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hysicochemical characterisation of glycoconjugateaccines for prevention of meningococcal diseases
ngela Bardotti, Giovanni Averani, Francesco Berti, Stefania Berti,aleria Carinci, Sandro D’Ascenzi, Barbara Fabbri, Sara Giannini,ldo Giannozzi, Claudia Magagnoli, Daniela Proietti, Francesco Norelli,ino Rappuoli, Stefano Ricci, Paolo Costantino ∗
ovartis Vaccines and Diagnostics Srl, Via Fiorentina 1, 53100 Siena, Italy
eceived 5 November 2007; received in revised form 23 December 2007; accepted 11 January 2008vailable online 15 February 2008
KEYWORDSGlycoconjugatevaccines;Physicochemicalcharacterisation;
Summary Bacterial capsular polysaccharides covalently linked to an appropriate carrier pro-tein represent the best tool to induce a protective immune response against a wide range ofbacterial diseases, such as meningococcal infections. We describe here the physico-chemicalcharacterisation of glycoconjugate molecules designed to prepare a vaccine against Neisseriameningitidis serogroups A, C, W135 and Y. The use of a selective conjugation chemistry resulted
Neisseriameningitidis
in well characterised, reproducible and traceable glycoconjugate that can be consistentlymanufactured at large scale.
A pool of physical and spectroscopic methods was used to establish glycosylation ratio, iden-tity, molecular weight profiles, integrity of carrier protein and sites of glycosylation, assuring
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ntroduction
acterial meningitis, an infection of the membranes anderebrospinal fluid surrounding the brain and spinal cord,
s a major cause of death and disability worldwide. Threerganisms are responsible for most cases of bacterial menin-itis: Neisseria meningitidis, Haemophilus influenzae type bnd Streptococcus pneumoniae. N. meningitidis is classified∗ Corresponding author. Tel.: +39 0577 243324;ax: +39 0577 245307.
E-mail address: [email protected] (P. Costantino).
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264-410X/$ — see front matter © 2008 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2008.01.022
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nto serogroups based on the immunological reactivity ofhe capsular polysaccharide. Serogroups A, B, C, W135 andand recently X account for the majority of meningococ-
al disease [1—4]. Vaccines composed of purified capsularolysaccharides have been available since 1976, howeverhey have not been widely used because they are notmmunogenic among infants or young children, the age groupt highest risk of disease; for this reason conjugate vaccinesave been developed during the last 25 years [5—9].
In our laboratories we have developed and brought to thearket glycoconjugate vaccines against meningitis causedy H. influenzae type b (Vaxem-Hib®) and N. meningitidisroup C (Menjugate®). We used a similar methodology toevelop three further glycoconjugate vaccines against N.
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meningitidis group A, W135 and Y with the aim to finallyproduce a combination that could protect infants againstmeningococcal disease caused by serogroups A, C, W135 andY.
In all cases the vaccines are based on oligosaccha-rides conjugated to the carrier protein CRM197 a non-toxicmutant of diphtheria toxin [10]. Extensive physicochemi-cal characterisation of glycoconjugates has been performedusing a number of technologies: NMR spectroscopy toestablish the identity and precise evaluation of O-acetyldistribution of conjugated CPSs [11,12], size exclusion chro-matography coupled to multi-angle light laser scatteringdetection (SEC—MALLS) for evaluation of molecular massand size distribution [13], circular dichroism (CD) and flu-orescence spectroscopy for carrier conformational analysis[14], glycosylation sites mapping by LC—MS to assess thecovalent linkage for each lysine involved in the conjugationreaction.
Materials and methods
Samples
CRM197 and meningococcal serogroups A (MenA), C (MenC),W135 (MenW) and Y (MenY) glycoconjugates were producedby Manufacturing facilities of Novartis Vaccines and Diagnos-tics (Siena, Italy).
Circular dichroism
300 �l of sample were transferred into Amicon Ultra-15 spincolumn (Millipore, 10,000 Da cut-off) with 14 ml of 10 mMsodium phosphate, pH 7.2 and spun at 3400 g at 15 ◦C, for20 min. The sample volume after-spin was about 250 �l. Thespin process was repeated 5 times by adding 14 ml sodiumphosphate 10 mM pH 7.2 to the sample each time. Sampleswere diluted to a protein concentration of approximately1.0 mg/ml for near UV CD and 0.3 mg/ml for far-UV CD. CDmeasurements were performed with a spectropolarimeter(JASCO J-710). For near UV CD the samples were scannedeight times from 260 to 180 nm at the rate of 100 nm/minand averaged. The sample compartment was kept at roomtemperature. The light path of quartz cuvette was 0.05 cm.A blank run, using 10 mM sodium phosphate buffer pH 7.2,was subtracted from the experimental spectra for cor-rections. All spectra were converted into molar ellipticity(Mol. Ellip. in degree × cm2 × dmol−1 × 103), by using a meanresidue molecular mass of 109 Da. The fractional percentageof secondary structure was calculated using three differentalgorithms, referred to as CONTIN, SELCON and CDSSTR bycomputer fitting to a library of CD spectra of proteins ofknown structure and then averaging the results. Near UV CDmeasurement were performed scanning eight times from 320to 250 nm at the rate of 100 nm/min and averaged. The lightpath of quartz cuvette was 1.0 cm.
Quantification of MenW and MenY by HPAEC-PAD
MenW and MenY samples (saccharide concentration of1—2 �g/ml) were treated with trifluoroacetic acid (TFA)
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ith a final concentration of 2 M. Samples were heated at00 ◦C for 2 h in a closed screw-cap test tube, then cen-rifuged to combine the condensate with the bulk liquid andried on a Speed Vac concentrator fitted with a refriger-ted condensation trap (Savant SC110) under vacuum forh. Samples to be analysed were first dissolved in distillednd degassed water and then filtered (0.22 �m). Analysisf the hydrolysed products was performed using a DionexX-500 or DX-600 chromatography system. The instrumentas equipped with a CarboPac PA1 column (4 mm × 250 mm)
n combination with a CarboPac PA1 guard column. Sepa-ation was performed with a flow rate of 1 ml/min usingsocratic elution of NaOH 15 mM of 20 min, following by
regeneration step with NaOAc 50 mM/NaOH 200 mM for0 min. The effluent was monitored using an electrochemi-al detector in the pulsed amperometric mode with a goldorking electrode and an Ag/AgCl reference electrode. A
riple-potential waveform was applied using the follow-ng settings: E1 = 0.05 V, t1 = 400 ms; E2 = 0.75 V, t2 = 200 ms;3 = 0.15 V, t3 = 400 ms. Integration occurs from 200 to 400 msuring E1 application. The resulting chromatographic dataere integrated and processed using Chromeleon software.alibration curves, treated as samples, were set up withalactose and with glucose (Fluka), respectively, for MenWnd MenY, in the range of 0.5—4.0 �g/ml.
MR
amples of glycoconjugates MenA, MenC, MenW and MenYor NMR analysis were freeze—dried and dissolved in deu-erium oxide (D2O—–Aldrich) to a uniform concentration.ata acquisition procedure: 1H NMR experiments wereecorded at 25 ± 0.1 ◦C on Bruker 600 MHz spectrometer andsing 5-mm NMR probes (Bruker). 1H NMR spectra werebtained in quantitative matter using a total recycle timeo ensure a full recovery of each signal. For data acquisi-ion and processing XWINNMR version 3.5 software packageBruker) was used. Mono-dimensional (1D) proton NMR spec-ra were collected using a standard one-pulse experiment.
EC—MALLS
etermination of dn/dcn/dc was measured with an interferometric refractometerOptilab DSP, Wyatt Technology Corp.) previously cali-rated with NaCl (Merck). A series of five different sampleoncentrations ranging between 0.1 and 0.5 mg/ml (mgaccharide + mg protein/ml) were prepared and injectedhrough the refractometer, starting with the lowest concen-ration. Protein content was determined using bicinchoniniccid assay [15]. The saccharide content was determinedsing the resorcinol assay [16] in the case of MenC gly-oconjugate, a chromatographic method [17] in the casef MenA while for MenW and MenY the chromatographicethod described in Quantification of MenW and MenY by
PAEC-PAD was used. All sample solutions were prepared inodium phosphate buffer 10 mM pH 7.2. Values of dn/dc werealculated using the software DNDC supplied by the manu-acturer. The procedure was repeated on four different lotsor MenC and one lot for MenA, MenW and MenY.
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hromatographic conditions and determination ofolecular mass by SEC—MALLShe SEC—MALLS system consisted of an Alliance 2695 solventelivery module (Waters, Millipore) and Ultrahydrogel 1000nd 250 analytical columns connected in series. The glyco-onjugate samples were analysed undiluted, at a flow rate of.8 ml/min. Scattered light intensities were measured usingDawn EOS multi-angle light scattering photometer (Wyatt
echnology Corp). Data were analysed using the softwareupplied by the manufacturers (ASTRA; Wyatt technologyorp.). Dual detection with in-line mass and light scatteringetectors allows determination of absolute molecular massccording to R(�) = K*McP(�)[1 − 2A2McP(�)], where R(�) ishe excess Rayleigh ratio, K* the polymer constant for aarticular scattering system, M the molecular mass, c theolute concentration (g/ml), P(�) the form factor related tohe mean square radius rg of the particle and A2 is the secondirial coefficient, a measure of solute—solvent interaction,hich to a first approximation can be taken as zero.
luorescence spectroscopy
luorescence spectra were obtained with a LS50B spectroflu-rimeter (PerkinElmer) at room temperature, in 1-cm quartzell (Hellma GmbH). Samples were diluted in 10 mM sodiumhosphate pH 7.2 at a protein concentration ranging from 20o 40 �g/ml in the case of CRM197, and from 40 to 80 �g/mlor the glycoconjugates. Excitation wavelength of 280 or95 nm were used, with a band pass of 4.25 nm for both exci-ation and emission monochromators. At � = 295 nm, due toignal saturation, samples were diluted three times further.luorescence spectra were corrected by subtracting the cor-esponding base lines obtained with buffer alone. Values ofmax obtained were accurate to ±1.0 nm.
lycosylation sites
everse-phase chromatography of tryptic digest ofRM197 and conjugates0 �l of enzymatic digest (see Trypsin digestion) werenjected in a Vydac C4 column, 5 �, 300 angstrom,50 mm × 2.1 mm, at a flow rate of 0.2 ml/min. The elu-nt were: A = water + 0.1% formic acid; B = acetonitrile + 0.1%ormic acid. The gradient was: B from 2% to 32% in 60 min,rom 32% to 62% in 10 min, from 62% to 95% in 5 min, 95%or 5 min, return to initial conditions. The instrument was aaters Alliance 2690 with a 996 PDA detector. The process-
ng software was MassLynx.
eduction and carboxymethylation of cysteinehe samples were diluted at protein concentration ofmg/ml in 10 mM sodium phosphate pH 7.2 and trans-
erred into a Microcon YM-30 (Millipore) device (30,000 Daut-off); spun at 4000 g for 20 min at 20 ◦C. 500 �l of guani-ine HCl 6 M buffer and 30 �l of dithiothreitol (DTT) 1 Mere added and samples were flushed with N2 for 1 min to
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xclude oxygen reaction. Reduction was carried out at 60 Cor 1 h and stopped by cooling samples at 4 ◦C. Then sam-les were added with 60 �l of iodoacetic acid 1 M, flushedith N2 for 1 min, maintained at room temperature, in theark, for 30 min. Purification of samples from reagents wasuti1c
A. Bardotti et al.
erformed by ultrafiltration with Vivaspin (Sartorius Viva-cience) (5000 Da MWCO) reconstituting with ammoniumcetate buffer 5 mM pH 6 at initial protein concentrationf 1 mg/ml.
rypsin digestionhe samples were digested with Porcine Pancreas Modifiedrypsin (Promega), in Tris 1 M pH 8 buffer, in a ratio of 1/50w/w) between enzyme and protein, at a temperature of7 ◦C for 18 h. Digestion was terminated by freezing theamples.
sp-N digestionhe samples were digested with Asp-N (Boehringerannheim), in 30 mM Tris, pH 8, in a ratio of 1/50 (w/w)etween enzyme and protein, at a temperature of 37 ◦C for8 h. Digestion was terminated by freezing the samples.
nrichment of oligosaccharides-containing peptides byEC—MS analysis0 �l of digested samples (dried by vacuum and re-dissolvedn 65 �l of ammonium acetate buffer 5 mM pH 6) werenjected in a Superdex Peptide PC 3.2/30, running with aow of 50 �l/min, with 30% ammonium acetate 5 mM pH+ 70% acetonitrile as eluent. Q-Tof (Micromass, Manch-ster, UK) mass spectrometer was tuned as follows: function= ESI source in positive mode, capillary 3000 V, cone 30 V,ass range 520—2000 m/z; function 2 = capillary 3000 V,
one 200 V, mass range 100—500 m/z. Fractions containingon fragments corresponding to MenC (316 m/z), MenW eenY (274 m/z) or MenA (284 m/z) oligosaccharides wereollected. The pools of glyco-peptides mixture were driedy vacuum.
cid hydrolysis and RP-MS analysislyco-peptides were acid hydrolysed with HCl 0.1 M, at0 ◦C for 4 h. Then they were dried by vacuum and dis-olved in 250 �l of ammonium acetate 5 mM pH 6. Thehromatography was performed with Vydac C4 column,�, 300 angstrom, 250 mm × 2.1 mm, at a flow rate of.2 ml/min, injecting 50 �l of samples. The eluents were:= water + 0.1% formic acid; B = acetonitrile + 0.1% formiccid. Gradient with eluents was: B from 5% to 60% in 30 min.he flow was splitted to Q-Tof (Micromass, Manchester,K) mass spectrometer (20 �l/min) and tuned as follows:SI source in positive mode, capillary 3000 V, cone 30 V,ass range 200—4000 m/z. For MS/MS experiments colli-
ion energy was set according to a program of charge stateecognition (from 10 to 35 V).
ass spectrometric analysis of CRM197 inon-denaturing conditionssample of CRM197 at 1 mg/ml (BCA concentration) in wateras ultrafiltered on a Microcon 30 (Millipore) for 15 min at0 ◦C and 10 000 g. The treatment was repeated three times,
sing ammonium acetate 5 mM pH 6.8 buffer to reconstitutehe retentate. The sample was then injected with directnfusion in a Q-Tof micro (Micromass), at a flow rate of0 �l/min. The mass was tuned as follows: ESI positive mode,apillary 3000 V, sample cone 30 V, desolvation temperature
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Glycoconjugate vaccines for prevention of meningococcal d
150 ◦C, source temperature 80 ◦C, scan range from 1000 to7000 Da.
Results
Conjugation strategy
The described glycoconjugate antigens consist of MenA,MenY, MenW and MenC oligosaccharides of interme-diate chain length covalently linked to the proteincarrier CRM197 via an adipic acid spacer. The meningo-coccal capsular polysaccharides have the followingchemical structure: MenA: →6)-�-D-ManpNAc(3/4OAc)-(1 → OPO3→; MenC: →9)-�-D-Neup5Ac(7/8OAc)-(2→;MenY: →6)-�-D-Glcp-(1 → 4)-�-D-Neup5Ac(9OAc)-(2→;MenW: →6)-�-D-Galp-(1 → 4)-�-D-Neup5Ac(9OAc)-(2→ [7].The steps used to generate the glycoconjugate vaccineare shown in Figure 1Fig. 1. The oligosaccharides aregenerated through mild acid hydrolysis followed by sizingvia tangential flow ultrafiltration and/or ionic exchangechromatography aimed at restricting the polydispersion.In the case of MenW and MenY it has been demonstratedthat the acid hydrolysis generates only NeuNAc residues atreducing ends, indicating that involves selectively the �2—6 linkages between sialic acid and galactose or glucose,respectively [18]. Subsequently they are activated viareductive amination followed by active ester derivatization[19—21]. In the final step, the activated oligosaccharidesreact with available amino groups on the surface of theprotein (� amino groups of lysine and the N-terminal aminogroups) to form the conjugate vaccine, which consists of apopulation of glycoforms for which the average propertiescan be measured.
Physicochemical characterisation
Degree of glycosylationThe degree of glycosylation expressed as saccharide toprotein ratio (w/w) has been determined using a combi-nation of colorimetric and chromatographic assays. In allglycoconjugates the protein content was determined usingbicinchoninic acid assay [15]. The saccharide content inMenC glycoconjugate was determined using the resorcinolassay [16], while more specific chromatographic assays weredeveloped for MenA [17], MenY and MenW. The saccharideto protein ratios resulted to be in the range of 0.5—0.6 forMenA and MenC conjugates, 0.6—0.7 for MenY while in thecase of MenW we obtained values of 1.0. The higher degreeof glycosylation in Men W conjugates was confirmed also bySEC—MALLS analysis.
NMR: identity and O-acetylation patternThe structure of the saccharide component of the glyco-conjugates was investigated by use of 1H NMR. In Fig. 2acomparison between different lots of glycoconjugates ofMenA, MenC, MenW135 and MenY is shown, demonstrating
profile consistency. All NMR proton assignments are consis-tent with published data [12]. Comparison of the protonchemical shift values used for identity analysis of MenA,MenC, MenW and MenY components shows good lot to lotconsistency (Table 1). In addition to NMR spectra and tabularfbtci
igure 1 Conjugation strategy of MenA, C, W and Y glycocon-ugates: steps used to generate the vaccines are shown.
omparison of the chemical shift values, the O-acetylationercentage of glycoconjugates was evaluated using differ-nt approaches [11]. In MenA conjugate we have consideredhe integral, as compared to H1, of the proton at position C-2hose chemical shift is influenced by the O-acetylation sta-
us of the position C-3 (HManNAc-3OAc2 , HManNAc-4OAc
2 , HManNAc-deOAc2 ).
able 2, panel A shows the distribution of the MenA-acetylation as determined by the percentage of the dif-
erent H2 species. The total O-acetylation status is givenManNAc-3OAc ManNAc-4OAc
y the sum of H2 and H2 signal integrationshat ranges between 76.6% and 84.6% indicating that theonjugation process consistently retains the O-acetyl groupsn the saccharide structures.
2288 A. Bardotti et al.
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In the MenC conjugate O-acetylation was estimatedy the integration of −NHCOCHNeuNAc-7OAc
3 signal (methylrotons of N-acetyl groups at position C-5 of the-acetyl-neuraminic residues O-acetylated at C-7) andNHCOCHNeuNAc-8OAc
3 signal (methyl protons of N-acetylroups at position C-5 of the N-acetyl-neuraminic residues-acetylated at C-8), in comparison to H3ax (proton atosition C-3 axial of the N-acetyl-neuraminic residues).he total O-acetylation level is obtained by the sumf NCOCHNeuNAc-7OAc
3 and NCOCHNeuNAc-8OAc3 peak integrations,
anging between 67.9% and 70.5% (Table 2, panel B).n MenW and MenY O-acetylation was estimated by thentegration of HNeuNAc-7OAc
7 signal (proton at position C-7f the N-acetyl-neuraminic residues O-acetylated at C-7)nd HNeuNAc-9OAc
9 signal (one of the two protons at posi-ion C-9 of the N-acetyl-neuraminic residues O-acetylatedt C-9), in comparison to HGal
1 or HGlc1 (proton at position
-1 of the galactose, for MenW, or glucose, for MenY,esidues). The total O-acetylation level is obtained by theum of HNeuNAc-7OAc
7 and HNeuNAc-9OAc9 signal integrations, rang-
ng between 41.0% and 41.7% for MenW and 34.9% and
7.3% for MenY (Table 2, panels C and D). The measured-acetylation levels are consistent with those reportedor the un-conjugated polysaccharide indicating that thedentity and structural integrity of the different saccha-ide components was retained after conjugation. Moreover
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lots of glycoconjugates of MenA, MenC, MenW and MenY.
atch-to-batch comparison indicated a high degree of con-istency in the conjugates manufacturing process.
olecular mass distribution by SEC—MALLSimensional characterisation was performed by SEC—MALLS,technique for the determination of absolute molecularass that is no longer dependent on the elution behaviour
f these macromolecules and capable to detect very lowmount of high molecular weight substances. In light scat-ering detection the amount of light scattered is directlyroportional to the product of the polymer molecular mass,he concentration and to the square of the specific refrac-ive index value (dn/dc). In order to have accurate resultsf molecular mass dn/dc has to be determined for eacholymer. Values obtained for the glycoconjugates of MenA,enC, MenW and MenY are reported in Table 3. The results
howed different dn/dc values for different conjugates, con-rming the importance of the experimental determinationf dn/dc for each antigen.
Molecular mass values and polydispersity of all glyco-
onjugates are also reported in Table 3. MenA, MenC andenY glycoconjugates had average molecular mass between0 and 90 kDa, while MenW had a higher average molecu-ar mass (110 kDa), which is in agreement with its higherlycosylation ratio.
Glycoconjugate vaccines for prevention of meningococcal diseases 2289
Table 1 Chemical shift values (ppm) of the meningococcal conjugates saccharide moieties
Peaks Lots Reference values, ıM
A B C
MenAH1 5.412 5.429 5.457 5.459HManNAc-3OAc
3 + HManNAc-4OAc4 5.146 5.157 5.192 5.185
HManNAc-3OAc2 4.538 4.551 4.586 4.582
OCOCH3 2.048 2.061 2.095 2.091NCOCH3 2.017 2.030 2.063 2.060
MenCH3eq 2.786 2.769 2.768 2.744OCOCH3 2.184 2.166 2.166 2.164NCOCHNeuNAc-8OAc
3 2.086 2.069 2.067 2.072NCOCHNeuNAc-7OAc
3 1.988 1.970 1.970 1.971H3ax 1.711 1.685 1.694 1.698
MenWHGal
1 5.058 5.059 5.058 5.044HNeuNAc
3eq 2.874 2.875 2.874 2.889OCOCH3 2.150 2.151 2.149 2.149NCOCH3 2.089 2.090 2.089 2.039HNeuNAc
3ax 1.669 1.669 1.668 1.709
MenYHGlc
1 5.012 5.034 5.043 5.081HNeuNAc
3eq 2.869 2.887 2.882 2.898OCOCH3 2.135 2.152 2.157 2.173NCOCH3 2.025 2.042 2.047 2.113HNeuNAc
3ax 1.690 1.707 1.708 1.696
The reference values of the NMR analysis were defined for five NMR peaks for each antigen, which are used to confirm the identity of) wentern
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the saccharide residues. The chemical shift reference values (ıMthey are the sample mean obtained from analysis of several lots i
Conformational analysis by CD and fluorescence ofCRM197 in conjugatesThe attachment of highly charged polysaccharide chainson the surface of the protein may induce some confor-mational changes in the protein carrier altering secondaryand tertiary structure. CD and fluorescence spectroscopyare well known methods to establish secondary and ter-tiary structure of the proteins. The CD profile obtainedin the far-UV region represents the averaged spectrum ofall the secondary structural elements (�-helix or �-sheet,turns and unordered) present in a protein, each one pro-ducing a distinct spectrum. Mathematical deconvolution isthen usually applied to establish the secondary structuralcomponents. On the other hand, near UV CD spectra showthe microenvironment around aromatic residues (Trp, Tyrand Phe) providing overall information on the protein’s ter-tiary structure. Fluorescence of a protein is related to thepresence of aromatic amino acids (Trp, Tyr and Phe). Excita-tion at 280 nm causes a fluorescence emission spectrum thatis indicative of the degree of solvent exposure of the sidechains of tryptophan residues and, to a lesser extent, tyro-
sine residues. Tryptophan fluorescence can be selectivelyevaluated using an excitation at 295 nm [14]. Both proper-ties relate with the tertiary structure of the protein. Wehave compared far and near UV CD and fluorescence prop-ebjC
re selected based on technical experience on NMR analysis andally produced.
rties of CRM197 and MenC glycoconjugate, while for MenA,enW and MenY glycoconjugates we have evaluated onlyuorescence properties. Far-UV CD for CRM197 and CRM-enC samples was recorded from 260 to 180 nm. The CRM197
pectra had a positive band centered at around 192 nm andpositive to negative crossover at 201 nm (Table 4). Two
roughs were observed at 209 and 222 nm, respectively. TheD ratio of 209—222 was slightly above 1. All these charac-eristics CD features suggest the protein is an ‘‘� + �’’ typef protein made of separated �-helix and �-sheet struc-ure region. Far-UV CD spectra for samples of CRM-MenCots were slightly different only for CD ratio because the09 nm band was less intense than the 222 nm band. Esti-ation of secondary structure content using the averaged
esults from three different algorithms (CONTIN, SELCONnd CDSSTR) for both CRM197 and CRM-MenC is shown inable 5. The CRM197 lots contained about 24—29% �-helicaltructure, 20—25% �-sheet structure; these results are inood agreement with those obtained from the X-ray crys-allographic analysis in which diphtheria toxin was foundo contain about one-third �-helical structure [22]. No rel-
vant differences in the secondary structure were foundetween CRM197 and CRM component of the MenC glycocon-ugate. Near UV CD was recorded from 250 to 320 nm. All theRM-MenC samples exhibited the same characteristic pro-
2290 A. Bardotti et al.
Table 2 O-Acetylation degree of different meningococcal conjugates
Signals Lots
A B C
(A) MenA: calculated assuming the H1 signal as reference at 100%HManNAc-3OAc
2 66.4 69.2 72.2HManNAc-4OAc
2 10.2 8.7 12.4HManNAc-deOAc
2 21.9 27.8 12.4
Total O-acetylation (HManNAc-3OAc2 + HManNAc-4OAc
2 ) 76.6 77.9 84.6
(B) MenC: calculated assuming the HNeuNAc3ax signal as reference at 100%
NCOCHNeuNAc-7OAc3 36.0 36.2 38.1
NCOCHNeuNAc-8OAc3 33.2 31.7 32.4
Total O-acetylation (NCOCHNeuNAc-7OAc3 + NCOCHNeuNAc-8OAc
3 ) 69.2 67.9 70.5
(C) MenW: calculated assuming the HGal1 signal as reference at 100%
HNeuNAc-7OAc7 8.3 8.1 7.9
HNeuNAc-9OAc9 32.7 33.6 33.8
Total O-acetylation (HNeuNAc-7OAc7 + HNeuNAc-9OAc
9 ) 41.0 41.7 41.7
(D) MenY: calculated assuming the HGlc1 signal as reference at 100%
HNeuNAc-7OAc7 5.3 2.6 3.1
HNeuNAc-9OAc9 32.0 33.3 31.8
Total O-acetylation (HNeuNAc-7OAc7 + HNeuNAc-9OAc
9 ) 37.3 35.9 34.9
file: a Trp-absorbing band around 292 nm, Tyr-Trp-absorbingbands around 270—280 nm, and a Phe-absorbing band around260 nm. This indicates that all analysed lots had compara-ble conformation. The intensities of the bands were slightlylower than those recorded for CRM197 alone, suggestingthat residues were more exposed to the environment inthe conjugate. The fluorescence spectra for MenA, MenC,MenW and MenY conjugates at excitation wavelengths of280 and 295 nm were recorded. Fluorescence maxima for allproducts were recorded (Table 6): the emission maxima ofglycoconjugates were higher than CRM197, confirming moreexposed aromatic residues in glycoconjugates with respectto carrier alone.
Mass spectrometry characterisation: determination ofglycosylation sites by LC—MS and LC—MS/MSIn order to analyse the distribution of glycosylation sites inCRM197 we have analysed our glycoconjugates with LC-MS
Table 3 Specific refractive indices (dn/dc), weight aver-age molecular masses (Mw) of conjugates and Mw/Mn valuesdetermined by SEC—MALLS
Sample dn/dc Mean, Mw (kDa) Polydispersity,Mw/Mn
CRM-MenA 0.163 88.5 1.012CRM-MenC 0.190 85.2 1.011CRM-MenW 0.159 110.1 1.049CRM-MenY 0.201 84.6 1.050
after trypsin digestion. Trypsin is expected to cleave pro-teins at level of lysines and arginin on the C-terminal side,except when they are chemical modified or followed by aproline. Considering that our conjugation chemistry involveslysine residues of the carrier protein, the idea was to iden-tify those peptides bearing a glycosylated lysine. In orderto guide this search we generated a list of all possible pep-tides with a modified lysine that could be formed based onCRM197 sequence and the specificity of the enzyme (Table 7first column). The primary structure of CRM197 was con-firmed by peptide mapping. All tryptic digests were analysedby LC—MS. The analysis resulted in sequence coverage of89%, reaching 100% overlapping results from Asp-N diges-tion. Comparison between tryptic digests of CRM197 andglycoconjugates confirmed that trypsin worked well in allcases. The strategy applied to identify the glycopeptides isdescribed in Figure 3Fig. 3.
Table 4 Main features of far-UV CD for MenC conjugateand CRM197 samples
Sample Lots Crossover(nm)
Positivemax. (nm)
CD ratio209/222
CRM-MenC A 201 192 0.91B 201 192 0.91C 201 192 0.90
CRM197 A 201 192 1.03B 201 192 1.03C 201 192 1.03
Glycoconjugate vaccines for prevention of meningococcal diseases 2291
Table 5 Secondary structure of MenC glycoconjugate in comparison with CRM197, estimated with far-UV CD data
Sample Lots % �-helix % �-sheet % turn % unordered
CRM-MenC A 22 25 20 34B 25 21 20 34C 24 21 19 35
CRM197 A 24 25 22 29
csgaafmMbbCftfimcntpoapmaotlsot
B 29C 29
The first step consisted in trypsin digestion of conju-gates, followed by a SEC—MS purification of glycopeptides.Conjugated peptides elute earlier than the majority ofthe normal peptides because the average size of anoligosaccharides-containing peptide varies from 2000 to5000 Da, whereas the average size of the majority of thenominal tryptic peptides is less than 3000 Da. In additionthe significantly larger hydrodynamic volume of the pep-tides linked to oligosaccharides facilitates size separation.The SEC—MS chromatograms were acquired under conditionsthat produced fragmentation of the oligosaccharides to yieldcharacteristic fragment ions at 316 m/z, representing sialicacid for MenC, 274 m/z representing sialic acid (with loss of2 water molecules) for MenW and MenY, and 284 m/z rep-resenting mannosammine-6-phosphate for MenA (with lossof 1 water molecule). Accordingly, these fractions were col-lected and pooled for further analysis. Sample heterogeneityarising from the polydispersion of oligosaccharide chainswas then reduced by mild acid hydrolysis to remove alloligosaccharide repeating units except the last one, whichis covalently attached to the linker through an amide boundresistant to hydrolysis conditions. The expected structure ofthe linker-carbohydrate adducts that are generated by thishydrolysis step is shown in Figure 4Fig. 4 and the expectedmass increment value for the corresponding glycopeptidesis 412.1 Da for MenA, 420.4 Da for MenC (with loss of 1 watermolecule), 582.2 Da for MenW and MenY. The mixture ofpeptides bearing the linker-carbohydrate adducts was thenanalysed by RP-LC—MS and glycopeptides with the expectedmass value increment were identified. In Figure 5Fig. 5 an
example of peptide extraction for MenY conjugate is shown:the triple charged ion 1016.5 m/z, representing the massof peptide 494—516 where the lysine 498 is coupled to thelinker-carbohydrate adduct, is extracted from the total ionctil
Table 6 Fluorescence emission maxima at two different excitati
Sample Emission maximum atexcitation, � = 280 nm;n = 3
S
CRM197 331.3 0CRM-MenA 336.3 2CRM-MenC 337.8 1CRM-MenW 334.2 0CRM-MenY 334.2 0
n: number of determinations; S.D.: standard deviation.
22 22 2820 22 28
urrent (TIC) chromatogram (Fig. 5 window) and the masspectrum of this peptide is obtained. All other expectedlycopeptides have been extracted from the chromatogramnd their mass spectra obtained. As an example of results,ll peptides with the expected mass increments found inour different lots of CRM-MenW are shown in Table 7. Theethod has been applied also to the other glycoconjugates,enA, MenC and MenY, obtaining similar results. A num-er of lysines ranging between 29 and 34 were found toe statistically available for conjugation (the number ofRM197 lysines is 39), however about 19 were commonlyound in all lots and in all antigens, meaning that probablyhese residues are more available for conjugation. To con-rm the number of more reactive lysines, mass spectrometryeasurement of CRM197 were conducted in non-denaturing
onditions, to maintain native structure and evaluate theumber of positive charges, mainly due to lysines, availableo give the mass ions. After infusion in Q-Tof of a sample atH 6.8, without organic solvent and ion source temperaturef 80 ◦C, we observed three peaks corresponding to 16, 17nd 18 positive charges (Figure 6Fig. 6). The other minoreaks correspond to a distribution of complex with sucroseolecules (sucrose was used as stabilizer and remain also
fter extensive ultra filtration of the sample). The numberf lysines available to take positive charges in mass spec-rometry analysis is in good agreement with the number ofysines always found conjugated in site glycosylation analy-is, confirming their more reactive behaviour. Experimentsf MS/MS were performed on some glycopeptides, to confirmhe covalent linkage between the protein and the oligosac-
haride. As an example of methodology, MS/MS analysis ofhe N-terminal peptide of CRM-MenA is reported, reveal-ng the ion 413.2 m/z, due to a molecule formed by theinker plus N-acetyl-mannosammine-6-phosphate, beyondon wavelength (280 and 295 nm) for conjugates and CRM197
.D. Emission maximum atexcitation, � = 295 nm;n = 3
S.D.
.37 334.6 0.46
.91 339.5 0.65
.15 340.5 1.68
.96 337.1 0.31
.98 337.5 0.90
2292 A. Bardotti et al.
Table 7 Peptides with mass increment found in four different lots of CRM-MenW conjugate
MenW Observed (m/z)
Residue no. Peptide Theoretical (m/z) Lot A Lot B Lot C Lot D
G1(N-t) 1—10 787.82+ 788.3 788.3 788.3 787.3K10 1—33 1045.54+ 1046.0 1046.0 1046.0 1045.5K24 11—33 1069.13+ 1069.7 1069.7 1069.7 ndK33 11—39 964.44+ 965.5 965.7 965.7 964.8K37 34—39 626.82+ 626.2 626.2 626.2 626.8K39 34—51 874.13+ 874.5 874.5 874.5 874.1K59 52—76 1123.43+ 1123.7 1123.8 1124.1 ndK76 60—82 965.63+ nd nd nd ndK82 77—90 658.03+ 659.3 659.3 659.3 659.0K90 83—95 993.02+ nd nd nd ndK95 91—103 998.52+ 998.4 998.4 998.4 ndK95/103 91—104 709.03+ 709.4 709.4 709.4 709.1K103 96—104 800.42+ 800.8 800.8 800.8 800.4K104/125 104—126 1077.83+ 1078.2 1078.5 1078.1 1077.2K104 104—125 1025.73+ 1026.1 1026.1 1026.0 1025.2K125 105—126 1035.03+ 1035.4 1035.7 1035.7 1034.5K157 134—170 1189.24+ 1189.7 1189.7 1189.9 1188.8K172 171—173 942.5+ 942.9 942.9 942.8 942.4K212 211—214 537.32+ nd nd nd ndK214 213—216 530.32+ nd nd nd ndK216 215—221 700.92+ nd nd nd ndK221 217—227 611.63+ nd nd nd ndK227 222—229 752.92+ 752.8 752.8 752.8 752.5K229 228—236 808.92+ nd nd 808.8 ndK236 230—242 1024.92+ 1025.5 1025.5 1025.0 1024.5K242 237—244 737.32+ 737.7 737.7 737.7 737.4K244 243—264 806.84+ 807.1 807.0 807.0 806.4K264 245—299 1662.04+ nd nd nd ndK299 245—385 1956.68+ nd nd nd ndK299 265—385 1889.77+ nd nd nd ndK385 300—407 1734.17+ nd nd nd ndK419 408—440 1319.43+ 1320.0 1319.9 1320.0 1319.0K440 420—445 1090.13+ 1090.5 1090.8 1090.5 ndK445 441—447 693.32+ 693.7 693.7 693.7 693.3K447 446—455 840.92+ 841.4 841.4 841.4 ndK456 456—458 998.5+ nd nd nd ndK474 463—493 1005.54+ 1006.0 1006.0 1006.0 1005.3K498 494—516 1017.03+ 1017.3 1017.3 1017.3 1016.5K516 499—522 1070.83+ 1071.8 1071.8 1071.7 ndK522 517—526 855.92+ 855.9 855.9 855.9 nd
tt
D
PpacWo[
ctcoco
K526 523—534 1004.52+
K534 527—535 833.42+
he complete fragmentation pattern of the N-terminal pep-ide (Figure 7Fig. 7).
iscussion
olysaccharide-protein conjugate vaccines have beenroved to be an effective way to elicit protective immunity
gainst encapsulated bacteria [4,6,23]. The introduction ofonjugate vaccines against meningococcal serogroups A, C,135 and Y offers the real prospect to prevent large partf meningococcal disease in infants and other classes of age2,3].
oooap
1005.1 1005.1 1005.0 1004.1833.9 833.9 833.8 nd
Semi-synthetic glycoconjugates are complex moleculesomposed of protein and carbohydrate moieties linkedogether by chemical reactions which, depending on theonjugation chemistry, can involve different linking sitesn the surface of the protein and along the saccharidehain. Moreover the carbohydrate moiety is generally anligo or polysaccharide characterised by a polydispersion
f molecular weights which increases the heterogeneityf the resulting glycoconjugates. While biological assaysn these vaccines provide limited information on theirbility to induce protective immunity in humans, modernhysicochemical approaches can provide detailed structural
Glycoconjugate vaccines for prevention of meningococcal diseases 2293
Fgf
fitaa
tipbcbccgptp
Figure 3 Flow chart of glycosylation site analysis of meningo-coccal conjugates.
information on both the saccharide and protein moietiesand are suitable to confirm the consistency of manufactur-ing and identity with materials used in clinical trials thusallowing a tool to establish a link between structure andvaccine immunogenicity. The application of a selective con-jugation chemistry that avoids the formation of lattice likestructures and the use of carbohydrate moieties having arestricted distribution of chain length, reduces the complex-
ity of the glycoconjugates resulting in well defined productsthat can be carefully characterised. Our conjugation strat-egy is based on chain length reduction of the polysaccharidesmolecules followed by molecular weight selection of thefiohe
igure 4 Expected structures of linker-carbohydrate adductsenerated by sample treatment of meningococcal conjugatesor glycosylation analysis.
nal pool of oligosaccharides which are then activated athe reducing end and conjugated. The resulting conjugatesnd the carrier protein are well-defined products that aremenable to physico-chemical characterisation.
One of the key chemical parameters that has been showno influence the immunogenicity of glycoconjugate vacciness the degree of glycosylation [24]. We have shown that ourrocess is able to provide consistent degree of glycosylationetween different lots of each meningococcal serogrouponjugate. The structure of the carbohydrate antigens cane altered by the chemical conditions applied during theonjugation to the protein carrier and a potent tool toonfirm their structural identity on the resulting glycoconju-ates is 1H NMR spectroscopy. We have established a fingerrint for each meningococcal saccharide using as referencehe chemical shift value of five characteristic signals of theroton NMR spectrum of the respective un-conjugated puri-
ed capsular polysaccharides. When the proton NMR spectraf different lots of conjugates was compared we found aigh degree of consistency within the same serogroup and anxcellent matching of the chemical shift values with those
2294 A. Bardotti et al.
F ted fm chros sine
osdt[s
acg
aocoT
Fis
igure 5 Mass spectrum of the 494—516 glycopeptide extracild acid hydrolysis. In the window, TIC chromatogram (A) and
enting the mass of peptide 494—516 with a linker-containing ly
f the reference finger print, indicating also a high level oftructure identity. The retention of O-acetylation has beenemonstrated critical for the conservation of protective epi-opes in the case of meningococcal serogroup A conjugates25] while for other meningococcal conjugates has not beenhown relevant [26].
By 1H NMR spectroscopy we have monitored the O-cetylation status of the different meningococcal oligosac-harides showing a consistent retention of this functionalroup after conjugation.
gita
igure 6 CRM197 mass spectrum recorded in non-denaturing conons have been observed, corresponding to 18, 17 and 16 total posucrose molecules (present as protein stabilizer).
rom the RP-LC—MS chromatogram of MenY glycopeptides aftermatogram of the triple charged ion at 1016.5 m/z (B), repre-(K498).
The physicochemical properties of the protein carrier,lone and conjugated, have been studied mainly usingptical spectroscopy methodologies like fluorescence andircular dichroism. These techniques provide informationn the secondary and tertiary structure of the protein.he analysis of far-UV CD spectra indicated that conju-
ation to meningococcal group C oligosaccharides did notnduce significant conformational changes on CRM197. Onhe other hand fluorescence data indicated more exposedromatic residues in all the glycoconjugates with respectditions (ammonium acetate buffer at pH 7): only three majoritive charges. The minor peaks correspond to complexes with
Glycoconjugate vaccines for prevention of meningococcal diseases 2295
indicmpl
A
DcW
R
[
[
Figure 7 MS/MS analysis of N-terminal peptide of CRM-MenA;the linker plus an N-acetyl-mannosammine-6-phosphate; the coand attributed by ProteinLynx software (Micromass).
to carrier alone as a consequence of a more open con-formation adopted by CRM197 after conjugation. Thisobservation is in line with results of previous studiesperformed on various CRM197-oligosaccharides conjugates[14,27].
To increase the level of characterisation of our con-jugates we addressed the question whether there was apreferential involvement of some lysines of CRM197 in thereaction with the activated oligosaccharides. At this aim amodified peptide mapping strategy was developed to char-acterise the sites of covalent linkage between carrier andoligosaccharides. A range from 29 to 34 lysines per car-rier molecule resulted available for conjugation and amongthese about 19 were commonly found as the more reactive.
By applying tandem mass spectrometry (MS/MS) to ourglycosylated peptides it has been possible to reveal thepresence of the adducts formed by the linker plus thesugar residue involved in the conjugation thus confirming,as showed in the case of MenA, the covalent nature of thelinkage between the oligosaccharides and the carrier pro-tein.
In this paper we have described well-defined conjugatesof Men A, C, W and Y oligosaccharides to the carrier proteinCRM197. The selective conjugation chemistry and the appli-cation of advanced analytical techniques allowed a detailedcharacterisation of conjugates. The results obtained by thisset of analyses indicated a high degree of consistency ofthe manufacturing process for the meningococcal glycocon-jugates as well as the excellent retention of the structuralidentity of the oligosaccharides following conjugation. Thedescribed meningococcal A, C, W135, Y oligosaccharides-
protein conjugates are now in advanced clinical trial in theform of a quadrivalent combination. Results obtained indi-cate that the vaccine is immunogenic in all classes of ageincluding infants and induces protective titres of serum bac-tericidal antibodies [28—30].[
ated by arrow the ion 413.2 m/z, due to a molecule formed byete fragmentation pattern of the N-terminal peptide is shown
cknowledgement
edicated to Angela Bardotti a great scientist and wonderfulolleague who will remain for ever in our mind and heart.e are grateful to Giorgio Corsi for graphical work.
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