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Vaccine 33 (2015) 2646–2654

Contents lists available at ScienceDirect

Vaccine

j o ur na l ho me page: www.elsev ier .com/ locate /vacc ine

Development and characterization of Haemophilus influenzae type bconjugate vaccine prepared using different polysaccharide chainlengths

R. Rana, J. Dalal, D. Singh, N. Kumar, S. Hanif, N. Joshi, M.K. Chhikara ∗

MSD Wellcome Trust Hilleman Laboratories Pvt. Ltd., 2nd Floor, Nanotechnology Building, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India

a r t i c l e i n f o

Article history:Received 16 February 2015Received in revised form 4 April 2015Accepted 11 April 2015Available online 20 April 2015

Keywords:Conjugate vaccineHaemophilus influenzae type bPRP sizeReductive aminationImmunogenicity

a b s t r a c t

Capsular polysaccharide conjugates of Haemophilus influenzae type b (Hib) are important componentsof several mono- or multi-valent childhood vaccines. However, their access to the most needy people islimited due to their high cost. As a step towards developing a cost effective and more immunogenic Hibconjugate vaccine, we present a method for the preparation of Hib capsular polysaccharide (PRP)–tetanustoxoid (TT) conjugates using optimized PRP chain length and conjugation conditions. Reactive aldehydegroups were introduced into the polysaccharides by controlled periodate oxidation of the native polysac-charide, which were subsequently covalently linked to hydrazide derivatized tetanus toxoid by means ofreductive amination. Native polysaccharides were reduced to average 100 or 50 kDa polysaccharide and10 kDa oligosaccharides in a controlled manner. Various conjugates were prepared using Hib polysaccha-ride and oligosaccharide yielding conjugates with polysaccharide to protein ratios in the range of 0.25–0.5(w/w) and free saccharide levels of less than 10%. Immunization of Sprague Dawley rats with the conju-gates elicited specific antibodies to PRP. The low molecular weight PRP–TT conjugates were found to bemore immunogenic as compared to their high molecular weight counterparts and the PRP–TT referencevaccine.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Invasive diseases due to Haemophilus influenzae type b (Hib)infections include pneumonia, sepsis and meningitis with a highincidence in infants less than one year age [1]. Vaccines derivedfrom Hib capsular polysaccharides (Hib-PRP) are T-cell indepen-dent (TI) antigens and have been found to be poorly immunogenicin children less than two years of age [2–4]. In 1929, Avery andGoebel have demonstrated that after conjugation to a carrier pro-tein, antibodies were induced to polysaccharide in an animalmodel in a T-dependent (TD) manner [5]. These TD antigens areimmunogenic early in infancy, the immune response induced canbe boosted, enhanced by adjuvants, and is characterized by anti-body class switch and production of antigen-specific IgG [4,6–8].However, the first application of this concept to a vaccine was in1980 with the development of the first conjugate vaccine againstHib that was later licensed [9–12]. Many other glycoconjugate

∗ Corresponding author. Tel.: +91 11 30997755; fax: +91 11 30997711.E-mail address: [email protected] (M.K. Chhikara).

vaccines have since been developed against other bacterialpathogens [13–16].

Several different conjugation chemistries have been tested formaking commercial Hib vaccines [17–20], however, two mainapproaches based on different chemistry of conjugation have beentraditionally used. One is based on the random chemical activationof the saccharide chain followed by covalent binding with the pro-tein carrier obtaining a cross-linked structure. A second approachis based on the generation, by controlled fragmentation, of appro-priately sized polysaccharides which are then activated at theirterminal groups, usually with a linker molecule, and subsequentlyconjugated to the carrier protein obtaining a radial structure.

The optimal length of the carbohydrate chain remains a matterof considerable debate for developing glycoconjugate vaccine andvarious lengths have been reported to be required for generatingoptimal immune response for various vaccine candidates [21–24].However, the size of the saccharide must be sufficiently large toexpress epitopes representative of the native antigen. There are fewreports on the use of different Hib-PRP sizes in preparing Hib conju-gate vaccines. Diphtheria toxoid–coupled PRP of mean chain length8 or 20 repeat units (Dpo8 and Dpo20) were tested for immuno-genicity, and Dpo8 elicited poorer anti-PRP response in infants than

http://dx.doi.org/10.1016/j.vaccine.2015.04.0310264-410X/© 2015 Elsevier Ltd. All rights reserved.


R. Rana et al. / Vaccine 33 (2015) 2646–2654 2647

Dpo20 [12]. However, in a separate study it was shown that nodifferences in the human immune response was found for conju-gates made with varying chain lengths of Hib oligosaccharides thatwere monoterminally activated [25]. Hib dimer (four saccharide-units) conjugated to tetanus toxoid or to synthetic peptides is apoor immunogen in rabbit while Hib trimer (six saccharide-units) isimmunogenic [26]. A method had been described for fractionationof Hib oligosaccharides of varying length, which permits removal ofshort fragments unsuitable for conjugate vaccine preparation [27].However a detailed correlation of oligo- and poly-saccharide Hibconjugate immunogenicity is not much evident.

In this study we tested the effect of molecular size of polysac-charide used for conjugation, and amount of conjugate injected,on immunogenicity of Hib PRP–tetanus toxoid conjugates. We pre-pared and evaluated conjugates using oligosaccharides of average10 kDa as well as polysaccharides of average 50 kDa and 100 kDamolecular size. The immunogenicity of these conjugates has beenassessed in a rat model in comparison with two different licensedHib conjugate vaccines.

2. Materials and methods

Hib capsular polysaccharide (PRP) was prepared using meth-ods described previously [28] with few modifications (processcommunicated separately for publication). Tetanus toxoid (TT)was procured from Fabtech Technologies Ltd., India. 2-(N-morpholino) ethanesulfonic acid (MES), hydrazine monohydrate,N-(3-D-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-ride (EDC), sodium chloride, sodium cynoborohydride solution, d-glucose, d-ribose, iron chloride hexahydrate, 2,4,6-trinitrobenzenesulfonic acid (TNBS), copper sulfate, sodium dodecyl sulfate,sodium deoxycholate, sodium nitrate, bovine serum albumin(BSA) protein standards, and boric acid were purchased fromSigma Aldrich. Sodium hydroxide pellets, hydrochloric acid, FolinCiocalteu’s phenol reagent, disodium tartarate dihydrate, andacetohydrazide were purchased from Merck Inc. Sodium car-bonate anhydrous was purchased from S D Fine Chemicals Ltd.Sodium metaperiodate was purchased from SRL Chemicals. Orci-nol monohydrate was purchased from Across Organics Ltd. Hiboligosaccharide-human serum albumin (HbO-HA) conjugate waspurchased from National Institute for Biological Standards and Con-trol (NIBSC), United Kingdom. Amicon filters were purchased fromMillipore.

2.1. Preparation of PRP–TT conjugates

Three steps were used in the preparation of the PRP–TT conju-gate; derivatization of TT, derivatization of PRP and conjugation ofderivatized PRP to derivatized TT.

2.1.1. Derivatization of tetanus toxoidTetanus toxoid was diafiltered against 0.1 M MES buffer con-

taining 0.2 M NaCl, pH 6.5 using 50 kDa molecular weight cutoff(MWCO) membrane. Hydrazine was incorporated into the proteinby a dehydration reaction. TT (4.2 mg/ml) was reacted with 0.4 Mhydrazine and 30 mM EDC as their final concentrations. After 4 hmixing at room temperature, the pH of the reaction mixture wasraised to 8.5 with 1 N NaOH to stop the reaction. The solution wasdiafiltered against 3 mM Na2CO3 buffer containing 30 mM NaCl (pH10.5). Hydrazide labelling of TT was determined by TNBS assay [29]using acetohydrazide as a reference; and protein concentration wasdetermined by Lowry’s assay [30] using BSA as a reference. Thedegree of activation (DOA, number of hydrazide per TT molecule)was calculated by dividing the moles of hydrazides generated bymoles of protein assuming 150,000 for molecular weight of TT.

The hydrazide derivatized TT (TT-H) was stored at pH 10.5 ± 0.1at –20 ◦C for up to one week.

2.1.2. Depolymerization and derivatization of PRPGiven the high molecular weight of fermentation-derived PRP,

different experimental conditions were used for depolymeriza-tion of the polysaccharide. Native PRP (10 mg/ml) was reactedwith sodium metaperiodate in defined molar ratio (PRP repeatingunit to periodate) of 1:0.2 for 12 ± 1 and 18 ± 1 min to gener-ate activated polysaccharides of an approximate molecular size of100 kDa (range 80–120 kDa PRP) and 50 kDa (range 40–60 kDa PRP),respectively. To prepare oligosaccharides of approximate 10 kDamolecular size (range 6–16 kDa PRP), native PRP was reacted withsodium metaperiodate in molar ratio of 1:3 for 5 ± 1 min. Afterthe prescribed incubation time, the reaction mixture was purifiedby Sephadex G-25 column equilibrated with 0.15 M MES buffercontaining 0.2 M NaCl, pH 6.5. The concentration of the resultingpurified PRP was determined by orcinol assay [31] using riboseas a reference; the aldehyde content of the derivatized PRP wasdetermined by BCA assay [32] using glucose as a reference. Deriva-tization of PRP was expressed as the degree of activation (DOA,number of saccharide repeats per aldehyde) which was calculatedby dividing the moles of monomer present in polysaccharide bymoles of aldehyde generated after oxidation with sodium metape-riodate. Derivatized PRP was stored at −20 ◦C as a dry powder afterevaporation.

2.1.3. Conjugation of derivatized PRP to derivatized TTDerivatized hydrazide-containing TT was diafiltered against

0.15 M MES buffer containing 0.2 M NaCl, pH 6.5. Derivatizedaldehyde-containing PRP was dissolved in 0.15 M MES buffer con-taining 0.2 M NaCl, pH 6.5. For conjugation of polysaccharides,derivatized PRP (10–15 mg/ml) and derivatized TT (5–10 mg/ml)were mixed in a molar ratio (PRP to TT) of 3:1 (for both 100 kDaand 50 kDa molecular size PRP); whereas for conjugation of 10 kDaoligosaccharides, derivatized PRP (10–15 mg/ml) and derivatizedTT (5–10 mg/ml) were mixed in a 20:1 molar ratio (PRP to TT). A1–1.5 equivalent of sodium cyanoborohydride to that of TT wasadded to the reaction mixture. The reaction mixture was incubatedat 20–25 ◦C for 14–16 h and then treated with sodium borohydride(at least a 10 fold molar equivalent to the initial aldehyde contentin the derivatized PS) for 2–3 h. The 50 and 100 kDa PRP–TT conju-gates were purified by ammonium sulfate precipitation to removeunconjugated PRP and further washed by 10 kDa MWCO Amiconfilter against 0.15 M MES, 0.2 M NaCl, pH 6.5 to remove small impu-rities from the purified conjugate. The 10 kDa PRP–TT conjugateswere purified by diafiltration against 0.15 M MES buffer containing0.2 M NaCl, pH 6.5 (50–60 volumes) through 50 kDa MWCO Ami-con filter and stored at 2–8 ◦C. Purified conjugates were analyzedby the Lowry assay [30] for protein content and Orcinol assay [31]for PRP content.

2.2. High performance size-exclusion liquid chromatography(HPSEC)

Samples of derivatized protein, derivatized PRP and conjugateswere eluted on a TSK gel 5000 PWXL (7.8 × 300 mm, particle size7 �m, TOSOH) column connected in series with a TSK gel 4000PWXL (7.8 × 300 mm, particle size 7 �m, TOSOH) with TSKgel PWXLguard column (6.0 × 40 mm, TOSOH). The mobile phase was 0.1 Msodium nitrate, pH 7.2 ± 0.1, at the flow rate of 1.0 ml/min (iso-cratic method for 30 min). Void and total column volume weredetermined with dextran, MW 50, 00,000–400,00,000 (HIMEDIA)and deuterium oxide (D2O, Merck), respectively. PRP peaks weredetected by dRI, while UV detection at 280 nm was used for free pro-tein and conjugate detection. The calibration curve was prepared


2648 R. Rana et al. / Vaccine 33 (2015) 2646–2654

using Pullulan standards (Shodex Standard P-82) and the resultingchromatographic data was processed using Empower® softwareversion 2.

2.3. Determination of unconjugated saccharide content inglycoconjugate preparations

Sodium deoxycholate precipitation was used for estimation ofunconjugated saccharides in conjugate preparations [33]. To 900 �lof conjugate sample (approximately 100 �g PS content), 80 �l of1% (w/v) aqueous sodium deoxycholate solution, pH 6.8 ± 0.2 wasadded. The reaction mixture was kept at 2–8 ◦C for 30 min, 50 �lof 1 N HCl was added, and the sample was centrifuged at 6000 × gfor 15 min. The supernatant was collected and the free saccharidecontent estimated by orcinol assay [31].

2.4. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE)

SDS–PAGE was performed using discontinuous gel/buffer sys-tem of Laemmli [34] using 4% stacking and 6% separating gel.The samples (5–15 �l with a protein content of 5–10 �g) weremixed with sample buffer containing 10 mM �-mercaptoethanol.The mixtures were heated at 100 ◦C for 2–3 min. The gel was elec-trophoresed at 40 mA in Tris–glycine SDS running buffer (25 mMTris, 200 mM glycine, 0.1% (w/v) SDS) and stained with Brilliantblue R electrophoresis reagent (Sigma).

2.5. Immunogenicity of PRP–TT conjugates

Various studies on the PRP–TT conjugates were conductedat a contract research organization in India. These studies weredesigned to understand the effect of different variables on immuneresponse. The facility was registered for breeding and experi-ment of animals with the Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA), Ministry ofEnvironment and Forest, Govt. of India. Study was approved byInstitutional Animal Ethics Committee and the husbandry condi-tions were maintained as per CPCSEA recommendations. Five-eightweek old female Sprague Dawley rats were used in all animalimmunogenicity studies and were acclimatized for at least 5 daysbefore initiation of study, following which they were randomizedinto groups of 6–10 animals each on the basis of their body weight.

Two studies were conducted to test the immune response withPRP–TT conjugates of varying PRP size (100 kDa, 50 kDa or 10 kDa).In total 2, 2 and 3 different lots of 100, 50 and 10 kDa PRP–TTconjugates, respectively were used in the immunogenicity studies.Thereafter, the 10 kDa PRP–TT conjugates were tested at differ-ent dose levels (2 �g, 1 �g, 0.5 �g and 0.2 �g of PRP) to see thedose response. Further, different lots of 10 kDa PRP–TT conjugateswere compared with two different licensed monovalent PRP–TTvaccines at 1 �g dose level (6 independent studies in comparisonto Licensed Vaccine-1 from a multi-national company and 2 studiesin comparison to Licensed Vaccine-2 from an Indian manufacturer).Rats immunized with normal saline were used as negative controlin all the studies. Two hundred �l of each PRP–TT conjugate wasadministered subcutaneously by single injection per animal on Day0, 28 and 42 of the experiment. Approximately 400–800 �l bloodfrom each animal was drawn from the retro-orbital plexus on Day0 (pre bleed), 28, 42 and on the day of terminal collection (Day 49),maximum possible blood was withdrawn. Serum was separatedby centrifugation at approximately 5000 × g for 15 min and thenstored at −15 to −20 ◦C until analysis.

2.6. Detection of anti–PRP IgG by enzyme-linked immunosorbentassay (ELISA)

The 96-well microtiter plate (Nunc Maxisorp) was coated with1 �g/ml of HbO-HA as described previously [35]. The plate wasinitially incubated at 37 ◦C for 90 min and then kept at 2–8 ◦Covernight. The plate was washed with PBS (phosphate-bufferedsaline, pH 7.3 ± 0.2) containing 0.05% Tween 20 (v/v), 10 mM EDTAand blocked with 1% BSA (w/v). Two fold serial dilutions of qualitycontrol sera and test sera were added and incubated for 90 min atroom temperature. The plate was then washed and incubated for90 min at room temperature with peroxidase labelled anti-rat IgGantibodies in PBS containing 0.3% Tween 20, 10 mM EDTA, 1% BSA.Plate was washed and incubated for 10 min at room temperaturewith 100 �l peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine-H2O2 in sodium acetate buffer. The reaction was stopped by adding50 �l of 2 M H2SO4. The absorbance at 450/630 nm was measuredusing a Tecan multimode reader and the data transferred to an Excelfile for analysis using the Combistat software. A Quality Control(QC) serum (pool of hyper-immune sera from rats immunized withthe licensed Hib conjugate vaccine) was given an arbitrary anti-PRP IgG concentration of 5000 ELISA units/ml (EU/ml) and used inevery assay plate as standard. This was used to generate a standardELISA curve for extrapolating optical density values of IgG in thetest rat sera dilutions. The assay had buffer blank in which antibodydilution buffer was used in place of serum.

2.7. Statistical analysis

The geometric mean concentrations (GMCs) and 95% confidenceinterval (95% CI) of the individual animal sera IgG concentrationsbelonging to a formulation group were calculated. The IgG con-centrations of two independent formulation group animals werecompared by unpaired t-test.

3. Results

3.1. Preparation of PRP–TT conjugates

Several lots of PRP–TT conjugate vaccines were prepared at5–100 mg PRP scales to ascertain reproducibility and scalabilityof conjugation method. As described in Section 2, native PRP wasdepolymerized and activated by oxidation with sodium metape-riodate to generate activated PRP of defined size. The reactionconditions for PRP activation were optimized (Table 1) to yielddesired PRP sizes. The derivatized PRP of average 100 kDa, 50 kDaand 10 kDa molecular size was found to have an average degree ofactivation of about 70–90, 30–50 and 4–10 saccharide repeatingunits per aldehyde group, respectively (Table 2). The HPSEC pro-file revealed that periodate derivatized polysaccharides (average100 and 50 kDa) and oligosaccharide (average 10 kDa) had lowermolecular weights than the native PS (Fig. 1A), as expected.

On the other hand, the carboxyl groups of tetanus toxoids werefirst substituted with hydrazine in the presence of EDC underacidic conditions. The average degree of activation of TT was foundto be 50 ± 5 hydrazine groups per TT molecule (Table 2). TheHPSEC profiles of native and derivatized TT and the PRP–TT con-jugate indicated that upon derivatization, the size of derivatizedTT remained similar to the native TT (Fig. 1B), suggesting that lit-tle or no aggregation occurred. After conjugation, a high molecularweight peak appeared (Fig. 1B), indicating the formation of PRP–TTconjugates. The conjugates being large in size eluted in the voidvolume whereas the free PRP and TT are getting separated at a verydifferent retention time. SDS-PAGE analysis of PRP–TT conjugates



Table 1Experimental conditions for depolymerization and activation to achieve defined average molecular size of PRP.

Initial molecular weight of Hibpolysaccharide

PRP:NaIO4 molar ratio foroxidation

Experimental conditions foroxidation of PRP

Resulting average molecularweight

450–600 kDa 1:0.2 At 4 ◦C in dark for 12 ± 1 min ∼100 kDa1:0.2 At 4 ◦C in dark for 18 ± 1 min ∼50 kDa1:3 At 4 ◦C in dark for 5 ± 1 min ∼10 kDa

Table 2Degree of activation (DOA) for various lots of derivatized TT and derivatized PRP.

Lot no. of activated TT DOA % Recovery (scale of experiment) Lot no. of activated PRP Average molecular size DOA % Recovery (scale of experiment)

01 50.6 82% (25 mg) 01 10 kDa 5 70% (25 mg)02 51.7 76% (50 mg) 02 10 kDa 7 66% (25 mg)03 53.4 85% (50 mg) 03 10 kDa 5 49% (100 mg)04 53.5 80% (100 mg) 04 45 kDa 30 62% (74 mg)05 54.2 82% (100 mg) 05 55 kDa 45 68% (20 mg)

06 100 kDa 80 71% (10 mg)07 90 kDa 69 72% (20 mg)08 107 kDa 89 55% (25 mg)

also suggests that derivatized TT had been successfully coupled toderivatized high as well as low molecular weight PRP (Fig. 1C).

Purified conjugates were also characterized for their PRP con-tent, protein content and for presence of unconjugated PRP. Thesaccharide to protein ratio (w/w) and the free saccharide percentfor conjugates prepared are reported in Table 3. The PRP to proteinratio of these conjugates ranged from 0.25 to 0.50 depending onthe size of the PRP and the mixing ratio of activated PRP to acti-vated TT. The free PRP varied from 1% to 10%. The overall yield ofconjugates for the whole process varied from 15% to 25%. Compar-atively higher yields were observed for 100 kDa PRP–TT conjugatesof oligosaccharides as compared to the 50 and 10 kDa PRP–TT con-jugates.

3.2. Immunogenicity of Hib PRP–TT conjugates

In total, eight independent animal immunogenicity studies inSprague Dawley rat model were conducted to study various devel-opmental parameters of the PRP–TT conjugate vaccine, that is, fordetermination of optimum PRP size required for best immuno-genicity (lead candidate), dose ranging of the lead candidate andeffect of number of doses required to achieve best immunogenicityof the lead candidate and comparison of the lead candidate with2 licensed PRP–TT vaccine comparators (Licensed vaccine-1 andLicensed vaccine-2). Each of the study included normal saline asvehicle (negative) control.

To observe the impact of the PRP size used for conjugation onthe immunogenicity of the PRP–TT conjugates two studies wereconducted. Rats were immunized with three 1 �g doses of conju-gates prepared using average 100 kDa, 50 kDa and 10 kDa on day 0,28 and 42 and sera samples were collected from each animal andtested for anti-PRP IgG titres on day 28 (post 1), 42 (post 2) and 49(post 3) of the study. The post 1 dose IgG GMCs were very low (datanot shown). The post 2 dose data showed 10 kDa PRP–TT to givehigher immune response in comparison to 50 and 100 kDa PRP–TTconjugates in 2 studies. The differences were statistically high inone study (p = 0.02 vs 50 kDa PRP–TT and 0.01 vs 100 kDa PRP–TT)but not in the other (p = 0.2 vs 50 kDa PRP–TT and 0.8 vs 100 kDaPRP–TT). The GMCs of the anti-PRP IgG values and 95% CI were cal-culated and the results for post 3 dose are presented in Fig. 2A.The data indicates that 10 kDa PRP–TT conjugate showed higherimmunogenicity than the 50 and 100 kDa PRP–TT conjugates after3 doses in both the studies. The post 3 dose differences of IgG GMCsbetween 10, 50 and 100 kDa PRP–TT conjugates were not statisti-cally different. The post 2 and 3 dose data suggested average 10 kDaPRP as most optimum size of polysaccharide than the other 2 sizes

tested for conjugation. Rest of the studies were focused on ∼10 kDaPRP–TT conjugates.

In the dose ranging study the 10 kDa PRP–TT conjugates weredosed at 2 �g, 1 �g, 0.5 �g and 0.2 �g dose level on day 0, 28 and42 and sera samples were collected from each animal and testedfor anti-PRP IgG titres on day 0 (pre-bleed), 28 (post 1), 42 (post 2),49 (post 3) and rats were maintained upto day 70 of the study toobserve the longevity of the response. Anti-PRP IgG GMCs and 95%CI for different sera per formulation including the vehicle controlare presented as histograms (Fig. 2B). The data indicates that theimmune response increased gradually after first dose (day 28) tosecond dose (day 42) and gave maximum IgG titres post 3 dose(day 49). The immune response in all groups declined to variedlevels at day 70 of the study but maintained to above one third ofhighest response except with 2 �g dose.

Three different lots of 10 kDa PRP–TT conjugates were used tocompare the immunogenicity against reference licensed vaccine-1 in six different studies at 1 �g dose per rat and the results ofpost 3-dose anti-PRP–TT IgG responses were compiled in termsof GMC and 95% CI (Table 4, Fig. 2C). Table 4 indicates that10 kDa PRP–TT conjugate showed higher immunogenicity than theLicensed vaccine-1 in 5 out of 6 studies, however, the differencewas not stasitically significant in any of the study. The antibodyconcentrations with 0.5 �g of 10 kDa PRP–TT conjugate, as testedin 3 different studies were also comparable to reponse from 1 �glicensed vaccine (data not shown). Further, two immunogenicitystudies involved comparison of 10 kDa PRP–TT conjugates withlicensed vaccine-2 at 1 �g dose and the results (Fig. 2C) indicatethat the former was superior to the later with statistically higherIgG GMCs in one study.

4. Discussion

Glycoconjugate vaccines are among the safest and most effectivevaccines developed over the past 30 years. Polysaccharide vac-cines are T-cell independent, poorly immunogenic in infants andyoung children less than 2 years of age [36,37] and do not induceimmunological memory [38]. The development of glycoconjugatevaccines has allowed the investigation of their immunogenicityin preclinical and clinical studies in relation with their chemicaland physical properties. In addition, glycoconjugates have beenintroduced in the form of combination vaccines that are now part ofhuman immunization schedules [39–41]. Hib conjugate vaccines,the archetype of successful conjugate vaccines, have resulted in thevirtual eradication of Hib induced disease in much of the developedworld [42]. Glycoconjugate vaccines developed so far have different



Fig. 1. High-performance size exclusion chromatography profiles of HibPRP, TT and PRP–TT conjugates on TSK 4000–5000 PWXL columns: (A) native and depolymerizedPRP. Data were recorded using RI detector, (B) TT and PRP–TT conjugates. Data were recorded using PDA detector (MV: Millivolt; Hib: Haemophilus influenzae; PRP: poly-ribosyl-ribitol-phosphate; AU: absorption unit; TT: tetanus toxoid); Vtot 23.01 min; V0 10.39 min, (C) SDS-PAGE analysis of native TT (lane 1); activated TT (lane 2); 10 kDaPRP–TT conjugate (lane 3); 100 kDa PRP–TT conjugate (lane 4); 5 �g of protein loaded per each sample.

Table 3Characterization of various lots of PRP–TT conjugates.

PRP–TT conjugate Lot no. Size of activated PRP PRP:protein ratio (w/w) Free PRP % yield

01 10 kDa 0.31 1.3% 1502 10 kDa 0.26 1.0% 1603 10 kDa 0.36 1.1% 1604 10 kDa 0.29 2.6% 1905 45 kDa 0.48 8.4% 1606 55 kDa 0.38 6.3% 2107 90 kDa 0.43 9.5% 2508 100 kDa 0.32 3.5% 22



Fig. 2. Anti-HibPRP IgG geometric mean concentrations (±95% confidence interval). Each rat was immunized subcutaneously with 3 doses of defined concentration on Day0, 28 and 42 and the blood sera tested for total anti-HibPRP IgGs on either day 0 (pre-bleed), 28 (post 1 dose), 42 (post 2 dose), 49 (post 3 dose) or 70 (to study longevity ofthe immune response) as required. (A) Post 3 dose response to HibPRP–TT conjugates prepared using PRP of average 10, 50 and 100 kDa size. (B) Response to 10 kDa PRP–TTconjugates at different doses compared to the vehicle control. (C) Post dose 3 response for comparison of 10 kDa PRP–TT conjugate with two different licensed comparators(p = 0.0826 and 0.2999 for Licensed vaccine-1 vs 10 kDa PRP–TT and p = 0.0545 and 0.0416 for Licensed vaccine-2 vs 10 kDa PRP–TT in study 1 and 2, respectively).

characteristics depending on their carbohydrate antigen, the car-rier protein and the conjugation chemistry. All these aspects conferto the different physico-chemical characteristics in the glycocon-jugate that may result in varying immunological profiles.

Our approach to developing novel Hib conjugated vaccinesincluded depolymerizing the PRP and using a simple couplingchemistry. Highly polymerized repeat epitopes are believed toaccount for the T-cell independent character of polysaccharide anti-gen [43], and thus the presentation of lower molecular weightoligosaccharides on the carrier protein is thought to make the

conjugate more susceptible to the effects of T-helper cells.Empirically, Makela and others reported that protein conjugatesmade with low molecular weight dextran gave higher secondaryresponses in mice than conjugates made with macromoleculardextran [44]. Other authors have also studied the impact of polysac-charide size on conjugate immunogenicity with varied inferences.Vi polysaccharide–protein conjugates composed of higher molec-ular weight Vi have higher immunogenicity compared to lowermolecular weight Vi when tested in mice and rhesus monkeys[23]. The molecular size of the polysaccharide used for conjugation,



Table 4Geometric mean for anti-HibPRP IgG concentrations on day 49 (±95% confidence interval) from different rat immunogenicity studies 7 days post 3 doses of vaccine/vehicleformulations.

Study number Vehicle controla Licensed vaccine-1a,b 10 kDa PRP–TTa,b p-Valuec

Study 1 39 (46,33) 1200 (2489,579) 2007 (5120,787) 0.268Study 2 230 (362,146) 661 (1077,406) 2607 (6029,1128) 0.100Study 3 59 (134,26) 2385 (3851,1477) 3546 (6020,2088) 0.343Study 4 176 (220,141) 5451 (10179,2919) 3376 (7347,1552) 0.577Study 5 114 (131,98) 2507 (5468,1150) 9867 (21310,4568) 0.083Study 6 90 (128,63) 1580 (3468,720) 2389 (6465,883) 0.299

a n = 4–6 for vehicle control; n = 6–10 for Licensed Vaccine-1 and 10 kDa PRP–TT conjugates.b Each rat was immunized with 1 �g conjugated PRP.c p-Value for Licensed vaccine versus 10 kDa PRP–TT from unpaired t-test.

and extent of polysaccharide-to-protein cross linking influencethe immunogenicity and protective efficacy of streptococcal typeIII polysaccharide–TT conjugate vaccines and intermediate-sizeoligosaccharide was found superior to the smaller or larger sizeoligosaccharide in eliciting specific antibodies when tested inrabbits [22,24].

The objective of this study was to investigate the impact ofcarbohydrate chain length on immunogenic potential of the conju-gates generated from them. In this attempt we made immunogenicPRP–TT conjugates with capsular polysaccharide (∼100 kDa and∼50 kDa) and oligosaccharides (∼10 kDa) in reproducible way. Theintention was to create a conjugate with a PRP:TT ratio of ∼0.3while at the same time maximizing the yield of both PRP and TTand minimizing the free PS content using reductive amination. Theanimal immunogenicity experiments were designed to study theeffect of different doses of lead conjugates on eliciting antibodyresponse. The lead conjugates were also compared with two differ-ent licensed Hib conjugate vaccines as positive control.

Periodate oxidation was found to be a suitable method fordepolymerization of polysaccharide as it leads to its simulta-neous derivatization. Aldehyde molecules generated during theperiodate oxidation can readily be reacted with amine containingprotein carrier (TT). It was observed that different molar concen-tration of sodium metaperiodate and different exposure time ofsodium metaperiodate with polysaccharide were required to gen-erate polysaccharide and/or oligosaccharide with different averagemolecular size (Table 1). Glucose might not be a proper standard(due to its hemiacetal form in solution) for estimation of aldehydesgenerated by periodate oxidation of PRP. In this work, BCA assaywas used to establish relative values of degree of activation for dif-ferent sized PRP using glucose as a standard. Moreover, relativemolecular sizes of PRP reported in this work were determined fromcalibration curve plotted by running known molecular weight Pul-lulan standards on HPSEC but not calculated from the reducing endgroups generated by periodate treatment.

It was observed that highly derivatized TT-H tends to precipitatein reaction mixture which led to lower yield of TT. To counter thiseffect, the reaction mixture was observed during incubation andreaction was quenched if precipitation was observed. The precipi-tation is most likely due to change in isoelectric point of TT, whichis normally between 6.2 and 6.5. The isoelectric point may increasedue to loading of hydrazide on the TT molecule which may have ledto precipitation of protein at a lower pH [45].

HPSEC profile and SDS-PAGE analysis indicates the coupling ofPRP and TT. The bivalency of PRP generated with periodate chem-istry may give rise to cross-linked species, which will result in apopulation of diverse molecular weight conjugates as is evidentfrom the HPSEC chromatogram and SDS-PAGE. The purificationof PRP–TT conjugate by ammonium sulfate precipitation wouldremove unconjugated PRP and high molecular weight peak rep-resents conjugate only. Further, no free TT was observed afteranalyzing conjugates on HPSEC and SDS-PAGE. The degree of

activation of protein was found to be inversely correlated with freePS content, i.e. greater the number of hydrazide moieties incor-porated into TT-H, the lower the free PS in conjugate. Increasingthe number of reactive sites on TT should improve reactivity andmore reactive sites would increase the probability of an interac-tion with the terminal aldehyde of activated PRP; thus decreasingthe free PS. The free PS content was below set limit of 10% with amaximum of 9.5%. The difference observed on the free saccharidecontent could have been due to different behaviour of the conju-gates, depending on the size and purification method employed toremove unconjugated PRP. Further, the conjugation yield was quitesatisfactory i.e. up to 19% for the 10 kDa, up to 21% for the 50 kDa andup to 25% for the 100 kDa PRP–TT conjugates. This can be explainedby the fact that the activated TT carries fixed number of reactivehydrazide groups for conjugating high as well as low molecularsize PRP. Hence lesser number of moles of PRP could be conjugatedon TT when PRP chain length is shorter which leads to lower PS toprotein ratio and yield in comparison of long chain PRP. However,experimental conditions can further be optimized to increase theconjugation yield for different sized PRP–TT conjugates.

The PRP–TT conjugates were shown to elicit a high antibody(total IgG) titre by ELISA as compared to vehicle control. In allthe immunogenicity studies conducted, the 10 kDa PRP–TT con-jugates gave rise to comparable or better antibody titres after threeinjections as compared to both the higher molecular weight in-house conjugates (50 and 100 kDa PRP–TT) when tested at 1 �gdose level (Fig. 1A) after third dose. This may be explained byextent of glycosylation in various conjugates. Within a similar rangeof polysaccharide to protein ratio, the lower size PRP conjugateswould have higher degree of glycosylation on a molar basis whichmight potentially influence the antibody response of glycoconju-gate vaccine. The dose ranging study on 10 kDa PRP–TT conjugateshowed that the highest response was achieved with 1 �g dosewhich declined gradually with 0.5 and 0.2 �g doses as expected,however the IgG GMCs with 2 �g were also lower than 1 �g dosesuggesting 1 �g dose to be preferred for animal test model. Thistype of reduction in antibody titres with higher doses as comparedto optimal dose has also been reported earlier for PRP–TT [35].

Also the 10 kDa PRP–TT conjugates gave rise to an equivalentor better antibody titres as compared to both the licensed vaccines(Table 4, Fig. 2C). The differences in 10 kDa PRP–TT conjugates andlicensed vaccine-1 immune responses in terms of IgG GMCs werenot statistically significant by t-test comparisons, however in 5 outof 6 studies, the former was more immunogenic (1.5–4 fold higherIgG GMCs) than the later after third dose. Similarly, the differencesbetween the 10 kDa PRP–TT versus 50 or 100 kDa PRP–TT conjugateimmune responses were not statistically different. The lack of sta-tistical difference may be due to the inherent very high variation inthe animal to animal responses as observed by other authors also[35]. There was no defined trend observed for post 1 (day 28) andpost 2 (day 42) IgG antibody responses for 10 kDa conjugate andlicensed vaccine (data not shown). Interestingly, in three of the



four studies, an equivalent or better response for 10 kDa PRP–TTwas observed at lower dose level of 0.5 �g in comparison to 1 �gof the licensed vaccine-1 (data not shown). The data was furtherboosted by comparison of the lead 10 kDa PRP–TT candidate withanother comparator (licensed vaccine-2) in two studies and bothof them showed a higher response to the former with statisticallysignificant difference in one study.

Theoretical considerations and limited experimental evidencesuggest that saccharide fragments of shorter chain length, asopposed to high-molecular-weight saccharides, may be bet-ter able to elicit T-cell dependent antibody responses [24],where the authors found short chain length (average 14.5 kDa)oligosaccharide–TT conjugate to be an immunogen superior tolonger chain length (average 27 kDa) oligosaccharide–TT conju-gates. However presence of conformational epitopes may be animportant determinant of the optimal size of the derivatizedoligosaccharides used in conjugate vaccines. The conjugation yieldsin this study were found marginally higher for high kDa RPP–TTconjugates in comparison to 10 kDa conjugates.

In conclusion, we report here the highly immunogenic PRP–TTconjugates generated from shorter chain PRP as compared to theirhigh molecular weight counterparts from the same laboratoryand also to the licensed vaccines. We have optimized methods toprepare more immunogenic low molecular weight PRP–TT conju-gate in a reproducible manner. The higher reactivity of hydrazidegroups as compared to the lysine �-amino group resulted in areduced conjugation time and an optimal yield. The conjugates thusproduced are significantly immunogenic in rats. Hallmark of thisresearch is the development of highly immunogenic PRP–TT con-jugates using short chain PRP which forms the basis for furtherdevelopment of oligosaccharide conjugates as successful vaccinecandidates alone or in combination formats.

Acknowledgements

Authors thank Dr. Davinder Gill and Dr. Zimra Israel for their ableguidance and Sandeep Sharma, Madhu Madan for their invaluabletechnical help.

Conflict of interest statement: The authors declare no conflict ofinterest.

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