DOI: 10.1126/scitranslmed.3002234, 91ra62 (2011);3 Sci Transl Med

, et al.Maria ScarselliImmunityRational Design of a Meningococcal Antigen Inducing Broad Protective

 Editor's Summary

 All for One and One for All

   like HIV that show a high degree of antigenic variation.structure-based approach to vaccine design may be useful not only for meningococcus B but also for other pathogensall strains of meningococcus B, suggesting that it could be used to produce a broadly protective vaccine. This bacterial strains. One chimeric immunogen, called G1, was capable of inducing bactericidal antibodies that could killthis, they injected the immunogens into mice and assayed mouse sera in vitro for bactericidal activity against multiple chimeric immunogens could elicit bactericidal antibodies against many different strains of meningococcus B. To donatural variation of variant 2 and 3 strains of meningococcus B. They then tested which of the 54 engineered single

theand 3. They then introduced groups of point mutations into the amino acids of these transplanted patches to mimic these three major variants, they engineered variant 1 to carry patches of amino acids from the surfaces of variants 2300 sequence variants of factor H binding protein into three major groups. Using detailed structural information about one vaccine that contains all of these variants. Rappuoli and his colleagues have tackled this problem by dividing theprotein should be a valuable immunogen, but because it has at least 300 sequence variants, it is impractical to make onslaught of the human immune system's complement pathway. Because it is essential for survival, factor H bindingprotein is essential for the survival of meningococcus B in the human host because it protects the pathogen from the degree of variation that has stymied attempts to use them as vaccine immunogens. For example, factor H bindingthis pathogen has yielded excellent targets that could be used in a vaccine, many of these antigens show a high against meningococcus B, the bacterial pathogen that causes meningitis. Although mining of the genome sequence ofmusketeer. That is precisely the approach that Rappuoli and his colleagues have taken with their design of a vaccine The three musketeers were a formidable team, but imagine combining all of their skills and valor into just one

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R E S EARCH ART I C L E

VACC INE DES IGN

Rational Design of a Meningococcal Antigen InducingBroad Protective ImmunityMaria Scarselli,1* Beatrice Aricò,1* Brunella Brunelli,1 Silvana Savino,1 Federica Di Marcello,1

Emmanuelle Palumbo,1 Daniele Veggi,1 Laura Ciucchi,1 Elena Cartocci,1

Matthew James Bottomley,1 Enrico Malito,2 Paola Lo Surdo,1 Maurizio Comanducci,1

Marzia Monica Giuliani,1 Francesca Cantini,3 Sara Dragonetti,3 Annalisa Colaprico,1

Francesco Doro,1 Patrizia Giannetti,1 Michele Pallaoro,1 Barbara Brogioni,1 Marta Tontini,1

Markus Hilleringmann,1 Vincenzo Nardi-Dei,1 Lucia Banci,3† Mariagrazia Pizza,1 Rino Rappuoli1†

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The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseriameningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigenfactor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified intothree distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to gen-erate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. Toachieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them inmice for the induction of protective immunity. We identified and determined the crystal structure of a leadchimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variantstrains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containingspecificities for all three variant groups. We demonstrate that the structure-based design of multiple immuno-dominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to createbroadly protective vaccines.

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INTRODUCTION

The development of vaccines against many pathogens is often limitedby the sequence variability of their protective antigens. When the an-tigenic variability is limited, as in the case of poliovirus or Streptococcuspneumoniae, vaccines have been developed by including up to 23 dif-ferent antigenic variants in a single vaccine vial. However, when theantigenic variability is high, as is the case for influenza viruses, a newvaccine is needed every year. In other cases exemplified by the bacte-rium Neisseria meningitidis serogroup B [meningococcus B (MenB)],the protozoan parasite causing malaria, African trypanosomes, rhino-virus, and HIV, the extreme antigenic variability of these pathogenshas stymied the development of a successful vaccine (1, 2). In all ofthese cases, the challenge for modern vaccinology is to develop anti-gens that are able to induce broad protective immunity against allnatural antigenic variants of the pathogen. To date, this has been ap-proached principally by trying to increase the immunogenicity of themost conserved regions of the antigen; however, these attempts havemet with limited success (3–5).

Here, we used a different approach to develop a vaccine againstMenB, an obligate human pathogen that can cause severe, often fatal,septicemia and meningitis. Poor immunogenicity, as well as the risk ofcross-reactivity of type B capsular polysaccharide with human tissues,has greatly limited the development of a glycoconjugated vaccineagainst serogroup B meningococcal strains. Mining the genome of this

1Novartis Vaccines and Diagnostics S.r.l., Via Fiorentina 1, 53100 Siena, Italy. 2GenomicsInstitute of Novartis Research Foundation, 10672 John Jay Hopkins Drive, San Diego,CA 92121, USA. 3Magnetic Resonance Center (CERM) and Department of Chemistry,University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy.*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail: rino.rappuoli@novartis.com(R.R.); banci@cerm.unifi.it (L.B.)

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bacterium recently led to the discovery of factor H binding protein(fHBP), a powerful protective antigen that binds to human factorH. Human factor H is a serum regulatory protein that provides thefirst line of defense of the innate immune system for protecting thehuman body against invading organisms. Antibodies elicited by inject-ing fHBP into mice are able to kill the bacterium in the presence ofcomplement, a property that is known to correlate with protection inhumans (6, 7). Sequencing the fhbp genes in almost 2000 strains (8, 9)of MenB revealed that more than 350 different nucleotide sequencesexist, coding for almost 300 different polypeptides (http://neisseria.org/nm/typing/fhbp/). The sequence types with less than 10%sequence variability were found to induce some cross-protective im-munity, whereas no cross-protection could be observed among moredivergent sequences (10). Using this information, we divided the en-tire family of fHBPs into three major antigenic variants (variants 1, 2,and 3), which are present in 65, 25, and 10% of the MenB global pop-ulation, respectively (8, 9, 11). The fHBP variant 1 antigen is an im-portant component of a vaccine currently in phase III clinical trialscontaining five different antigens (4CMenB) recently discovered byreverse vaccinology (that is, a method for selection of vaccine candi-dates based on in silico analysis of bacterial genomes) (6). A secondvaccine containing a mixture of the main fHBP antigenic variants1 and 2 (or families A and B according to the different nomenclatures)is in phase II clinical trials (12).

Here, we designed a panel of chimeric fHBP antigens with the aimof combining in a single molecule the complete antigenic repertoire ofthe three major variant groups. When injected into mice, one of theseengineered fHBP antigens was able to elicit cross-protective immunityagainst meningococcal strains carrying different fHBP alleles. Furtherengineering of this candidate led to a final molecule with increasedefficacy.

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RESULTS

Rational design of fHBP mutantsThe three-dimensional (3D) structure of fHBP determined by nuclearmagnetic resonance (NMR) (13, 14) and x-ray crystallography (15)showed that the protein is essentially composed of two b barrelsconnected by a short linker (Fig. 1). Sequence comparison of the threedifferent variants suggested that the protein architecture is well con-served among different members of the fHBP family. Analysis of theepitopes recognized by variant-specific monoclonal antibodies (mAbs)(16–20) revealed that fHBP amino acids important for recognition bythe antibodies against variant 1 were Asp25, His26, Lys27, Thr56, Tyr57,Gly121, Glu146, Gly147, Gly148, Arg149, and Arg204 (colored red in Fig.1). In contrast, the amino acids important for recognition of variants 2and 3 were Ala174 (Lys in variants 2 and 3), Lys180 (Arg in variants 2and 3), Asp192 (Glu in variants 2 and 3), and Gln216 (Ser or Gly invariant 2, Ser in variant 3) (colored purple in Fig. 1). In the primarysequence Arg204, one of the most important residues for epitope rec-ognition of variant 1 falls in the middle of a region also important forvariants 2 and 3 that spans Asp192 to Gln216 (yellow ribbon in Fig. 1).From the analysis of the 3D structures, it is clear that amino acidscontributing to the immunogenicity of variant 1 or variants 2 and 3are located in non-overlapping regions. This important observationsuggested that the immunodominant regions of the three variantgroups are distinct and that an alternative immunogenic epitopefeaturing variant 2 or 3 residues could be grafted onto the variant1 backbone.

With the aim of generating a single antigen that is able to induce abroad antibody response against all of the fHBP variants of MenB, weinitially generated, purified, and immunized mice with mutants of var-iant 1 containing single, double, or triple amino acid mutations

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derived from the sequences of variants 2 and 3. In no case were thenew antigens found to induce a broad immune response, indicatingthat changing just a few amino acids was not sufficient to create im-munodominant epitopes that could induce immune responses to var-iants 2 and 3. Therefore, we decided to engineer portions of theantigen surface potentially recognizable by an antibody as a whole,instead of making single amino acid mutations. We focused on theC-terminal b barrel domain of fHBP, which contains most of theamino acids recognized by antibodies against variants 2 and 3 (17).Taking into account that conformational epitopes of proteins typicallyrange in size from about 900 to 2000 Å2 (21–23), we divided the entireC-terminal b barrel domain into 11 partially overlapping areas of sizelarge enough to hold at least one conformational epitope (table S1).Within each distinct area, residues of the variant 1 serobase protein[that is, the protein of variant 1 included in 4CMenB vaccine (7)and selected as a reference molecule] were replaced with the corre-sponding amino acids of variants 2 and 3 regardless of their posi-tion in the primary sequence. To preserve folding, we introducedamino acid substitutions only for residues whose side chains werewell exposed to the solvent, leaving the internal core of the proteinunaltered.

The different surface areas created by this structure-based muta-tional approach were assigned letters A to K (Fig. 2). Within each zone,groups of point mutations were introduced using the multiple sequencealignment of 85 different alleles (9) as a guideline. Depending on thenumber of hypervariable residues, distinct sets of substitutions were in-troduced within each area to reproduce all of the concurrent variationsobserved in the natural bacterial population. A numeric suffix was usedto indicate each single mutant characterized by a specific combinationof such hypervariable substitutions. A total of 54 different antigenicvariants were designed (figs. S1 and S2), expressed in Escherichia coli,purified, and then used to immunize mice, using protocols establishedpreviously (6). Mouse sera were collected and tested in vitro for theirability to kill bacteria in the presence of complement. The screeningassay for measuring protection induced in the mice was performedagainst a panel of seven meningococcal strains expressing divergentsequences of fHBP. These divergent sequences included subvariants1.10, 1.14, 2.1, 2.4, 2.10, and 3.1 that had 93.4, 91.6, 74.1, 73.7, 69.7,and 62.8% amino acid identity compared to the serobase protein, re-spectively. Strain MC58 of MenB expressing the serobase protein sub-variant 1.1 was used as a positive control (Table 1).

Of the 54 mutants tested, 18 resulted in a more than 10-fold re-duction in bactericidal activity of the mouse sera against the MC58strain and were therefore discarded. Among the remaining 36 mole-cules, 15 showed at least a 10-fold increase in bactericidal titer againstthe prototypic strain of variant 2 or were also positive against variant3. This group of 15 proteins carried mutations within 6 of the 11 dif-ferent engineered areas, and so, we consequently selected a represent-ative panel of six mutants (D5, E2, F3, G1, G2, and H9; fig. S3, A toG). Two additional mutants, B4 and J1, showing wide coverage againststrains of variant 1 were also included. The results of the completescreening are reported in table S2.

Biochemical characterization of selected candidatesTo verify that the mutations introduced did not cause major altera-tions in protein folding or stability, we analyzed the selected mutantsto evaluate different biochemical and biophysical properties when com-pared with the serobase fHBP subvariant 1.1. In particular, the tendency

Fig. 1. 3D structure of fHBP from MenB strain MC58 (subvariant 1.1). Rib-bon diagrams show the distribution of the amino acids recognized by

mAbs elicited by variant 1 (red) and variants 2 and 3 (purple) as reportedpreviously (16–20). Amino acid positions 174, 180, and 192 in variants 2 and3 correspond to Lys, Arg, and Glu, respectively. In position 216, strains ofvariant 2 have Gly and strains of variant 3 have Ser. Ribbons showing theN- and C-terminal domains are colored blue and green, respectively. Theregion containing Arg204 is shown in yellow.

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to aggregate, secondary structure, and folding were monitored by sizeexclusion high-pressure liquid chromatography (SEC-HPLC), circu-lar dichroism (CD) spectroscopy, and NMR, respectively. Analysis ofthe SEC-HPLC retention times of the serobase protein and respec-tive mutant proteins showed that all fHBP mutants eluted as singlepeaks co-migrating with the original serobase protein. Comparisonwith retention times of reference molecules indicated that the mutantswere present as monodisperse monomers and did not form aggregates(table S3A). As a second approach to confirm the quality of the pro-teins overall, far-ultraviolet (UV) CD spectroscopy of the same sam-ples suggested that all of the fHBP proteins have the same secondarystructure (table S3B). Subsequently, protein folding and conformationwere checked in detail by expressing the selected mutants in 15N-enrichedmedium and analyzing them by NMR spectroscopy, which enabledanalysis at the residue-specific level. Heteronuclear single-quantumcorrelation (1H-15N HSQC) spectra for each mutant indicate foldedproteins with well-dispersed amide chemical shifts and whose total peaknumber matched the predicted one (Fig. 3). Most of the 1H-15N crosspeaks in the 15N HSQC spectrum of each mutant overlapped those ofthe wild-type protein. In contrast, the major chemical shift variationswere confined to the region harboring mutations on the C-terminal b

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barrel (residues 138 to 255). The new peaks that appeared due to pointmutations also had chemical shift values indicative of a well-foldedenvironment. Collectively, these data indicated that the mutations in-troduced within each mutant did not alter the overall architecture ofthe protein nor did they introduce regions of disorder, suggesting thatthe designed conformational epitopes were successfully grafted withinthe correct molecular scaffold.

Bactericidal activity of selected candidatesTo further investigate the extent of cross-protection that could be in-duced by the mutants, we immunized mice with the selected mutantsformulated with an adjuvant suitable for human use composed of amix of aluminum hydroxide and IC31 (a Toll-like receptor 9 agonist)recently used in clinical trials (24, 25). The bactericidal activity of theresulting sera was tested against an enlarged panel of MenB strainscontaining the serobase protein; the subvariants 1.10, 1.12, and 1.14that are the most divergent sequences of variant 1; and the subvariants2.1, 2.4, 2.7, 2.10, and 3.1 (Table 2). Bactericidal antibody titers elicitedin mice by each mutant were compared to those obtained by im-munizing mice with the homologous fHBPmolecule purified from eachstrain. As expected, the serobase subvariant 1.1 elicited bactericidal

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Fig. 2. Mutation of fHBP. The surface of the variant 1 fHBP reference pro-tein is shown in gray. Backbones of the N- and C-terminal domains are

extensive figure with fHBP shown in three different orientations to bettervisualize the mutated surfaces is provided in fig. S2. A detailed representa-

colored blue and green, respectively. The different engineered regionsare represented as solid surfaces. Residues conserved among the three var-iants are colored light blue. Residues conserved between variants 2 and 3are colored dark red. Hypervariable residues are colored yellow. A more

tion of the eight selected candidates is also reported in fig. S3, A to G. Aminoacid sequences of all the mutants generated are reported in fig. S3. Proteinrendering was realized using UCSF (University of California, San Francisco)Chimera software (http://www.cgl.ucsf.edu/chimera/).

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titers >1000 against subvariants 1.1, 1.10, and 1.12, and titers of 256or lower against distantly related 1, 2, and 3 variants. In marked con-trast, the wild-type subvariant 2.1 induced no bactericidal antibodytiters against variant 1, titers >1000 against all variant 2 strains, anda titer of 512 against variant 3 strains. When mice were immunizedwith each of the fHBP natural subvariants, immune sera had titers of>1000 against the homologous strain. The mutated molecules induced

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broader immunity with respect to thatelicited by the fHBP subvariant 1.1. Inparticular, sera raised by G1, G2, and H9were bactericidal against all of the strainstested. G1 elicited titers >1000 againstmost of the strains tested. Arg204, origi-nally described as part of a bactericidal epi-tope of variant 1 (17), was mutated in G1to serine without destroying variant 1 im-munogenicity. This suggested that it wasthe larger surface area and not the individ-ual amino acids that was important forimmunogenicity.

Crystal structure of G1To characterize further the lead candidateantigen, we determined the x-ray crystal-lographic structure of the fHBP mutantG1 refined to 1.9 Å resolution (Fig. 4 andtable S5). A comparison of the G1 struc-ture with the crystal structure of fHBP var-iant 1, which was determined in complexwith factor H, shows that the backbonestructures are virtually identical (root-mean-square deviation of 0.67 Å for 239equivalent main-chain carbon atoms shar-ing 89% sequence identity). Only small dif-

ferences in a few loop regions were observed, most notably for theloops connecting b strands b5-b6, b7-b8, and b15-b16 (Fig. 4A). Theseminor differences may be due to an intrinsic flexibility in the exposedloop regions, differences in crystal packing, or to the absence of factorH in the G1 structure. Electron density was clearly visible for the sidechains of most of the mutated residues (colored green in Fig. 4B), con-firming that the substitutions changed the exposed surface without

Fig. 3. NMR spectra of fHBP. Shown are 1H-15N HSQC NMR spectra acquired at 25°C for fHBP subvariant1.1 and mutants. The two panels on the left show the spectra acquired for wild-type (wt) fHBP in 20 mM

sodium phosphate at pH 7.2 (upper panel) and 4 M urea at pH 2 (lower panel). In the remaining panels,NMR spectra acquired for each fHBP mutant are overlaid on the spectrum of the fHBP wild-type protein.Each peak represents a signal from the NH group of the protein. The wild-type spectrum is colored blueand mutant spectra are colored pink (B4), red (D5), aquamarine (E2), cyan (F3), hot pink (G1), light green(G2), sable (H9), and green (J1). The concentration of the protein samples was 1.0 mM in a 90% H2O/10%2H2O mixture containing 20 mM sodium phosphate buffer at pH 7.2.

Table 1. Principal features of the meningococcal strains used in this study.Meningococcal strains were selected to sample the fHBP genetic diversity ob-served in the bacterial population. Expression and exposure of fHBP on the

bacterial cell surface was tested by Western blotting and fluorescence-activatedcell sorting (FACS) analysis, respectively (figs. S1 and S2). na, not assigned; Cpx,clonal complex; ST, sequence type; NZ, New Zealand; AUS, Australia; C, Canada.

Strain*

Cpx ST Year† Country† Typing‡

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Amino acid identityto variant 1.1 (%)

MC58§

32 74 1985 UK B:15:P1.7,16b 1.1 100

UK149

41/44 41 2001 UK B:4:P1,7,4 1.4 95.9

UK185§

11 11 2001 UK B:2a:P1.5,10 1.10 93.4

NZ98/254§

41/44 42 1998 NZ B:4:P1.4 1.14 91.6

961-5945§

8 153 1996 AUS B:2b:P1.21,16 2.1 74.1

M3153§

41/44 5906 1996 USA B:4,7:P1.4 2.4 73.7

M2552§

103 103 1996 USA B:NT:NT 2.10 69.7

M1239§

41/44 437 1994 USA B:14:P1.23,14 3.1 62.8

M4030

na 178 1993 USA B:17:P1.19,15 1.12 93.4

C11

na 345 1965 C C:NT:P1.1 2.7 70.8

M6208

103 2006 1999 USA B:NT:P1.5-1,10-4 2.10 69.7

*Strains used during the initial screening. †Year in which the strain was isolated and where (country). ‡PorA/PorB typing scheme and subvariant are reported for each strain. §fHBPsubvariants are named in accordance with the public fHBP database (http://pubmlst.org/neisseria/).

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inducing any local disorder. Comparison of the G1 structure (Fig. 4B)to the published structure of fHBP variant 1 in complex with factor H(Fig. 4C) shows an extreme similarity of the global structure com-posed of conserved amino acids and a distinct surface structure

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containing variant 2 and 3 specificities (in green) grafted onto theC-terminal portion of the molecule.

Analysis of the G1 structure also shows that the region interactingwith factor H, covering ~1500 Å2 of the surface, was minimally

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Table 2. Bactericidal titers in mice. Shown are the bactericidal titers elicitedin mice by selected fHBP mutants formulated with aluminum hydroxideplus IC31 adjuvant. The recombinant antigens were administered intra-peritoneally to groups of eight mice at days 1, 21, and 35. Bactericidal ac-tivities in mouse serum are defined as the reciprocal of serum dilution that

resulted in a 50% reduction in numbers of CFUs surviving compared withthe results for an equivalent negative control containing heat-inactivatedcomplement. The serobase protein from the MC58 strain (fHBP variant 1.1wild type) and the homologous alleles of each strain have been included aspositive controls. Titers of 512 or higher are in bold.

MC58

UK185 M4030 NZ98/254 961-5945 M3153 C11 M2552 M1239

Antigen

1.1 1.10 1.12 1.14 2.1 2.4 2.7 2.10 3.1

fHBP var.1.1 wt

4096 2048 1024 128 16 256 <16 <16 <16

fHBP var.2.1 wt

16 16 <16 <16 32768 >8192 1024 2048 512

11

fHbp homolog 4096 4096 8192* >_8192 32768 8192 2048 1024 >8192

20

D5

2048 64 2048 <16 2048 512 <16 <16 <16

15,

F3

8192 256 8192 1024 2048 1024 <16 <16 <16

ly

B4

32768 256 8192 1024 2048 1024 16 512 <16

Ju

J1

16384 4096 8192 256 256 2048 <16 <16 <16

on

E2

65536 256 2048 4096 1024 128 <16 <16 <16

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G2 16384 8192 4096 512 1024 >8192 32 64 128

ag

H9 4096 512 4096 256 2048 2048 128 128 128

em

G1 8192 512 >_8192 1024 8192 8192 1024 1024 2048

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ci

en

*This bactericidal assay titer was obtained using antibodies against a protein derived from the closely related strain UK185 that has a 97.8% amino acid sequence identity with the protein of theM4030 strain.

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Fig. 4. Structural comparison of wild-type fHBP and the G1 mutant. Com-parison of the structures of wild-type fHBP variant 1 and the chimeric G1mutant reveals that the conserved fold displays a different immunogenicsurface. (A) Ribbon display of the superimposed a carbon traces of G1(N-terminal domain in blue, C-terminal domain in green) and fHBP variant1 (yellow; from PDB entry 2W80) (13) reveals minor variations restricted to afew exposed loop regions (labeled). Spheres show a carbon atoms of G1where mutations were inserted. The alignment reveals a very slightdifference in domain orientation, such that b strands 13, 14, 15, and 16are shifted by 0.2 to 1.0 Å. (B) Surface display of the G1 structure showingthe newly introduced specificities in green. The green sites mutated in G1are I134→L*, A135→G, S140→A, D142→N*, K143→Q*, E146→D*,R148→K, T150→E, A172→T*, A173→K, D191→E, A194→S, D196→E*,

P199→A*, R203→S*, S208→L*, S210→D*, L212→R*, N214→G, Q215→S,A216→E*, and K229→R* (asterisks mark residues visible in this image). Theunchanged portion of the molecule is colored as follows: (i) residues con-served among all three variants, blue; (ii) residues conserved between var-iants 2 and 3 but differing from variant 1, red; (iii) hypervariable residues,yellow. The G1 mutation scheme eliminates a Gly at position 147, with acorresponding shift of “−1” in the numbering scheme from this point on-ward; consequently, the residue S203 corresponds to R204 in wild-typefHBP (see also fig. S1). (C) Structure of the complex between wild-type fHBPvariant 1 and human factor H (13) showing the surface of fHBP and a car-bon trace of human factor H (green). Regions of the fHBP surface arecolored according to the variability profile using the same color code asin (B). Figures were prepared using PyMOL (http://www.pymol.org).

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changed in G1. Only three residues were substituted in this region(namely, I134L, S208L, and S210D), corresponding to a total surface-accessible area of 250 Å2. Indeed, the binding of G1 to factor H wasfound to be similar to that of the native molecule (table S6).

Further engineering of G1To further optimize the immunogenicity of the engineered G1 mole-cule and generate a final vaccine candidate, we produced a fusion pro-tein, a general strategy previously shown to improve vaccineperformance (26). Thus, two copies of G1 were fused to GNA2091,a meningococcal antigen that enhances the stability and efficacy offHBP variant 1, although it does not itself induce bactericidal anti-bodies (26). The new fusion antigen, GNA2091-G1-G1, was formu-lated with the same adjuvant as described above, and thebactericidal assays were performed in the presence of rabbit and hu-man complement. When rabbit complement was used, bactericidal ti-ters induced against MC58 and NZ98/254 strains by GNA2091-G1-G1 (Table 3A) were comparable to those previously reported for the4CMenB vaccine currently in phase III clinical trials (7). Moreover,the fusion molecule was able to promote the killing of each meningo-coccal strain at levels comparable to those observed when mice wereimmunized with the homologous alleles. A positive bactericidal titeragainst all variants tested was also obtained by performing the assay in

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the presence of human complement (Table 3B), which is the moststringent test available for vaccine potency (27). In this assay, whichtypically gives lower titers, positive titers are predictive of efficacy inhumans. Collectively, these data provide a solid rationale for the use ofthis molecule in a vaccine that recognizes all of the antigenic variantsof fHBP and thus should provide efficacious and broad protectionagainst infection with MenB.

DISCUSSION

We have successfully engineered a single molecule of fHBP to displaysimultaneously two independent immunodominant regions that in-duce protective immunity against all fHBP antigenic variants of theMenB serotype. In so doing, we have demonstrated that the structure-based design and engineering of the entire conformational surface ofthe epitope is essential to increase the immunogenicity of the mole-cule, whereas a change of only one or a few amino acids of the epitopeis not sufficient. Notably, the most efficacious antigens present in theinitial group of 54 proteins, G1 and G2, carry substitutions within thesame region, one of the largest among those modified. Therefore,among all of the regions modified, the area defined as G is the onethat best accommodated heterologous epitopes within the serobase

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Table 3. Bactericidal titers elicited by the GNA2091-G1-G1 fusion protein.Bactericidal titers elicited in mice immunized with the GNA2091-G1-G1fusion protein in the presence of rabbit (A) or human (B) complement.Antigens were formulated in aluminum hydroxide and IC31 adjuvant.The bactericidal activities induced against each strain by wild-typestrains of subvariants 1.1 and 2.1 and the homolog fHBP alleles are also

shown. In the assay with rabbit complement, titers of 512 or higher areshown in bold. We also compared the bactericidal assay titers elicited byG1 and GNA2091-G1-G1 with rabbit complement, repeating the mea-surements three times (fig. S6). In the assay with human complement(B), which is much more stringent, sera dilutions greater than 1:8 areshown in bold.

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MC58 UK149 M4030 UK185 NZ98/254

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fHBP var.1.1 wt >32768 4096 8192 256 512 256 64 32 32 <16

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fHBP var.2.1 wt 64 <16 128 <16 64 >8192 >8192 1024 4096 8192

GNA2091-G1-G1

16384 2048 4096 4096 4096 8192 4096 1024* 4096 1024

Homologs

>65536 4096 >8192 4096 >8192 32768 8192 2048 1024 >8192

B

MC58

UK149 M4030 UK185 NZ98/254 961-5945 M3153 C11 M6208† M1239

1.1

1.4 1.12 1.10 1.14 2.1 2.4 2.7 2.10 3.1

Antigen

fHBP var.1.1 wt

2048 256 512 16 128 <8 <8 <8 <8 <8

fHBP var.2.1 wt

<8 <8 <8 <8 <8 64 <8 <8 128 <8

GNA2091-G1-G1

1024 512 >512 256 256 64 256 256 512 256

Homologs

2048 >1024 512‡ >1024 >1024 256 128 128 512 1024

*Bacteriostatic titer. †Because human complement was toxic for the M2552 strain, the M6208 strain belonging to the same subvariant was used instead for the bactericidal assay. ‡This bactericidalassay titer was obtained using antibodies against fHBP derived from the closely related UK185 strain that has a 97.8% amino acid sequence identity with the fHBP protein of M4030.

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protein structure without detrimental effects on the original repertoireand without interfering with the ability of the molecule to bind tofactor H. Moreover, modifications in this region seem to modulateoptimally the immunodominance among variants 1, 2, and 3. Graftingnew epitopes into a single protein to make a single vaccine moleculethat is able to induce immunity against all antigenic variants of a path-ogen is a challenging task in vaccinology. Here, we have proposed ageneral strategy that is summarized in Fig. 5. For this type of ap-proach, the critical steps are (i) the structure of the antigen necessaryto define surface amino acids; (ii) epitope mapping by variant-specificmAbs, which provides the initial indication that distinctive epitopessegregating on the protein surface can be recognized; (iii) structure-based design of “transplanted” amino acids mimicking the newlyintroduced epitopes; and (iv) testing the biochemical and structuralintegrity of the generated molecules.

Traditionally, vaccines have been developed using or mimickingnatural antigens, but here, we have shown that natural antigens canbe improved to provide better immunity. Previous approaches tomaking universal vaccines against pathogens showing a degree ofantigenic variation relied on the identification of conserved epi-topes (3–5). Although this approach would be the most logical, sofar it has met with limited success, probably because the conserved

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regions of the antigens have been naturally selected during evolutionto be poorly immunogenic. Our results suggest an alternative approachin which structural knowledge is applied to graft multiple immuno-dominant epitopes onto a single molecule. We have engineered thefHBP protective antigen of MenB to contain an artificial combinationof epitopes that is able to expand markedly the coverage of the originalantigen. This success suggests that a similar approach could, in prin-ciple, be used in other cases where sequence variability is the mainobstacle to vaccine development.

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MATERIALS AND METHODS

StrainsE. coli DH5a and BL21 (DE3) star were used as cloning strain andexpression host, respectively, and used as recommended by the man-ufacturer (Invitrogen).

Cloning, expression, and purification in E. coliThe starting sequence of the fhbp gene where mutations were intro-duced is a version of MC58 fhbp devoid of the sequence coding for theleader peptide and glycine stretch sequence, starting with the Val codon.The DNA sequence encoding each mutant was obtained by modifyingthe codons to encode for the new amino acids avoiding the use of tworare AGG and AGA arginine codons. Nde I and Xho I restriction siteswere added at 5′ and 3′ ends, respectively. Synthetic genes encoding forthe 54 mutants were purchased from GeneArt. Each gene was digestedwith Nde I and Xho I and cloned into the Nde I/Xho I sites of thepET21b(+) expression vector (Novagen). The use of Nde I and Xho Isites resulted in the insertion of a starting Met codon and the additionof leucine-glutamate amino acids at the end of the sequence, respectively.Recombinant proteins were expressed as His-tag fusions.

Antigen formulationAll formulations were performed in sterile conditions under flow hood.Each recombinant protein was adsorbed onto aluminum hydroxide atprotein, aluminum (alum), and NaCl concentrations of 100 mg/ml,3 mg/ml, and 9 mg/ml, respectively, in 10 mM histidine (pH 6.5).Water for injection and histidine buffer were premixed. Sodium chlo-ride was added to result in a final formulation osmolality of 0.308mosmol/kg. Alum addition was calculated on the basis of the concen-tration of the alum stock to obtain a final concentration of 3 mg/ml.Antigens at respective concentrations were added to the mix and left for15 min under stirring at room temperature and then stored overnightat 4°C before the immunization. IC31-containing formulations wereprepared by adding an equal volume of IC31 (1000 nmol KLK and40 nmol ODN1a) to the alum-adjuvanted formulations just before im-munization. In this case, the immunization volume was doubled tomaintain a constant antigen dose (20 mg). Final formulations were iso-tonic and at physiological pH. All alum and alum-IC31 formulationswere characterized soon after immunization, antigen adsorption was>90%, and adsorption profile was similar for all antigens and adju-vants tested.

Mice immunizationTo prepare antisera, we used 20 mg each of recombinant protein toimmunize 6-week-old CD1 female mice (Charles River). Each an-tigen was used to immunize a group of eight mice. The recombi-

Fig. 5. A structure-based approach for designing fHBP mutants. The firststeps of rational structure-based design are as follows: identification of

surface-exposed residues, mapping of sequence variability onto the pro-tein structure, and planning of point mutation groups to be introducedinto the native molecule. These steps are followed by the experimentalevaluation of candidate antigen performance measured by an in vitrobactericidal assay using human or rabbit complement with serum frommice immunized with the candidate antigens.

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nant proteins were administered intraperitoneally, together withaluminum hydroxide (3 mg/ml) or IC31 plus aluminum hydroxideat days 0, 21, and 35. Blood samples of each group were taken onday 49 and pooled for serological analyses.

Heat-inactivated whole-cell preparationsN. meningitidis wild-type strains and DfHBP mutant strains weregrown overnight on agar chocolate plates at 37°C in 5% CO2. Col-onies from each strain were collected and used to inoculate 7 ml ofMueller-Hinton broth containing 0.25% glucose to an initial OD620

(optical density at 620 nm) of 0.05 to 0.06. The culture was incu-bated for ~2.5 hours at 37°C with shaking until an OD620 of 0.5 wasreached and then centrifuged for 10 min at 3500 rpm. The super-natant was discarded and the pellet was resuspended in 500 ml ofphosphate-buffered saline (PBS). Heat inactivation was performedat 56°C for 30 min.

SDS–polyacrylamide gel electrophoresis andWestern blot analysisFor Western blot analysis, 15 ml of each total whole extract samplein 1× SDS–polyacrylamide gel electrophoresis (SDS-PAGE) loadingbuffer (Invitrogen) was separated by SDS-PAGE and transferredonto a nitrocellulose filter by standard procedure. Filters wereblocked for 1 hour at room temperature by agitation in blockingsolution (10% skim milk, 0.1% Triton X-100 in PBS) and incubatedfor another hour with a 1:1000 dilution of the anti-fHBP proteinserum in washing solution (3% skim milk, 0.1% Triton X-100 inPBS). After washing, the filters were incubated in a 1:2000 dilutionof peroxidase-conjugated anti–mouse immunoglobulin (Dako) inblocking solution for 1 hour, and the resulting signal was detectedwith the SuperSignal West Pico chemiluminescent substrate (Pierce).Results are reported in fig. S4.

Fluorescence-activated cell sorting analysisThe ability of polyclonal anti-fHBP sera to bind to the surface oflive meningococci was determined with a FACScan flow cytometeron strains representative of the subvariants. Antibody binding wasdetected with a fluorescein isothiocyanate (FITC)–conjugated sec-ondary antibody to mouse (whole molecule) (Sigma). The positivecontrol included SEAM 12, a mAb specific for the MenB capsularpolysaccharide (9). Negative control was the unrelated cytoplasmicprotein NMB1380. Results are reported in fig. S5.

Complement-mediated bactericidal activitySerum bactericidal activity against N. meningitidis strains was eval-uated as previously described (8) using as complement source 25%(12.5 ml of complement in 50 ml of total volume reaction) of pooledbaby rabbit serum (Pel-Freeze) or human serum. Serum bactericidaltiters were defined as the serum dilution resulting in 50% decrease incolony-forming units (CFUs) per ml after 60-min incubation ofbacteria with reaction mixture compared to control CFU per ml attime 0. Typically, bacteria incubated with the negative control anti-body in the presence of complement showed a 150 to 200% increasein CFU per ml during the 60-min incubation. Because of the highnumber of mutants tested during the initial screening, pooled serawere tested only once against each of the seven reference strains.

Data reported in Table 3 are instead representative on averageof three different immunization experiments in which pooled sera

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were tested. The experiments were repeated on different days withthe same complement source, and results were comparable and re-producible. Bactericidal titers elicited by G1 and GNA2091-G1-G1were also compared by repeating the measurements three times.Results of comparisons are reported in fig. S6.

Analytical SECRecombinant fHBP wild type and mutants (in 10 mM KPPi, pH7.5) were analyzed by SEC-HPLC to study their state of aggrega-tion. Samples were applied onto a Superdex 200 PC 3.2/30 (GEHealthcare) column at a flow rate of 0.1 ml/min in PBS buffer sys-tem. For calibration, a gel filtration standard (670, 158, 44, 17, and1.35 kD; Bio-Rad) was applied (table S2).

CD spectroscopyFar-UV CD spectra from 195 to 260 nm of fHBP wild-type andmutant proteins containing equimolar mixtures of proteins in 10 mMKPPi (pH 7.5) buffer were acquired at 25°C on a CD spectrometer(Jasco J-810) equipped with a water-cooled Peltier System (PCB1500).KPPi (10 mM, pH 7.5) buffer was used as a blank, and respectivespectra were subtracted from all recorded CD spectra. Analysis ofsecondary structure of the constructs was evaluated by deconvolutionof the spectra using CDNN v.2.1 (11). Original CD data in milli-degrees were converted to De units.

NMR structural characterizationThe fHBP variant 1.1 wild-type and mutant clones E. coli BL21 (DE3)are grown in ISOGRO-15N provided by Sigma. In all cases, the pro-tein expression was induced by isopropyl-b-D-thiogalactopyranoside(IPTG). All samples were first purified by nickel-chelating affinity chro-matography followed by anion exchange chromatography, and thenall the proteins were dialyzed in 20 mM sodium phosphate (pH 7.2)before being subjected to NMR analysis. NMR measurements wereacquired at 900 MHz on an ADVANCE Bruker spectrometer.

Protein crystallizationCrystallization experiments were performed in a nanodroplet sitting-drop vapor-diffusion format with 480 condition screens using 96-welllow-profile Greiner plates and an Art Robbins Instruments Phoenixliquid handling robot. Crystals were grown at 277 K using sitting dropsformed by mixing equal volumes (0.25 ml) of fHBP-G1 (7 mg/ml) incrystallization buffer (50 mM tris, pH 8.0) and a reservoir solution con-sisting of 20% PEG 3350 (polyethylene glycol, molecular weight 3350)and 0.2 M ammonium formate (pH 6.6). Crystals of fHBP-G1 belongto space group P21, with unit cell dimensions of a = 47.5, b = 99.6, c =58.0, and b = 97.8. The asymmetric unit contains two monomers with asolvent content of 53% (Matthews coefficient, 2.62 Å3 dalton−1). Allcrystals were mounted in cryoloops and cooled to 100 K for data col-lection without use of cryo-protectant.

Data collection and structure determination and analysisDiffraction data were collected at beamline 5.0.3 of the Advanced LightSource and were indexed and integrated with iMosflm and reducedwith Scala (1, 2). The structure of fHBP-G1 was determined at 1.9 Åby molecular replacement in Phaser (3) using as search model coor-dinates from Protein Data Bank (PDB) entry 2W80 (4). A single solu-tion was obtained for the two monomers with RFZ = 16.5, TFZ = 12.5,LLG = −85, and RFZ = 20.7, TFZ = 34.8, LLG = 1652, respectively.

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Rigid body and restrained refinement were carried out with Phenix (5)and manual model building in COOT (6). All crystallographic ma-nipulations were carried out with the CCP4 package (7). Data col-lection and refinement statistics are shown in table S5. The structuralsimilarity of fHBP-G1 was compared with existing fHBP structuresusing the PDBe FOLD server (http://pdbe.org/FOLD). The surface-accessible areas and/or interfaces were measured using the CCP4package (7) and the PDBe PISA server (http://pdbe.org/PISA).

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www.sciencetranslationalmedicine.org/cgi/content/full/3/91/91ra62/DC1Fig. S1. Amino acid sequence alignment of all the fHBP mutants designed.Fig. S2. Overview of the engineered surfaces of fHBP analyzed in this study.Fig. S3. (A to G) Molecular localization of point mutations introduced in the best candidates.Fig. S4. Western blot analysis of the meningococcal strains used in this study.Fig. S5. FACS analysis of N. meningitidis strains carrying the different fHBP subvariants.Fig. S6. Comparison of bactericidal titers elicited by G1 and GNA2091-G1-G1.Table S1. Sizes of surface-accessible areas subjected to mutagenesis.Table S2. Bactericidal activity of all the fHBP mutants.Table S3. (A and B) Analytical size exclusion chromatography and circular dichroism spectros-copy of fHBP wild-type and mutant proteins.Table S4. Data collection and refinement statistics for G1.Table S5. Association rate, dissociation rate, and equilibrium dissociation constants of G1 andfHBP wild-type to fH deduced by surface plasmonic resonance.References

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2. A. G. Barbour, B. I. Restrepo, Antigenic variation in vector-borne pathogens. Emerg. Infect.Dis. 6, 449–457 (2000).

3. X. Wu, Z. Y. Yang, Y. Li, C. M. Hogerkorp, W. R. Schief, M. S. Seaman, T. Zhou, S. D. Schmidt,L. Wu, L. Xu, N. S. Longo, K. McKee, S. O’Dell, M. K. Louder, D. L. Wycuff, Y. Feng, M. Nason,N. Doria-Rose, M. Connors, P. D. Kwong, M. Roederer, R. T. Wyatt, G. J. Nabel, J. R. Mascola,Rational design of envelope identifies broadly neutralizing human monoclonal antibodiesto HIV-1. Science 329, 856–861 (2010).

4. C. C. Huang, M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L. Stanfield, D. S. Dimitrov,B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, P. D. Kwong, Structure of a V3-containingHIV-1 gp120 core. Science 310, 1025–1028 (2005).

5. U. Katpally, T. M. Fu, D. C. Freed, D. R. Casimiro, T. J. Smith, Antibodies to the buried Nterminus of rhinovirus VP4 exhibit cross-serotypic neutralization. J. Virol. 83, 7040–7048(2009).

6. M. Pizza, V. Scarlato, V. Masignani, M. M. Giuliani, B. Aricò, M. Comanducci, G. T. Jennings,L. Baldi, E. Bartolini, B. Capecchi, C. L. Galeotti, E. Luzzi, R. Manetti, E. Marchetti, M. Mora,S. Nuti, G. Ratti, L. Santini, S. Savino, M. Scarselli, E. Storni, P. Zuo, M. Broeker, E. Hundt,B. Knapp, E. Blair, T. Mason, H. Tettelin, D. W. Hood, A. C. Jeffries, N. J. Saunders, D. M. Granoff,J. C. Venter, E. R. Moxon, G. Grandi, R. Rappuoli, Identification of vaccine candidates againstserogroup B meningococcus by whole-genome sequencing. Science 287, 1816–1820(2000).

7. X. Bai, J. Findlow, R. Barrow, Recombinant protein meningococcal serogroup B vaccinecombined with outer membrane vesicles. Expert Opin. Biol. Ther. 11, 969–985 (2011).

8. E. Murphy, L. Andrew, K. L. Lee, D. A. Dilts, L. Nunez, P. S. Fink, K. Ambrose, R. Borrow,J. Findlow, M. K. Taha, A. E. Deghmane, P. Kriz, M. Musilek, J. Kalmusova, D. A. Caugant,T. Alvestad, L. W. Mayer, C. T. Sacchi, X. Wang, D. Martin, A. von Gottberg, M. du Plessis,K. P. Klugman, A. S. Anderson, K. U. Jansen, G. W. Zlotnick, S. K. Hoiseth, Sequencediversity of the factor H binding protein vaccine candidate in epidemiologically rele-vant strains of serogroup B Neisseria meningitidis. J. Infect. Dis. 200, 379–389 (2009).

9. S. Bambini, A. Muzzi, P. Olcen, R. Rappuoli, M. Pizza, M. Comanducci, Distribution and ge-netic variability of three vaccine components in a panel of strains representative of thediversity of serogroup B meningococcus. Vaccine 27, 2794–2803 (2009).

10. V. Masignani, M. Comanducci, M. M. Giuliani, S. Bambini, J. Adu-Bobie, B. Aricò, B. Brunelli,A. Pieri, L. Santini, S. Savino, D. Serruto, D. Litt, S. Kroll, J. A. Welsch, D. M. Granoff, R. Rappuoli,

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M. Pizza, Vaccination against Neisseria meningitidis using three variants of the lipoproteinGNA1870. J. Exp. Med. 197, 789–799 (2003).

11. P. T. Beernink, J. A. Welsch, L. H. Harrison, A. Leipus, S. L. Kaplan, D. M. Granoff, Prevalenceof factor H-binding protein variants and NadA among meningococcal group B isolatesfrom the United States: Implications for the development of a multicomponent group Bvaccine. J. Infect. Dis. 195, 1472–1479 (2007).

12. D. M. Granoff, Review of meningococcal group B vaccines. Clin. Infect. Dis. 50, S54–S65(2010).

13. A. Mascioni, B. E. Bentley, R. Camarda, D. A. Dilts, P. Fink, V. Gusarova, S. K. Hoiseth, J. Jacob,S. L. Lin, K. Malakian, L. K. McNeil, T. Mininni, F. Moy, E. Murphy, E. Novikova, S. Sigethy,Y. Wen, G. W. Zlotnick, D. H. Tsao, Structural basis for the immunogenic propertiesof the meningococcal vaccine candidate LP2086. J. Biol. Chem. 284, 8738–8746(2009).

14. F. Cantini, D. Veggi, S. Dragonetti, S. Savino, M. Scarselli, G. Romagnoli, M. Pizza,L. Banci, R. Rappuoli, Solution structure of the factor H-binding protein, a survivalfactor and protective antigen of Neisseria meningitidis. J. Biol. Chem. 284, 9022–9026(2009).

15. M. C. Schneider, B. E. Prosser, J. J. Caesar, E. Kugelberg, S. Li, Q. Zhang, S. Quoraishi, J. E. Lovett,J. E. Deane, R. B. Sim, P. Roversi, S. Johnson, C. M. Tang, S. M. Lea, Neisseria meningitidisrecruits factor H using protein mimicry of host carbohydrates. Nature 458, 890–893(2009).

16. J. A. Welsch, R. Rossi, M. Comanducci, D. M. Granoff, Protective activity of monoclonalantibodies to genome-derived neisserial antigen 1870, a Neisseria meningitidis candidatevaccine. J. Immunol. 172, 5606–5615 (2004).

17. M. M. Giuliani, L. Santini, B. Brunelli, A. Biolchi, B. Aricò, F. Di Marcello, E. Cartocci,M. Comanducci, V. Masignani, L. Lozzi, S. Savino, M. Scarselli, R. Rappuoli, M. Pizza,The region comprising amino acids 100 to 255 of Neisseria meningitidis lipoprotein GNA1870 elicits bactericidal antibodies. Infect. Immun. 73, 1151–1160 (2005).

18. P. T. Beernink, J. A. Welsch, M. Bar-Lev, O. Koeberling, M. Comanducci, D. M. Granoff, Fineantigenic specificity and cooperative bactericidal activity of monoclonal antibodiesdirected at the meningococcal vaccine candidate factor H-binding protein. Infect. Immun.76, 4232–4240 (2008).

19. M. Scarselli, F. Cantini, L. Santini, D. Veggi, S. Dragonetti, C. Donati, S. Savino, M. M. Giuliani,M. Comanducci, F. Di Marcello, G. Romagnoli, M. Pizza, L. Banci, R. Rappuoli, Epitope mappingof a bactericidal monoclonal antibody against the factor H binding protein of Neisseriameningitidis. J. Mol. Biol. 386, 97–108 (2009).

20. P. T. Beernink, D. M. Granoff, Bactericidal antibody responses induced by meningococcalrecombinant chimeric factor H-binding protein vaccines. Infect. Immun. 76, 2568–2575(2008).

21. D. R. Davies, G. H. Cohen, Interactions of protein antigens with antibodies. Proc. Natl. Acad.Sci. U.S.A. 93, 7–12 (1996).

22. N. D. Rubinstein, I. Mayrose, D. Halperin, D. Yekutieli, J. M. Gershoni, T. Pupko, Computa-tional characterization of B-cell epitopes. Mol. Immunol. 45, 3477–3489 (2008).

23. P. Chakrabarti, J. Janin, Dissecting protein–protein recognition sites. Proteins 47, 334–343(2002).

24. V. Schijns, J. Brewer, “Immunopotentiators in Modern Vaccines” (IMV-II) held in Malaga,Spain, May 18–20, 2005. Vaccine 24, 5391–5392 (2006).

25. J. T. van Dissel, S. M. Arend, C. Prins, P. Bang, P. N. Tingskov, K. Lignau, J. Nouta, M. R. Klein,I. Rosenkrands, T. H. Ottenhoff, I. Kromann, T. M. Doherty, P. Andersen, Ag85B–ESAT-6adjuvanted with IC31 promotes strong and long-lived Mycobacterium tuberculosis specificT cell responses in naïve human volunteers. Vaccine 28, 3571–3581 (2010).

26. M. M. Giuliani, J. Adu-Bobie, M. Comanducci, B. Aricò, S. Savino, L. Santini, B. Brunelli,S. Bambini, A. Biolchi, B. Capecchi, E. Cartocci, L. Ciucchi, F. Di Marcello, F. Ferlicca,B. Galli, E. Luzzi, V. Masignani, D. Serruto, D. Veggi, M. Contorni, M. Morandi, A. Bartalesi,V. Cinotti, D. Mannucci, F. Titta, E. Ovidi, J. A. Welsch, D. Granoff, R. Rappuoli, M. Pizza,A universal vaccine for serogroup B meningococcus. Proc. Natl. Acad. Sci. U.S.A. 103,10834–10839 (2006).

27. R. Borrow, G. M. Carlone, N. Rosestein, M. Blake, I. Fevers, D. Martin, W. Zollinger, J. Robbins,I. Aaberge, D. M. Granoff, E. Miller, B. Plykaytis, L. van Alphen, J. Poolman, R. Rappuoli,L. Danzig, J. Hackell, B. Danve, M. Caulfield, S. Lambert, D. Stephens, Neisseria meningitidisgroup B correlates of protection and assay standardization—International meeting reportEmory University, Atlanta, Georgia, United States, 16–17 March 2005. Vaccine 24, 5093–5107(2006).

28. Acknowledgments: We thank L. W. Mayer (Centers for Disease Control and Prevention,Atlanta) for the U.S. strains, R. Borrow (Health Protection Agency, Manchester) for the UKstrains, E. R. Moxon (University of Oxford) for the MC58 and C11 strains, D. R. Martin(Institute of Environmental Science and Research, Porirua) for the NZ98/254 strains,and G. Hogg (University of Melbourne) for the 9615945 strain. We also thank P. Dormitzer,E. De Gregorio, I. Ferlenghi, and E. Settembre for critical reading of the manuscript; S. Marchifor the purification of the G1 protein; G. Spraggon (Genomics Institute of Novartis Re-search Foundation, San Diego, CA) for continuous support in crystallography and helpful

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discussions; C. Mallia for assistance in manuscript editing; and G. Corsi for artwork. Funding:L.B., F.C., and S.D. were supported by Ministero dell’Istruzione, dell’Università e dellaRicerca (grant FIRB-Proteomica RBRN07BMCT). Author contributions: M.S. designed thefHBP mutants, analyzed the data, and contributed to the writing of the paper; B.A. designedthe expression strategy of the mutants and analyzed the data; B. Brunelli and M.M.G. per-formed serological analysis and analyzed the data; S.S. coordinated the protein purificationand biochemical characterization work on the recombinant proteins; F.D.M. and E.P. per-formed gene expression of the mutants; D.V., L.C., and E.C. purified the recombinant proteins;M.J.B. and E.M. solved the crystal structure of G1; P.L.S., B. Brogioni, and M.T. were responsiblefor surface plasmonic resonance experiments; F.C. and S.D. were responsible for NMRexperiments; L.B. supervised the NMR characterization and contributed to the writing of thepaper; M.C. was responsible for the meningococcal molecular epidemiology; A.C., F.D.,P.G., and M. Pallaoro were responsible for antigen formulations; M.H. and V.N.-D. performedbiochemical characterization of candidates; M. Pizza conceived the experiments andcontributed to the writing of the paper; R.R. conceived the experiments and wrote the paper.

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Competing interests: The following patents are associated with this work: WO2009/104097(meningococcal fHBP polypeptides) and WO2011/024072 (hybrid polypeptides includingmeningococcal fHBP sequences). Accession numbers: Atomic coordinates of G1 can befound as PDB 2Y7S code.

Submitted 7 February 2011Accepted 28 June 2011Published 13 July 201110.1126/scitranslmed.3002234

Citation: M. Scarselli, B. Aricò, B. Brunelli, S. Savino, F. Di Marcello, E. Palumbo, D. Veggi,L. Ciucchi, E. Cartocci, M. J. Bottomley, E. Malito, P. Lo Surdo, M. Comanducci, M. M. Giuliani,F. Cantini, S. Dragonetti, A. Colaprico, F. Doro, P. Giannetti, M. Pallaoro, B. Brogioni, M. Tontini,M. Hilleringmann, V. Nardi-Dei, L. Banci, M. Pizza, R. Rappuoli, Rational design of ameningococcal antigen inducing broad protective immunity. Sci. Transl. Med. 3, 91ra62 (2011).

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