Transcriptional Regulation of the nadA Gene in Neisseria meningitidisImpacts the Prediction of Coverage of a MulticomponentMeningococcal Serogroup B Vaccine

Luca Fagnocchi,a Alessia Biolchi,a Francesca Ferlicca,a Giuseppe Boccadifuoco,a Brunella Brunelli,a Sébastien Brier,a Nathalie Norais,a

Emiliano Chiarot,a Giuliano Bensi,a J. Simon Kroll,b Mariagrazia Pizza,a John Donnelly,a* Marzia Monica Giuliani,a Isabel Delanya

Novartis Vaccines and Diagnotics, Research Center, Siena, Italya; Departments of Paediatrics and Microbiology, Faculty of Medicine, Wright-Fleming Institute, ImperialCollege London, London, United Kingdomb

The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection.Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killingof MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadAin laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be differ-ent from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR.All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infantswhen the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant ratsfrom bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by othersto be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing.Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Ourresults suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levelsof NadA expression from any nadA-positive (nadA�) strain and therefore efficient killing by anti-NadA antibodies elicited by the4CMenB vaccine.

The human pathogen Neisseria meningitidis is an encapsulatedGram-negative diplococcus which asymptomatically colo-

nizes the naso- and oropharynx of 10% to 15% of healthy adults.For reasons not yet fully understood, it occasionally crosses themucosal epithelial barrier to cause severe septicemia and menin-gitis (1, 2). Each year, there are an estimated 1.2 million cases ofinvasive meningococcal disease and 135,000 deaths (http://www.who.int/mediacentre/en/), and infants represent the populationat highest risk of infection. Individuals surviving the disease oftensuffer from permanent disabilities, including brain damage re-sponsible for hearing loss or learning difficulties, as well as ampu-tation of limbs (1). Of the 12 known serogroups classified by theimmunochemistry of their capsular polysaccharides, six, A, B, C,X, Y, and W, regularly cause disease (3–5). Meningococcal diseaseprogresses rapidly, and in its early stages, it is easily misdiagnosed(1), making vaccination the best public health option and themost effective way to prevent it. Polysaccharide and glycoconju-gate vaccines are available against serogroups A, C, Y, and W, butthere is no broadly protective vaccine against meningococcus se-rogroup B (MenB).

A novel vaccine against MenB named 4CMenB has been devel-oped (6) and has progressed through clinical trials that have dem-onstrated its safety (7) and its efficacy in inducing a protectiveimmune response in infants, children, adolescents, and adults topotentially the majority of MenB strains (8, 9). The 4CMenB vac-cine is composed of the recombinant protein Neisserial adhesin A(NadA) (10), the factor H binding protein (fHbp) (11) and Neis-serial Heparin-Binding Antigen (NHBA) (12) fused with the me-ningococcal gene product GNA2091 or GNA1030, and OuterMembrane Vesicles (OMVs) from the meningococcus B NZ98/

254 strain in which PorA serosubtype 1.4 represents the majorantigen. In order to evaluate 4CMenB vaccine coverage, an assay,the Meningococcal Antigen Typing System (MATS), which as-sesses simultaneously the cross-reactivity and the expression ofthe antigens present on the surface of an unknown test strain withrespect to a reference MenB strain, has been developed (13). TheMATS relative potency (MATS RP), obtained by applying MATSto unknown strains, correlates with data from the human SerumBactericidal Antibody (hSBA) assay, the surrogate of protectionaccepted for meningococcal infection (14–17), and may predictwhether a strain would be killed due to antibodies elicited by the4CMenB vaccine (13). A MATS RP threshold value for comple-ment-mediated killing of MenB by antibodies against NadA,fHbp, and NHBA antigens was established and termed the Posi-tive Bactericidal Threshold (PBT). Using MATS, it has been esti-mated that 78% of circulating MenB strains in Europe would have

Received 5 October 2012 Returned for modification 22 October 2012Accepted 1 December 2012

Published ahead of print 10 December 2012

Editor: A. Camilli

Address correspondence to Isabel Delany, isabel.delany@novartis.com.

* Present address: John Donnelly, Program for Appropriate Technologies inHealth, Seattle, Washington, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01085-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01085-12

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at least one antigen rated above the PBT and therefore would becovered by the 4CMenB vaccine. However, the estimated contri-bution of the NadA antigen to the vaccine coverage appears to bevery low (18).

The nadA gene is carried by about 30% of pathogenic isolatescollected from patients in 5 European countries and the UnitedStates and is always present in members of three of four majormeningococcal hypervirulent lineages (ST8, ST11, and ST32 com-plexes) (10, 19). Despite the presence of the gene, the quantities ofNadA protein that are expressed by bacteria cultured in vitro differgreatly in different strains due to complex mechanisms of nadAregulation. The nadA gene shows growth-phase-dependent ex-pression, reaching a maximal level in the stationary phase (20). Itis also subject to phase variation, through the presence of a vari-able-length tetranucleotide repeat upstream of its promoter. It hasbeen shown that different strains comprising different phase vari-ants of nadA express the protein at different levels in vitro (20).However, the major mediator of the phase-variable expression ofnadA is NadR, which binds to two high-affinity sites on the pro-moter of nadA, repressing it. When nadR is knocked out (KO), thelevel of expression of NadA is induced to almost comparable levelsin all tested strains, suggesting that the differential ability of NadRto repress different phase variants of nadA is the cause of the vari-ability of NadA within and between strains (20).

NadR belongs to the MarR family of regulators, which areknown to respond to small-molecule inducers, often low-molec-ular-weight phenolic compounds (21). It has been demonstratedthat NadR responds to 4-hydroxyphenylacetic acid (4-HPA),which is able to alleviate the binding of the repressor on nadA,thereby inducing its expression (20). 4-HPA is a catabolite of ar-omatic amino acids and is commonly found in human saliva (22).It has also been recently reported that human saliva itself caninduce NadA expression as well as 4-HPA, suggesting that signalscapable of inducing NadA expression, which can be mimicked by4-HPA in vitro, are present in the oropharyngeal niche of menin-gococcal colonization (23). Moreover, it has also been demon-strated that sera from children convalescent after meningococcaldisease are able to recognize NadA, suggesting that NadA is ex-pressed during invasive human infection to a level which is suffi-cient to elicit anti-NadA antibodies (24). Importantly, NadA elic-ited strong reactivity in convalescent patients previously infectedwith nadA-positive (nadA�) strains relative to uninfected sub-

jects. Taken together, these observations suggest that the level ofexpression of NadA during invasive disease may be very differentfrom the levels that are measured under in vitro growth condi-tions.

In this report, we address the possibility that the contributionof the NadA antigen to 4CMenB vaccine coverage is underesti-mated due to the conditions used for performing the hSBA andthe MATS assay. We provide data showing that strains able tocause disease in humans, even with low MATS RP, are in fact ableto induce anti-NadA antibodies during infection. We demon-strate that once NadR repression is alleviated, strains carryingnadA express high levels of NadA and therefore have the potentialto be killed due to antibodies elicited by the 4CMenB vaccine.Finally, we have assessed our hypothesis in a case study, showingthat MenB strain NGP165, which exhibits a NadA MATS RP un-der the PBT and therefore is not killed by anti-NadA antibodies invitro, is killed in an in vivo infant rat model by passive protectionconferred by sera of infants immunized with 4CMenB.

MATERIALS AND METHODSEthics statement. The trials described in this paper were conducted fol-lowing good clinical practice and the principles outlined in the Declara-tion of Helsinki. The studies were approved in different countries by localethical committees. All animal experiments were performed in accor-dance with European and Italian guidelines regarding the protection ofanimals used for experimental and other scientific purposes.

Bacterial strains and culture conditions. The N. meningitidis strainsused in this study are listed in Table 1 and include the respective NadR nullmutant derivatives, which have been described elsewhere (23). TheNGP165 NHBA null mutant was generated as described previously (12).In addition, the previously reported clinical isolates Nm036, Nm037,Nm066, Nm067, Nm069, Nm081, Nm088, Nm100, Nm119, Nm145,Nm154, Nm156, Nm188, and Nm191 (24) were used. All strains wereroutinely cultured in GC-based medium (Difco) and stocked as previ-ously described (25). When required, indicated small molecules wereadded to culture media to achieve a final concentration of 2 or 5 mM inaqueous solution. All small molecules were obtained from Sigma-Aldrich,with the exception of the 3Br4-HPA, which was purchased from Chem-sigma.

Generation of lux reporter strains. To generate bacterial luciferasetranscriptional fusions of the promoter under study at a chromosomallocation between the two converging open reading frames (ORFs)NMB1074 and NMB1075, flanked on both sides with transcriptional ter-minators, plasmid pSL-LuxFla was constructed for allelic exchange in N.

TABLE 1 Selected strains used in this study

Strain Clonal complex(es) STa

Yr ofcollection Countryb Typing resultc fHbp IDd

No. of isolates

NHBANadAvariant

5/99 ST-8 complex/cluster A4 1349 1999 B:2b:P1.5,2 N 23 20 2961-5945 ST-8 complex/cluster A4 153 1996 AUS B:2b:P1.21,16 16 20 2LNP17094 ST-8 complex/cluster A4 153 1999 F B:2b:P1.10 16 22 2B3937 ST-18 complex 6344 1995 D B:22:P1.16 17 23 3M10574 ST-32 complex/ET5 complex 803 2003 USA B:NT:P1.7–2,13-1 76 3 1M14933 ST-32 complex/ET5 complex 32 2006 USA B:ND:P1.22-1,14 76 3 1MC58 ST-32 complex/ET5 complex 74 1985 UK B:15:P1-7,16b 1 3 1NGP165 ST11 complex/ET-37 complex 11 1974 N B:NT:P1.2 29 29 2a ST, multilocus sequence type.b AUS, Austria; D, Denmark; F, France; N, Norway; UK, United Kingdom; USA, United States of America.c No strains match the PorA P1.4 allele in the OMV_NZ vaccine component.d The fHbp allele identification numbers (ID) are reported here according to Oxford database nomenclature.

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meningitidis strains. Briefly, the promoterless luxCDABE operon and catcassette were subcloned from pSB1075 (21) into pBluescript II (26) as anEcoRI-BamHI fragment and then cloned as a 6.5-kb XhoI-BamHI frag-ment into pSL-furlacZ (27), replacing 4.7 kb containing an erythromycincassette and fur-lacZ fusion, generating pSL-LuxFla. The nadA promoterwas cloned as a 250-bp Xho-KpnI fragment upstream of the luxCDABEoperon, generating pSLPnadA-lux. The pSL-LuxFla and pSLPnadA-luxplasmids were used for transformation of the MC58 and MC58-�1843strains, generating the MC58-lux and MC58-PnadA-lux strains and theMC58-�1843-lux and MC58-�1843-PnadA-lux strains, respectively, forthe in vitro reporter analyses, and the 2996 strain, generating 2996-lux and2996-PnadA-lux, respectively, for the in vivo reporter analysis.

Western blot analysis. Liquid cultures were grown to mid-exponen-tial phase (optical density at 600 nm [OD600] � 0.4 to 0.5), harvested, andresuspended in GC, GC plus a 2 mM concentration of the indicated mol-ecule, or GC plus a 5 mM concentration of the indicated molecule or 10%,50%, or 90% (vol/vol) human saliva (prepared as described previously[23]) in GC containing EDTA-free protease inhibitor cocktail (Roche).After 1 h of incubation at 37°C, total protein extracts were prepared andWestern blot experiments were performed as previously described (23).After quantification of the signal of the bands, statistical analyses wereperformed to evaluate the significance of the results.

In silico docking experiments. The generation of a tridimensionalstructural model of NadR and the description of docking experimentswere fully reported by Brier and colleagues (28). The coordinates of the4-HPA molecule were obtained from the Protein Data Bank (PDB) website (identification [ID] no. 4HP). AutoDockTools 1.5.4 (ADT) softwarewas used to prepare the ligand for docking (29).

Immunization of mice. To prepare antisera, 20 �g of NadA, NHBA-GNA1030, or GNA2091-fHbp antigen or a combination of 20 �g each ofNHBA-GNA1030, GNA2091-fHbp, and NadA with or without 10 �g ofdeoxycholate-extracted OMVs derived from the NZ98/254 strain wasused to immunize 6-week-old CD1 female mice (Charles River). Five to10 mice per group were used. The antigens were administered intraperi-toneally (i.p.), together with aluminum hydroxide (3 mg/ml), on days 0,21, and 35.

MATS and Serum Bactericidal Assay (SBA). The MATS assay wasperformed as previously described (13). When required, bacteria weregrown overnight with 4-HPA and 3Cl4-HPA supplementation on Choc-olate Agar plates (bioMérieux). Raw data reduction and analysis wereperformed by StatLIA (Brendan Technologies).

Serum bactericidal antibody activity against N. meningitidis strainswith mice antisera was evaluated as previously described (30), with pooledbaby rabbit serum used as the complement source (rSBA). Serum bacte-ricidal antibody assays with human complement (hSBA) were performedas described by Borrow et al. (30). When required, 4-HPA and 3Cl4-HPAin aqueous solution were added to plates at a final concentration of 5 mM.

Human serum samples. Serum samples before and after immuniza-tion were obtained from the following clinical trials. Study 1 was a clinicaltrial conducted in healthy adults and laboratory workers. Pooled sera werederived from 23 subjects before and after 3 doses of 4CMenB adminis-tered at 0, 2, and 6 months. Study 2 was a clinical study evaluating thesafety, immunogenicity, and lot consistency of 4CMenB administered toinfants at 2, 4, and 6 months of age. Extensions of this clinical studyinvestigated a fourth (booster) dose at 12 months of age. Pooled sera werederived from 107 infants at 7 months of age who received the primaryseries of 3 doses of routine vaccine at 2, 4, and 6 months of age and from141 infants who received the primary series of 3 doses of 4CMenB at 2, 4,and 6 months of age plus a booster in the second year of life. Study 3 wasa clinical study evaluating the safety, tolerability, and immunogenicity of4CMenB administered to infants at 2, 4, and 6 months of age. Extensionsof this clinical study investigated a fourth (booster) dose at 12, 18, or 24months of age. Pooled sera were derived from 109 infants at 5 months ofage who received the primary series of 3 doses of routine vaccine at 2, 3,and 4 months of age and from 69 infants who received the primary series

of 3 doses of 4CMenB at 2, 4, and 6 months of age plus a booster in thesecond year of life.

Passive protection and in vivo imaging in infant rats. The ability ofanti-NadA antibodies to confer passive protection against N. meningitidisbacteremia was tested in infant rats challenged intraperitoneally (i.p.) aspreviously described (31). In vivo imaging of the bioluminescence of thePnadA-lux reporter and control strains was monitored in infant rats in-fected i.p.

On the morning of the challenge/infection, the bacteria were grown,washed, and diluted in phosphate-buffered saline (PBS) to obtain 105

CFU/ml. For passive protection, groups of 3 to 19 animals were treated i.p.at time zero with 100-�l doses of different dilutions of test or controlantisera. Three hours later, the animals were challenged i.p. with a 100-�ldose of 105 CFU of N. meningitidis strain NGP165 or NGP165 NHBA KO.Eighteen hours after the bacterial challenge, blood samples were obtainedby cheek puncture with a syringe containing 25 U of heparin withoutpreservative (American Pharmaceutical Partners), and CFU levels weremeasured. Rats were considered infected when �10 CFU were counted onplates carrying 100 �l of blood. Counts above the threshold were verifiedfor positivity by examining plates carrying 10- and 100-fold dilutions ofblood.

For in vivo imaging of bioluminescent reporters, groups of 5 animalswere inoculated i.p. at time zero with 100-�l doses of 104 CFU of the2996-lux or 2996-Pnad-lux strain. Rats were then anesthetized using aconstant flow of 2.5% isoflurane mixed with oxygen. Bioluminescencemeasurements of ventral views of each group of rats were taken at timezero and at 3 and 24 h, using an IVIS 100 system (Xenogen Corp., Ala-meda, CA) according to instructions from the manufacturer. Analysis andacquisition were performed using Living Image 3.1 software (XenogenCorp.). Quantification was performed using the photons emitted per sec-ond by each rat. Two rats infected with the 2996 wild-type strain under thesame conditions of acquisition were used for subtracting the background.At 24 h after infection, blood samples were obtained and CFU countsmeasured. Statistical analyses were performed to assess the relevance ofthe results obtained.

RESULTSStrains with MATS RP < PBT express NadA in an immunogenicform during invasive disease. Litt and colleagues (24) observedthat many protein antigens, including the NadA protein, wererecognized by antibodies present in sera of children convalescingafter meningococcal disease. Importantly, NadA was significantlymore strongly recognized by sera of convalescent patients infectedwith nadA-positive strains than by sera of uninfected control sub-jects (24). We have extended this study by subjecting the 14 nadA-positive isolates, matched to the sera of the Litt study, to Westernblot and MATS analysis in order to visualize the in vitro levels ofNadA expression of the infecting strain. In Fig. 1A, Western blotsreveal that while the regulator NadR is expressed comparably byall strains tested, the levels of NadA are variable, and some strains(Nm067, Nm100, and Nm188) failed to express the protein atdetectable levels under the in vitro growth conditions used. Whenanalyzed by MATS, the level of NadA expression, calculated asrelative potency compared to that of a reference strain (5/99),correlated well with Western blot results (Fig. 1B; see also Table S1in the supplemental material). As shown, only 5 strains (Nm036,Nm037, Nm081, Nm088, and Nm154) have RP values above thePBT of NadA, which is 0.009, while the remaining strains showRP � PBT. However, sera from children infected either by strainsthat failed to express NadA in vitro (MATS RP � 0) or by strainswith a NadA RP � PBT are nonetheless able to recognize at leastone form of the NadA recombinant proteins used in the dot blotexperiments of Litt and colleagues (24) (Fig. 1C) more efficiently

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than sera from subjects infected by nadA knockout meningococ-cal strains.

These data show that strains with MATS RP values below thePBT are nevertheless able to express the NadA protein in an im-munogenic form during invasive disease, driving a robust hu-moral response. This observation suggests that the levels of ex-pression of NadA under in vitro growth conditions may differfrom, and be lower than, those reached during infection in thehuman host.

All strains carrying the nadA gene can express high levels ofthe NadA protein and can therefore be killed by vaccine-in-duced bactericidal antibodies. The main mediator of various ex-pression levels of NadA within and between strains is the tran-scriptional regulator NadR, through its ability to differentiallyrepress the phase-variant promoter of nadA (20). To understandthe relevance of NadR repression to the variable expression levels

of NadA observed in different MenB strains under in vitro condi-tions, a representative panel of strains covering a range of NadAexpression levels was selected (Table 1) and the nadR gene wasdeleted in each of them. We evaluated the implications of thealleviation of NadR repression under in vitro conditions by West-ern blotting, MATS, and SBA analysis (Fig. 2 and Tables 2 and 3).

As previously shown (20), all the nadR knockout strains ex-pressed considerably more NadA than their wild-type forebears,confirming that deletion of nadR results in strong induction ofNadA. Furthermore, all nadR knockout strains expressed compa-rable high levels of the NadA antigen measured by Western blot-ting (Fig. 2A). The MATS assay performed on wild-type and nadRknockout strain pairs demonstrated that the ratio of the RP valuesfor NadA increased from 3-fold to up to 100-fold in the mutantstrains (Fig. 2B), indicating that all these strains can express highlevels of immunogenically relevant NadA antigen when NadR re-pression is abolished. The MATS assay correlates with the hSBA(13) at values of RP higher than the PBT. Therefore, we comparedthe ability of immune sera to kill the nadR knockout strains andtheir related wild types. Table 2 shows that sera from mice immu-nized with NadA alone or with the 4CMenB vaccine have an in-creased NadA-specific bactericidal activity on nadR knockoutstrains compared with wild-type strains. The only exception is forstrain 5/99, in which, as expected, there is no significant differencein SBA titers between the wild-type and the nadR knockoutstrains. In this strain, the NadR-mediated repression of NadA isminimal: NadA is highly expressed in the wild-type strain. Serafrom immunizations with NHBA and fHbp (Table 2) had invari-ant activity toward wild-type and nadR knockout strains, con-firming that the knockout of nadR does not alter the susceptibilityof these strains in the bactericidal assay and suggesting that neitherNHBA expression nor fHbp expression is regulated in a NadR-

FIG 1 Strains with MATS RP � PBT express NadA in an immunogenic formduring infection. (A) Western blot analyses of the wild-type nadA� strainsfrom the Litt study (24), showing NadA and NadR expression. (B) MATSrelative potency (RP) of NadA determined by the MATS ELISA. A blackdashed line represents the positive bactericidal threshold (PBT) for NadA. TheRPs of each strain are reported in Table S1 in the supplemental material. (C)Spot intensity of dot blot experiments adjusted from the data of Litt and col-leagues (24). Reactivities of sera from children infected with the reported iso-lated strains are reported (961 � full-length NadA, 961 cm and 961c2 � trun-cated forms comprising the extracellular portion of NadA). The average valuesfor 14 nadA� strains and 17 nadA knockout strains are reported (Avrg NadA�and Avrg NadA-, respectively).

FIG 2 NadA espression levels in a panel of wild-type and nadR knockoutstrains. (A) Western blot analyses of wild-type and relative nadR knockout(KO) strains. The NadA and NadR levels of expression are shown. (B) Ratio ofNadA MATS RPs of nadR knockout strains versus the wild-type strains. TheRP values of wild-type strains and the fold increase of NadA RP of the nadRknockout strain, calculated by dividing the RP value of nadR knockout strainsby the RP value of the relative wild-type strains, are reported in the graph.

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dependent way. The results of a hSBA assay performed on themutant strains demonstrated that sera from clinical trial subjectsof different age groups immunized with the 4CMenB vaccine for-mulations were efficiently able to kill all nadR knockout strainsand exhibited extremely high bactericidal titers (Table 3). Of note,antibodies present in sera from some age groups and apparentlyineffective in the killing of certain strains (e.g., B3937 andNGP165) show the ability to efficiently kill the equivalent recom-binant strains once NadR repression has been relieved.

Taken together these data demonstrate that all the strains car-rying nadA can potentially express NadA to a level which is suffi-cient to be recognized and to mediate killing by the bactericidalantibody elicited by the 4CMenB vaccine.

NadA expression can be induced in vitro by different physi-ologically relevant signals in strain NGP165. The 4CMenB vac-cine has been formulated in order to confer protection by target-ing multiple antigens on the surface of as many strains as possible.To evaluate the contribution of NadA to vaccine coverage and totest the hypothesis that analysis of the levels of NadA expression invitro could underestimate the predicted efficacy of bactericidalantibody in mediating the killing of NadA-positive strains duringinfection, we selected strain NGP165 for a case study. NGP165 and4CMenB are mismatched with respect to fHbp and PorA (carryingfHbp variant 3.29 and PorA serosubtype 1.2 and carrying fHbpvariant 1.1 and PorA serosubtype 1.4, respectively), and, with re-spect to NHBA, it had MATS RP below the PBT and almost neg-ative rSBA titers in the preclinical studies (Table 2). Thus, only theNadA antigen could plausibly contribute to 4CMenB-induced an-tibody-mediated killing of this strain.

It has been previously shown that NadR-mediated repressionof the nadA promoter can be alleviated by 4-HPA, a catabolite ofaromatic amino acids which is commonly found in human saliva(20, 22). Human saliva has been shown to induce NadA expres-sion to the same level as 4-HPA in strain MC58, suggesting that, invivo, the expression of the nadA gene might be induced by signalspresent in saliva (23). Mass spectrometry and hydrogen/deute-rium exchange analysis (MS-HDX) have recently identified a sub-

strate binding pocket which is involved in the interaction of the4-HPA ligand and the NadR repressor (28). We used the resultantinducer-NadR interaction model (see Fig. S1A in the supplemen-tal material) for in silico docking experiments to screen a numberof molecules structurally similar to 4-HPA in order to identifycandidates for other potentially physiologically relevant inducersof NadA expression. Any molecule that was identified in silico asable to dock in the binding pocket of NadR was tested for its abilityto induce NadA expression in MC58 in in vitro-grown cultures(see Fig. S1B in the supplemental material). Among the molecularspecies tested, some (2-HPA, 2,4-HPA, and 3,4-HPA) were unableto induce NadA expression, while others (3Cl4-HPA, 3Br4-HPA,NO2-4HPA, and, to a lesser extent, 3-HPA) increased expressionof NadA to a level comparable to that of 4-HPA itself. We thenverified which of the newly found inducers might have a signifi-cant role during meningococcal infection. Interestingly, 3Cl4-HPA, which is structurally similar to 4-HPA (Fig. 3A), has beenshown to be produced during inflammatory processes as a catab-olite of chlorinated aromatic amino acids (32) and therefore rep-resents a possible natural ligand that the meningococcus mightencounter during infection of the host. Due to their physiologicalrelevance, we accordingly decided to test both the 4-HPA and the3Cl4-HPA molecules in in vitro assays using the selected NGP165strain in order to assess the putative level of NadA expression inthe host.

Figure 3B shows that NadA expression is induced by 4-HPAand 3Cl4-HPA to similar levels, with no statistically significantdifference seen between the inductions achieved by the two mo-lecular species in three biological replicates. As previously re-ported for strain MC58 (23), human saliva from different donorsis able to induce NadA expression in a dose-dependent manner tothe same extent as 4-HPA (or 3Cl4-HPA; data not shown) inNGP165 (Fig. 3C).

These observations support the proposition that NadA expres-sion in NGP165 can be induced in vivo in the human host by

TABLE 2 rSBA performed with immunized mice sera and rabbitcomplement on wild-type and nadR knockout strainsa

Strain

rSBA titer

NHBA fHbp NadA 4CMenBb

5/99 512 �16 �65,536 �32,7685/99 nadR KO 128 �16 �65,536 �65,536961-5945 1,024 1,024 1,024 4,096961-5945 nadR KO 1,024 2,048 �65,536 �65,536LNP17094 1,024 �16 128 4,096LNP17094 nadR KO 512 �16 32,768 �65,536B3937 �16 1,024 512 2,048B3937 nadR KO �16 512 �65,536 �65,536M10574 4,096 128 �8,192 �8,192M10574 nadR KO 4,096 64 �32,768 �32,768M14933 4,096 �16 512 �8,192M14933 nadR KO 4,096 �16 �32,768 �32,768NGP165 128 �16 128 512NGP165 nadR KO 128 �16 �32,768 �32,768a Strains were considered killed if pooled mouse sera achieved an rSBA titer � 128.nadR KO, nadR knockout.b Vaccine formulation with both the three recombinant major antigens and the OMVs,as described in the text.

TABLE 3 hSBA performed on wild-type and nadR knockout mutantselected strainsa

Strain

Study 1 (adults) SBA titerStudy 2 (infants) SBAtiter

Preimmune4CMenBpost 3b Routine

4CMenBpost 4c

5/99 �4 256 �4 �512961-5945 �4 16 �2 16961-5945 nadR KO �4 �512 �4 �512LNP17094 �4 16 �2 16LNP17094 nadR KO �4 �512 �4 �512B3937 �4 8 �2 �2B3937 nadR KO �4 �512 8 �512M10574 �4 32 �2 64M10574 nadR KO �4 �512 �4 �512M14933 �4 16 �2 32M14933 nadR KO �4 �512 8 �512NGP165 �4 �4 2 4NGP165 nadR KO �4 �256 4 �256a Strains were considered killed if pooled sera from different age groups who receivedthe 4CMenB achieved an SBA titer � 8.b 4CMenB post 3, 3 doses of the vaccine given at 0, 2, and 6 months.c 4CMenB post 4, 3 doses of the vaccine given at 0, 2, and 6 months plus 1 boostbetween 12 and 24 months.

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alleviating NadR repression through the action of signals eitherpresent in saliva or produced during inflammation. The use ofHPA derivatives in in vitro assays achieves levels of NadA expres-sion similar to those seen with ex vivo human saliva and maymimic the predicted levels in the host.

hSBA and MATS analysis performed with 3Cl4-HPA predict4CMenB vaccine coverage of the NGP165 strain. We performedthe hSBA and the MATS assays on the NGP165 wild-type and thenadR knockout mutant strains grown in the absence or presenceof 3Cl4-HPA (Table 4) in order to evaluate the 4CMenB vaccinecoverage prediction for NGP165 by taking into account the puta-

tive level of NadA expression in the host. The NGP165 wild-typestrain expressing low levels of NadA in vitro (Fig. 3B) has a NadAMATS RP � 0.005, below the PBT for NadA (0.009). Results of thehSBA demonstrated that NGP165 was indeed resistant to killingby pooled sera from infants who received 4 doses of 4CMenB.When grown in the presence of 3Cl4-HPA, the MATS RP ofNGP165 increased to 0.028 (Table 4) and the strain was renderedsusceptible in the hSBA using the same infants’ sera. Bactericidaltiters increased from 4 with preimmune sera to 128 with immu-nized sera (Table 4). As seen for other strains tested, a more pro-nounced increase in NadA expression was seen in the nadR knock-out mutant, in which the nadA gene is fully derepressed (NadAMATS RP � 0.503). This situation correlates with positive bacte-ricidal titers of �256 in hSBA.

In conclusion, using a modified in vitro growth protocol (withHPA supplementation) that we consider more accurately reflectsthe level of NadA expression that occurs in vivo, the MATS andhSBA assays predicted that NGP165 would be efficiently killedduring infection by anti-NadA antibodies in sera of subjects im-munized with the 4CMenB vaccine.

Sera from 4CMenB-immunized infants protect infant ratsfrom infection with strain NGP165. To determine whetherNGP165 would be killed in vivo by anti-NadA antibodies, we per-formed a passive protection assay in the infant rat model (33).

FIG 3 Induction of NadA by different physiologically relevant signals in the NGP165 selected strain. (A) Representation of the chemical structures of the 4-HPAand the 3Cl4-HPA compounds. (B) Western blot analyses of the level of expression of NadA in the NGP165 strain. The experiment was repeated with threebiological replicates, and the band signals were quantified using a loading control as reference (fHbp). One representative Western blot is shown together witha histogram summarizing the results of three replicates. (** � P � 0.01; ns � not significant). (C) Induction of the NadA expression in the NGP165 strain withhuman saliva (HS). On the left, Western blot analyses of total protein from mid-log-phase cultures of NGP165 under the indicated conditions are shown. On theright, a histogram reporting the average values of NadA expression determined in 5 independent experiments is shown. Western blot bands were normalized fora nonspecific band (loading control) in order to avoid 4-HPA- or NadR-independent effects on NadA expression due to possible protein degradation in saliva.* � P � 0.05; ** � P � 0.01 (comparing all condition to the basal GC medium level). Levels of NadA induction comparable to those seen with 4-HPA and salivawere obtained with the 3Cl4-HPA molecule (data not shown). In addition, neither 4HPA nor 3Cl-4HPA had any effect on the expression level of NHBA or fHbpin NGP165 (data not shown).

TABLE 4 hSBA and MATS of the NGP165 strain performed with the3Cl4-HPA moleculea

Strain InducerNadAMATS RP

hSBA titer (study 3[infants])

Routine4CMenBpost 4

NGP165 None 0.005 2 4NGP165 3Cl4-HPA 0.028 4 128NGP165 nadR KO None 0.503 2 �256a Strains were considered killed if pooled sera from infants who received threeimmunizations plus one booster of 4CMenB achieved an SBA titer � 8.

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Figure 4 reports the results of these experiments. Groups of infantrats were inoculated i.p. with an infectious dose (105 CFU) ofNGP165 after being treated with control serum or pre- or postim-mune sera from mice and infants immunized with either NadA orthe 4CMenB vaccine. Administration of preimmune sera fromeither mice or human infants had no effect on the ability ofNGP165 to infect infant rats: 105 CFU of NGP165 led to sustainedinfection in all but 1 of the 19 animals tested. Sera from miceimmunized with the NadA antigen alone resulted in the protec-tion of 15 of 19 infant rats challenged with NGP165. Sera frommice or human infants immunized with the 4CMenB vaccine for-mulation conferred protection on the infant rats as well (15 of 16or 4 of 4 rats, respectively, protected in two experiments). Treat-ment with the same human sera resulted in protection of infantrats from infection with a NGP165 NHBA KO strain (4 of 5 rats),demonstrating that killing of the strain is not due to anti-NHBAantibodies present in the sera. Taken together, these data suggestthat in this in vivo model, NadA is expressed to a sufficient level to

be recognized by specific anti-NadA antibodies elicited by NadAin the 4CMenB vaccine and to mediate killing of the bacterium.

PnadA is activated in vivo during infection of the infant ratmodel. In order to directly evaluate the expression and inductionof nadA during infection, we generated reporter strains carryingthe promoterless luciferase operon (negative control) or carry-ing the operon under the control of the nadA promoter (PnadA-lux). The bioluminescence of the resulting strains was evaluated inin vitro experiments. Interestingly, the PnadA-lux strain is signif-icantly less bioluminescent than the negative control (data notshown), indicating that the nadA promoter is efficiently repressedunder in vitro conditions, and, as expected, 4HPA-specific induc-tion of PnadA-lux (10-fold) and the derepression of PnadA-lux inthe NadR KO background (385-fold) were observed (see Fig. S2 inthe supplemental material). The infective dose of 104 CFU of thenegative control and the PnadA-lux reporter strains was used toinfect groups of 5 infant rats, and images of ventral views of intra-peritoneally infected rats were collected either immediately or 3

FIG 4 Passive protection in the in vivo rat model. (A) Plot of the number of CFU counted for each infant rat alternatively injected with either preimmune mousesera or sera from immunized mice, as indicated below the chart. (B) Plot of the number of CFU counted for each infant rat, alternatively injected with eitherpreimmune human sera or sera from human immunized with the 4CMenB vaccine, as indicated. Infant rats were infected with either the NGP165 wild-type orNGP165 NHBA KO strain. Circles indicate single infant rats, while solid horizontal black lines indicate the average of CFU counted for each condition; error barsare also reported. A horizontal dashed line indicates the limit of quantification of the CFU. * � P � 0.05; ** � P � 0.01 (comparing data determined under alldescribed conditions to those determined for rats injected with preimmune sera). No statistical difference between data corresponding to the protection of infantrats from infection by either the NGP165 wild-type or the NHBA KO strain is present. (C) Table showing the results obtained in the in vivo passive protectionmodel.

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and 24 h after infection. Immediately after infection, both thenegative control and the PnadA reporter strains were poorly bio-luminescent (less than 1.5-fold change from the background)(dark gray and white circles, respectively, in Fig. 5A); however, at3 h postinfection, expression of the PnadA-lux reporter strain wassignificantly induced (6-fold on average) whereas the negativecontrol maintained low levels of bioluminescence (Fig. 5). It isworth noting that, in Pnad-luxA-infected rats, a widespread bio-luminescence was observed over the entire rat that is indicative ofbioluminescence from bacteria in a systemic infection. At 24 hafter infection, the bioluminescent signals from both the negativecontrol and the reporter strains were almost indistinguishablefrom the background (Fig. 5A). These data produced in the infantrat model of septicemia indicate that expression of the nadA pro-moter is induced during infection in vivo, suggesting that NadA isexpressed during invasive disease.

DISCUSSION

In the absence of an efficacious broadly protective vaccine, MenBis the leading cause of bacterial meningitis and septicemia in manyindustrialized countries. A novel multicomponent vaccine,4CMenB, is able to induce bactericidal antibodies against strainsexpressing vaccine antigens, but because MenB clinical isolates arediverse, it is necessary to evaluate the coverage of circulatingstrains and therefore the potential public health impact of thisvaccine. The MATS assay, which assesses the relative contribu-tions of the 4 major components present in the 4CMenB vaccine,predicts whether a given isolate can be killed or not (13). Usingthis assay, an evaluation of more than 1,000 MenB strains from 5European Union countries predicted that 73% to 87% would becovered by 4CMenB vaccination. However, the relative contribu-tion of NadA to this combined-coverage prediction is estimated tobe significantly lower than that predicted by the number of strainscarrying the nadA gene (18).

The role of NadA in eliciting bactericidal antibodies protectingagainst circulating strains has been unclear due to NadR-mediated

repression of NadA expression under the in vitro growth condi-tions used for MATS and hSBA assays, which are performed in theearly phase of growth, when NadR maximally represses nadA ex-pression (20). For this reason, it is reasonable to anticipate thatnadA expression in vitro could be different from the level reachedin the host. Litt and colleagues (24) previously showed the pres-ence of antibodies that recognized recombinant NadA in childrenconvalescing after meningococcal disease. Interestingly, we reporthere that sera from children infected by isolates that failed to ex-press NadA in culture (MATS RP � 0) or that expressed low NadAlevels (MATS RP value � PBT) are able to recognize NadA atsignificantly higher levels than sera from subjects infected by nadAknockout strains (Fig. 1). Taken together, these observations sug-gest that, despite the low levels of NadA expression in vitro, allthese strains express NadA in an immunogenic form in the settingof invasive disease. Furthermore, anti-NadA antibodies are alsofound in healthy individuals, with levels tending to increase withage (34), suggesting that NadA is expressed in vivo, at a level suf-ficient to drive the immune response. NadA expression is alsoinduced in the ex vivo model of human saliva (reference 23 andthis study), again suggesting high expression in the niche of me-ningococcal colonization, which can be mimicked in vitro by ad-dition of 4-HPA or 3Cl4-HPA, representing natural inducers ofNadA present in the host.

Using the recombinant nadR knockout strains and the HPAinducers as a model for in vivo expression, we have validated NadAas a potent immunogen for patients of all ages and a valid target forprotective responses. Once repression mediated by NadR is re-lieved and NadA is expressed at high levels, strains normally resis-tant to killing by 4CMenB immune sera are rendered highly sus-ceptible to killing in SBA. The molecular mechanism of bothNadR repression of nadA and induction mediated by HPA com-pounds is conserved in a wide panel of N. meningitidis strainsbelonging to different clonal complexes (Fig. 2 and reference 23).These observations suggest that any strain carrying nadA couldpotentially be targeted by bactericidal antibody elicited by the

FIG 5 Direct visualization of PnadA expression in the in vivo infant rat model. (A) Histogram representing the bioluminescence increment from the background(infant rats infected with the 2996 wild-type strain) of groups of 5 infant rats infected with 104 CFU of the 2996 negative control (dark gray circles) or the 2996nadA-lux reporter strain (white circles) as well as median values of bioluminescence increment of each group (solid horizontal red lines) at the time pointsindicated. * � P � 0.05; *** � P � 0.001; ns � not significant. (B) Panel of ventral views of groups of 5 infant rats infected with 104 CFU of the negative controlor the PnadA-lux reporter, as indicated, taken 3 h after infection. Red boxes indicates the regions of interest (ROI) that were taken into consideration by the LiveImaging software to quantify bioluminescent values. Blood samples were recovered 24 h after infection, and CFU counts confirm that the bacterial loads withininfant rats infected with different strains were similar.

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4CMenB vaccine when NadR repression is relieved during infec-tion.

The ability of passively administered vaccinees’ sera to protectmice from infection of NGP165, and to return protective results inthe modified MATS and hSBA assays when NGP165 is grownin vitro with added HPA, suggests that this treatment mimics thein vivo status of NGP165 with respect to NadA expression. It isnoteworthy that passively administered sera from mice immu-nized with NadA recombinant protein alone protect infant ratsfrom infection with NGP165, demonstrating that the level of ex-pression reached by NadA alone in vivo is sufficient to promotebacterial killing. Finally, a bioluminescent nadA-lux fusion dem-onstrates the induction of the nadA promoter 3 h after infection inthe infant rat model, demonstrating the expression of nadA dur-ing bacteremia in vivo.

The expression of NadA in vivo in the host is in line with itsputative role during meningococcal infection. Its adhesive role isimportant in the course of colonization of the human upper re-spiratory tract (10, 35). Following the passage across the epithe-lium, meningococcus invades tissues and blood, where NadA caninteract with human blood leukocytes and lead to enhanced im-mune stimulation (36–39). For these reasons, NadA is to be con-sidered an important meningococcal virulence factor, involved inprogressively invasive steps of infection. In accordance with thisstatement, we demonstrated here that different signals are able tomodulate the NadR-repressive activity on NadA (see Fig. S1 in thesupplemental material). Signals present in saliva, which we canmimic by using HPA molecules at a high concentration during invitro growth, may mediate NadA induction during the coloniza-tion of the oropharynx. However, because NadA could be re-quired during other steps of the pathogenesis, multiple signals inniches other than the pharynx could modulate NadA expressionas soon as the bacterium passes the epithelial barrier. We demon-strate indeed in the infant rat model of bacteremia that NadA isexpressed widely 3 hours postinfection and that this upregulationcould be due to molecules such as 3CI4-HPA and NO2-4HPA (seeFig. S1 in the supplemental material), which are produced by leu-kocytes during inflammatory processes (32, 40). Furthermore, wecannot exclude the possibility that other molecules can act onNadR by increasing its repressive action on its targets.

From our data, current methods used to predict coverage ofstrains by the 4CMenB vaccine are underestimating the contribu-tion of the NadA antigen, suggesting that all nadA� meningococ-cal strains may be susceptible to bactericidal anti-NadA antibodieselicited by vaccination. A more accurate prediction may be ob-tained by addition of physiologically relevant inducers to in vitro-grown bacteria, resulting in NadA expression levels similar tothose measured in ex vivo and in vivo models of infection. Thisreport provides new insights into the expression of the NadA an-tigen during infection which are fundamental for the implemen-tation of prophylactic strategies such as vaccines and evaluation oftheir impact on public health.

ACKNOWLEDGMENTS

We are thankful to the Active Bacterial Core surveillance team for provid-ing the Nm isolates M10574 and M14933. We thank Phil Boucher, DavideSerruto, Marirosa Mora, Maurizio Comanducci, Kate Seib, and Ana An-tunes for critical reading of the manuscript. We are grateful to Fabio Rigatand Giacomo Frosi for their support in evaluating the sample numerosityfor passive protection experiments. We also thank Mauro Agnusdei,

Isabella Simmini, Angela Spagnuolo, and the animal care facility at No-vartis Vaccines for technical support. We thank Giorgio Corsi for artwork.

L.F. is the recipient of a Novartis fellowship from the Ph.D. program inFunctional Biology of Molecular and Cellular Systems of the University ofBologna.

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