RELION and Xmipp to obtain a stable low–moderate resolution map suitable for interpretation.\nLastly, in this manuscript, we focused on reporting empirical data rather than using software for structure prediction, which typically requires experimental validation. It was not the primary scope of this work to validate the potential of such prediction software. Nevertheless, we agree with the reviewer that such integrative modelling (including MD simulations) is a logical and exciting follow-up for future higher-resolution studies.\nOur response. We fully agree with the reviewer's comment that the OMV-embedded NadA better mimics the protein’s natural state on the bacterial surface, as OMVs are naturally released from the bacterial outer membrane. Therefore, both antigen environments are expected to be similar. However, prior to our work, it was not predicted that the stronger functional immune response elicited by the OMV-embedded form of NadA, compared to the recombinant protein, could be attributed to a more open conformation in its native state, which enhances epitope exposure. This phenomenon has been reported for viral proteins but, to the best of our knowledge, our work is the first study to report such a conformational difference between a recombinant and a native bacterial antigen that directly correlates with an improved immune response. This finding highlights a novel mechanism for enhanced immunogenicity, distinct from other factors that could be hypothesized like antigen density or adjuvant effects of the membrane building block."}]

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