Development and characterization of Haemophilus influenzae type b conjugate vaccine prepared using different polysaccharide chain lengths.

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Development and characterization of Haemophilus influenzae type b conjugate vaccine prepared using different polysaccharide chain lengths 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 2015 Received in revised form 4 April 2015 Accepted 11 April 2015 Available online xxx Keywords: Conjugate vaccine Haemophilus influenzae type b PRP size Reductive amination Immunogenicity

a b s t r a c t Capsular polysaccharide conjugates of Haemophilus influenzae type b (Hib) are important components of several mono- or multi-valent childhood vaccines. However, their access to the most needy people is limited due to their high cost. As a step towards developing a cost effective and more immunogenic Hib conjugate vaccine, we present a method for the preparation of Hib capsular polysaccharide (PRP)–tetanus toxoid (TT) conjugates using optimized PRP chain length and conjugation conditions. Reactive aldehyde groups were introduced into the polysaccharides by controlled periodate oxidation of the native polysaccharide, which were subsequently covalently linked to hydrazide derivatized tetanus toxoid by means of reductive amination. Native polysaccharides were reduced to average 100 or 50 kDa polysaccharide and 10 kDa oligosaccharides in a controlled manner. Various conjugates were prepared using Hib polysaccharide 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 conjugates elicited specific antibodies to PRP. The low molecular weight PRP–TT conjugates were found to be more immunogenic as compared to their high molecular weight counterparts and the PRP–TT reference vaccine. © 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 high incidence in infants less than one year age [1]. Vaccines derived from Hib capsular polysaccharides (Hib-PRP) are T-cell independent (TI) antigens and have been found to be poorly immunogenic in children less than two years of age [2–4]. In 1929, Avery and Goebel have demonstrated that after conjugation to a carrier protein, antibodies were induced to polysaccharide in an animal model in a T-dependent (TD) manner [5]. These TD antigens are immunogenic early in infancy, the immune response induced can be boosted, enhanced by adjuvants, and is characterized by antibody class switch and production of antigen-specific IgG [4,6–8]. However, the first application of this concept to a vaccine was in 1980 with the development of the first conjugate vaccine against Hib 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 bacterial pathogens [13–16]. Several different conjugation chemistries have been tested for making commercial Hib vaccines [17–20], however, two main approaches based on different chemistry of conjugation have been traditionally used. One is based on the random chemical activation of the saccharide chain followed by covalent binding with the protein carrier obtaining a cross-linked structure. A second approach is based on the generation, by controlled fragmentation, of appropriately sized polysaccharides which are then activated at their terminal groups, usually with a linker molecule, and subsequently conjugated to the carrier protein obtaining a radial structure. The optimal length of the carbohydrate chain remains a matter of considerable debate for developing glycoconjugate vaccine and various lengths have been reported to be required for generating optimal immune response for various vaccine candidates [21–24]. However, the size of the saccharide must be sufficiently large to express epitopes representative of the native antigen. There are few reports on the use of different Hib-PRP sizes in preparing Hib conjugate vaccines. Diphtheria toxoid–coupled PRP of mean chain length 8 or 20 repeat units (Dpo8 and Dpo20) were tested for immunogenicity, and Dpo8 elicited poorer anti-PRP response in infants than

http://dx.doi.org/10.1016/j.vaccine.2015.04.031 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Rana R, et al. Development and characterization of Haemophilus influenzae type b conjugate vaccine prepared using different polysaccharide chain lengths. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.04.031

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Dpo20 [12]. However, in a separate study it was shown that no differences in the human immune response was found for conjugates made with varying chain lengths of Hib oligosaccharides that were monoterminally activated [25]. Hib dimer (four saccharideunits) conjugated to tetanus toxoid or to synthetic peptides is a poor immunogen in rabbit while Hib trimer (six saccharide-units) is immunogenic [26]. A method had been described for fractionation of Hib oligosaccharides of varying length, which permits removal of short fragments unsuitable for conjugate vaccine preparation [27]. However a detailed correlation of oligo- and poly-saccharide Hib conjugate immunogenicity is not much evident. In this study we tested the effect of molecular size of polysaccharide used for conjugation, and amount of conjugate injected, on immunogenicity of Hib PRP–tetanus toxoid conjugates. We prepared and evaluated conjugates using oligosaccharides of average 10 kDa as well as polysaccharides of average 50 kDa and 100 kDa molecular size. The immunogenicity of these conjugates has been assessed in a rat model in comparison with two different licensed Hib conjugate vaccines. 2. Materials and methods Hib capsular polysaccharide (PRP) was prepared using methods described previously [28] with few modifications (process communicated separately for publication). Tetanus toxoid (TT) was procured from Fabtech Technologies Ltd., India. 2-(Nmorpholino) ethanesulfonic acid (MES), hydrazine monohydrate, N-(3-D-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC), sodium chloride, sodium cynoborohydride solution, dglucose, d-ribose, iron chloride hexahydrate, 2,4,6-trinitrobenzene sulfonic acid (TNBS), copper sulfate, sodium dodecyl sulfate, sodium deoxycholate, sodium nitrate, bovine serum albumin (BSA) protein standards, and boric acid were purchased from Sigma Aldrich. Sodium hydroxide pellets, hydrochloric acid, Folin Ciocalteu’s phenol reagent, disodium tartarate dihydrate, and acetohydrazide were purchased from Merck Inc. Sodium carbonate anhydrous was purchased from S D Fine Chemicals Ltd. Sodium metaperiodate was purchased from SRL Chemicals. Orcinol monohydrate was purchased from Across Organics Ltd. Hib oligosaccharide-human serum albumin (HbO-HA) conjugate was purchased from National Institute for Biological Standards and Control (NIBSC), United Kingdom. Amicon filters were purchased from Millipore. 2.1. Preparation of PRP–TT conjugates Three steps were used in the preparation of the PRP–TT conjugate; derivatization of TT, derivatization of PRP and conjugation of derivatized PRP to derivatized TT. 2.1.1. Derivatization of tetanus toxoid Tetanus toxoid was diafiltered against 0.1 M MES buffer containing 0.2 M NaCl, pH 6.5 using 50 kDa molecular weight cutoff (MWCO) membrane. Hydrazine was incorporated into the protein by a dehydration reaction. TT (4.2 mg/ml) was reacted with 0.4 M hydrazine and 30 mM EDC as their final concentrations. After 4 h mixing at room temperature, the pH of the reaction mixture was raised to 8.5 with 1 N NaOH to stop the reaction. The solution was diafiltered against 3 mM Na2 CO3 buffer containing 30 mM NaCl (pH 10.5). Hydrazide labelling of TT was determined by TNBS assay [29] using acetohydrazide as a reference; and protein concentration was determined by Lowry’s assay [30] using BSA as a reference. The degree of activation (DOA, number of hydrazide per TT molecule) was calculated by dividing the moles of hydrazides generated by moles of protein assuming 150,000 for molecular weight of TT.

The hydrazide derivatized TT (TT-H) was stored at pH 10.5 ± 0.1 at –20 ◦ C for up to one week. 2.1.2. Depolymerization and derivatization of PRP Given the high molecular weight of fermentation-derived PRP, different experimental conditions were used for depolymerization of the polysaccharide. Native PRP (10 mg/ml) was reacted with sodium metaperiodate in defined molar ratio (PRP repeating unit to periodate) of 1:0.2 for 12 ± 1 and 18 ± 1 min to generate activated polysaccharides of an approximate molecular size of 100 kDa (range 80–120 kDa PRP) and 50 kDa (range 40–60 kDa PRP), respectively. To prepare oligosaccharides of approximate 10 kDa molecular size (range 6–16 kDa PRP), native PRP was reacted with sodium metaperiodate in molar ratio of 1:3 for 5 ± 1 min. After the prescribed incubation time, the reaction mixture was purified by Sephadex G-25 column equilibrated with 0.15 M MES buffer containing 0.2 M NaCl, pH 6.5. The concentration of the resulting purified PRP was determined by orcinol assay [31] using ribose as a reference; the aldehyde content of the derivatized PRP was determined by BCA assay [32] using glucose as a reference. Derivatization of PRP was expressed as the degree of activation (DOA, number of saccharide repeats per aldehyde) which was calculated by dividing the moles of monomer present in polysaccharide by moles of aldehyde generated after oxidation with sodium metaperiodate. Derivatized PRP was stored at −20 ◦ C as a dry powder after evaporation. 2.1.3. Conjugation of derivatized PRP to derivatized TT Derivatized hydrazide-containing TT was diafiltered against 0.15 M MES buffer containing 0.2 M NaCl, pH 6.5. Derivatized aldehyde-containing PRP was dissolved in 0.15 M MES buffer containing 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 kDa and 50 kDa molecular size PRP); whereas for conjugation of 10 kDa oligosaccharides, derivatized PRP (10–15 mg/ml) and derivatized TT (5–10 mg/ml) were mixed in a 20:1 molar ratio (PRP to TT). A 1–1.5 equivalent of sodium cyanoborohydride to that of TT was added to the reaction mixture. The reaction mixture was incubated at 20–25 ◦ C for 14–16 h and then treated with sodium borohydride (at least a 10 fold molar equivalent to the initial aldehyde content in the derivatized PS) for 2–3 h. The 50 and 100 kDa PRP–TT conjugates were purified by ammonium sulfate precipitation to remove unconjugated PRP and further washed by 10 kDa MWCO Amicon filter against 0.15 M MES, 0.2 M NaCl, pH 6.5 to remove small impurities from the purified conjugate. The 10 kDa PRP–TT conjugates were purified by diafiltration against 0.15 M MES buffer containing 0.2 M NaCl, pH 6.5 (50–60 volumes) through 50 kDa MWCO Amicon filter and stored at 2–8 ◦ C. Purified conjugates were analyzed by 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 conjugates were eluted on a TSK gel 5000 PWXL (7.8 × 300 mm, particle size 7 ␮m, TOSOH) column connected in series with a TSK gel 4000 PWXL (7.8 × 300 mm, particle size 7 ␮m, TOSOH) with TSKgel PWXL guard column (6.0 × 40 mm, TOSOH). The mobile phase was 0.1 M sodium nitrate, pH 7.2 ± 0.1, at the flow rate of 1.0 ml/min (isocratic method for 30 min). Void and total column volume were determined with dextran, MW 50, 00,000–400,00,000 (HIMEDIA) and deuterium oxide (D2 O, Merck), respectively. PRP peaks were detected by dRI, while UV detection at 280 nm was used for free protein and conjugate detection. The calibration curve was prepared




using Pullulan standards (Shodex Standard P-82) and the resulting chromatographic data was processed using Empower® software version 2.

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

2.4. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE was performed using discontinuous gel/buffer system of Laemmli [34] using 4% stacking and 6% separating gel. The samples (5–15 ␮l with a protein content of 5–10 ␮g) were mixed with sample buffer containing 10 mM ␤-mercaptoethanol. The mixtures were heated at 100 ◦ C for 2–3 min. The gel was electrophoresed at 40 mA in Tris–glycine SDS running buffer (25 mM Tris, 200 mM glycine, 0.1% (w/v) SDS) and stained with Brilliant blue R electrophoresis reagent (Sigma).

2.5. Immunogenicity of PRP–TT conjugates Various studies on the PRP–TT conjugates were conducted at a contract research organization in India. These studies were designed to understand the effect of different variables on immune response. The facility was registered for breeding and experiment of animals with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India. Study was approved by Institutional Animal Ethics Committee and the husbandry conditions were maintained as per CPCSEA recommendations. Five-eight week old female Sprague Dawley rats were used in all animal immunogenicity studies and were acclimatized for at least 5 days before initiation of study, following which they were randomized into groups of 6–10 animals each on the basis of their body weight. Two studies were conducted to test the immune response with PRP–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–TT conjugates, respectively were used in the immunogenicity studies. Thereafter, the 10 kDa PRP–TT conjugates were tested at different dose levels (2 ␮g, 1 ␮g, 0.5 ␮g and 0.2 ␮g of PRP) to see the dose response. Further, different lots of 10 kDa PRP–TT conjugates were compared with two different licensed monovalent PRP–TT vaccines at 1 ␮g dose level (6 independent studies in comparison to Licensed Vaccine-1 from a multi-national company and 2 studies in comparison to Licensed Vaccine-2 from an Indian manufacturer). Rats immunized with normal saline were used as negative control in all the studies. Two hundred ␮l of each PRP–TT conjugate was administered subcutaneously by single injection per animal on Day 0, 28 and 42 of the experiment. Approximately 400–800 ␮l blood from each animal was drawn from the retro-orbital plexus on Day 0 (pre bleed), 28, 42 and on the day of terminal collection (Day 49), maximum possible blood was withdrawn. Serum was separated by centrifugation at approximately 5000 × g for 15 min and then stored at −15 to −20 ◦ C until analysis.

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2.6. Detection of anti–PRP IgG by enzyme-linked immunosorbent assay (ELISA) The 96-well microtiter plate (Nunc Maxisorp) was coated with 1 ␮g/ml of HbO-HA as described previously [35]. The plate was initially incubated at 37 ◦ C for 90 min and then kept at 2–8 ◦ C overnight. The plate was washed with PBS (phosphate-buffered saline, pH 7.3 ± 0.2) containing 0.05% Tween 20 (v/v), 10 mM EDTA and blocked with 1% BSA (w/v). Two fold serial dilutions of quality control sera and test sera were added and incubated for 90 min at room temperature. The plate was then washed and incubated for 90 min at room temperature with peroxidase labelled anti-rat IgG antibodies in PBS containing 0.3% Tween 20, 10 mM EDTA, 1% BSA. Plate was washed and incubated for 10 min at room temperature with 100 ␮l peroxidase substrate, 3,3 ,5,5 -tetramethylbenzidineH2 O2 in sodium acetate buffer. The reaction was stopped by adding 50 ␮l of 2 M H2 SO4 . The absorbance at 450/630 nm was measured using a Tecan multimode reader and the data transferred to an Excel file for analysis using the Combistat software. A Quality Control (QC) serum (pool of hyper-immune sera from rats immunized with the licensed Hib conjugate vaccine) was given an arbitrary antiPRP IgG concentration of 5000 ELISA units/ml (EU/ml) and used in every assay plate as standard. This was used to generate a standard ELISA curve for extrapolating optical density values of IgG in the test rat sera dilutions. The assay had buffer blank in which antibody dilution buffer was used in place of serum.

2.7. Statistical analysis The geometric mean concentrations (GMCs) and 95% confidence interval (95% CI) of the individual animal sera IgG concentrations belonging to a formulation group were calculated. The IgG concentrations of two independent formulation group animals were compared by unpaired t-test.

3. Results 3.1. Preparation of PRP–TT conjugates Several lots of PRP–TT conjugate vaccines were prepared at 5–100 mg PRP scales to ascertain reproducibility and scalability of conjugation method. As described in Section 2, native PRP was depolymerized and activated by oxidation with sodium metaperiodate to generate activated PRP of defined size. The reaction conditions for PRP activation were optimized (Table 1) to yield desired PRP sizes. The derivatized PRP of average 100 kDa, 50 kDa and 10 kDa molecular size was found to have an average degree of activation of about 70–90, 30–50 and 4–10 saccharide repeating units per aldehyde group, respectively (Table 2). The HPSEC profile revealed that periodate derivatized polysaccharides (average 100 and 50 kDa) and oligosaccharide (average 10 kDa) had lower molecular weights than the native PS (Fig. 1A), as expected. On the other hand, the carboxyl groups of tetanus toxoids were first substituted with hydrazine in the presence of EDC under acidic conditions. The average degree of activation of TT was found to be 50 ± 5 hydrazine groups per TT molecule (Table 2). The HPSEC profiles of native and derivatized TT and the PRP–TT conjugate indicated that upon derivatization, the size of derivatized TT remained similar to the native TT (Fig. 1B), suggesting that little or no aggregation occurred. After conjugation, a high molecular weight peak appeared (Fig. 1B), indicating the formation of PRP–TT conjugates. The conjugates being large in size eluted in the void volume whereas the free PRP and TT are getting separated at a very different retention time. SDS-PAGE analysis of PRP–TT conjugates


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

PRP:NaIO4 molar ratio for oxidation

Experimental conditions for oxidation of PRP

Resulting average molecular weight

450–600 kDa

1:0.2 1:0.2 1:3

At 4 ◦ C in dark for 12 ± 1 min At 4 ◦ C in dark for 18 ± 1 min At 4 ◦ C in dark for 5 ± 1 min

∼100 kDa ∼50 kDa ∼10 kDa

Table 2 Degree 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 02 03 04 05

50.6 51.7 53.4 53.5 54.2

82% (25 mg) 76% (50 mg) 85% (50 mg) 80% (100 mg) 82% (100 mg)

01 02 03 04 05 06 07 08

10 kDa 10 kDa 10 kDa 45 kDa 55 kDa 100 kDa 90 kDa 107 kDa

5 7 5 30 45 80 69 89

70% (25 mg) 66% (25 mg) 49% (100 mg) 62% (74 mg) 68% (20 mg) 71% (10 mg) 72% (20 mg) 55% (25 mg)

also suggests that derivatized TT had been successfully coupled to derivatized high as well as low molecular weight PRP (Fig. 1C). Purified conjugates were also characterized for their PRP content, protein content and for presence of unconjugated PRP. The saccharide to protein ratio (w/w) and the free saccharide percent for conjugates prepared are reported in Table 3. The PRP to protein ratio of these conjugates ranged from 0.25 to 0.50 depending on the size of the PRP and the mixing ratio of activated PRP to activated TT. The free PRP varied from 1% to 10%. The overall yield of conjugates for the whole process varied from 15% to 25%. Comparatively higher yields were observed for 100 kDa PRP–TT conjugates of oligosaccharides as compared to the 50 and 10 kDa PRP–TT conjugates. 3.2. Immunogenicity of Hib PRP–TT conjugates In total, eight independent animal immunogenicity studies in Sprague Dawley rat model were conducted to study various developmental parameters of the PRP–TT conjugate vaccine, that is, for determination of optimum PRP size required for best immunogenicity (lead candidate), dose ranging of the lead candidate and effect of number of doses required to achieve best immunogenicity of the lead candidate and comparison of the lead candidate with 2 licensed PRP–TT vaccine comparators (Licensed vaccine-1 and Licensed vaccine-2). Each of the study included normal saline as vehicle (negative) control. To observe the impact of the PRP size used for conjugation on the immunogenicity of the PRP–TT conjugates two studies were conducted. Rats were immunized with three 1 ␮g doses of conjugates prepared using average 100 kDa, 50 kDa and 10 kDa on day 0, 28 and 42 and sera samples were collected from each animal and tested 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 (data not shown). The post 2 dose data showed 10 kDa PRP–TT to give higher immune response in comparison to 50 and 100 kDa PRP–TT conjugates in 2 studies. The differences were statistically high in one 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 kDa PRP–TT). The GMCs of the anti-PRP IgG values and 95% CI were calculated and the results for post 3 dose are presented in Fig. 2A. The data indicates that 10 kDa PRP–TT conjugate showed higher immunogenicity than the 50 and 100 kDa PRP–TT conjugates after 3 doses in both the studies. The post 3 dose differences of IgG GMCs between 10, 50 and 100 kDa PRP–TT conjugates were not statistically different. The post 2 and 3 dose data suggested average 10 kDa PRP as most optimum size of polysaccharide than the other 2 sizes

tested for conjugation. Rest of the studies were focused on ∼10 kDa PRP–TT conjugates. In the dose ranging study the 10 kDa PRP–TT conjugates were dosed at 2 ␮g, 1 ␮g, 0.5 ␮g and 0.2 ␮g dose level on day 0, 28 and 42 and sera samples were collected from each animal and tested for 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 to observe the longevity of the response. Anti-PRP IgG GMCs and 95% CI for different sera per formulation including the vehicle control are presented as histograms (Fig. 2B). The data indicates that the immune response increased gradually after first dose (day 28) to second dose (day 42) and gave maximum IgG titres post 3 dose (day 49). The immune response in all groups declined to varied levels at day 70 of the study but maintained to above one third of highest response except with 2 ␮g dose. Three different lots of 10 kDa PRP–TT conjugates were used to compare the immunogenicity against reference licensed vaccine1 in six different studies at 1 ␮g dose per rat and the results of post 3-dose anti-PRP–TT IgG responses were compiled in terms of GMC and 95% CI (Table 4, Fig. 2C). Table 4 indicates that 10 kDa PRP–TT conjugate showed higher immunogenicity than the Licensed vaccine-1 in 5 out of 6 studies, however, the difference was not stasitically significant in any of the study. The antibody concentrations with 0.5 ␮g of 10 kDa PRP–TT conjugate, as tested in 3 different studies were also comparable to reponse from 1 ␮g licensed vaccine (data not shown). Further, two immunogenicity studies involved comparison of 10 kDa PRP–TT conjugates with licensed vaccine-2 at 1 ␮g dose and the results (Fig. 2C) indicate that the former was superior to the later with statistically higher IgG GMCs in one study. 4. Discussion Glycoconjugate vaccines are among the safest and most effective vaccines developed over the past 30 years. Polysaccharide vaccines are T-cell independent, poorly immunogenic in infants and young children less than 2 years of age [36,37] and do not induce immunological memory [38]. The development of glycoconjugate vaccines has allowed the investigation of their immunogenicity in preclinical and clinical studies in relation with their chemical and physical properties. In addition, glycoconjugates have been introduced in the form of combination vaccines that are now part of human immunization schedules [39–41]. Hib conjugate vaccines, the archetype of successful conjugate vaccines, have resulted in the virtual eradication of Hib induced disease in much of the developed world [42]. Glycoconjugate vaccines developed so far have different


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Fig. 1. High-performance size exclusion chromatography profiles of HibPRP, TT and PRP–TT conjugates on TSK 4000–5000 PWXL columns: (A) native and depolymerized PRP. Data were recorded using RI detector, (B) TT and PRP–TT conjugates. Data were recorded using PDA detector (MV: Millivolt; Hib: Haemophilus influenzae; PRP: polyribosyl-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 kDa PRP–TT conjugate (lane 3); 100 kDa PRP–TT conjugate (lane 4); 5 ␮g of protein loaded per each sample. Table 3 Characterization 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 02 03 04 05 06 07 08

10 kDa 10 kDa 10 kDa 10 kDa 45 kDa 55 kDa 90 kDa 100 kDa

0.31 0.26 0.36 0.29 0.48 0.38 0.43 0.32

1.3% 1.0% 1.1% 2.6% 8.4% 6.3% 9.5% 3.5%

15 16 16 19 16 21 25 22


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Fig. 2. Anti-HibPRP IgG geometric mean concentrations (±95% confidence interval). Each rat was immunized subcutaneously with 3 doses of defined concentration on Day 0, 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 of the 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–TT conjugates 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 carrier protein and the conjugation chemistry. All these aspects confer to the different physico-chemical characteristics in the glycoconjugate that may result in varying immunological profiles. Our approach to developing novel Hib conjugated vaccines included depolymerizing the PRP and using a simple coupling chemistry. Highly polymerized repeat epitopes are believed to account for the T-cell independent character of polysaccharide antigen [43], and thus the presentation of lower molecular weight oligosaccharides 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 conjugates made with low molecular weight dextran gave higher secondary responses in mice than conjugates made with macromolecular dextran [44]. Other authors have also studied the impact of polysaccharide size on conjugate immunogenicity with varied inferences. Vi polysaccharide–protein conjugates composed of higher molecular weight Vi have higher immunogenicity compared to lower molecular weight Vi when tested in mice and rhesus monkeys [23]. The molecular size of the polysaccharide used for conjugation,


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

Vehicle controla

Licensed vaccine-1a,b

10 kDa PRP–TTa,b

p-Valuec

Study 1 Study 2 Study 3 Study 4 Study 5 Study 6

39 (46,33) 230 (362,146) 59 (134,26) 176 (220,141) 114 (131,98) 90 (128,63)

1200 (2489,579) 661 (1077,406) 2385 (3851,1477) 5451 (10179,2919) 2507 (5468,1150) 1580 (3468,720)

2007 (5120,787) 2607 (6029,1128) 3546 (6020,2088) 3376 (7347,1552) 9867 (21310,4568) 2389 (6465,883)

0.268 0.100 0.343 0.577 0.083 0.299

a b c

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

and extent of polysaccharide-to-protein cross linking influence the immunogenicity and protective efficacy of streptococcal type III polysaccharide–TT conjugate vaccines and intermediate-size oligosaccharide was found superior to the smaller or larger size oligosaccharide in eliciting specific antibodies when tested in rabbits [22,24]. The objective of this study was to investigate the impact of carbohydrate chain length on immunogenic potential of the conjugates generated from them. In this attempt we made immunogenic PRP–TT conjugates with capsular polysaccharide (∼100 kDa and ∼50 kDa) and oligosaccharides (∼10 kDa) in reproducible way. The intention was to create a conjugate with a PRP:TT ratio of ∼0.3 while at the same time maximizing the yield of both PRP and TT and minimizing the free PS content using reductive amination. The animal immunogenicity experiments were designed to study the effect of different doses of lead conjugates on eliciting antibody response. The lead conjugates were also compared with two different licensed Hib conjugate vaccines as positive control. Periodate oxidation was found to be a suitable method for depolymerization of polysaccharide as it leads to its simultaneous derivatization. Aldehyde molecules generated during the periodate oxidation can readily be reacted with amine containing protein carrier (TT). It was observed that different molar concentration of sodium metaperiodate and different exposure time of sodium metaperiodate with polysaccharide were required to generate polysaccharide and/or oligosaccharide with different average molecular size (Table 1). Glucose might not be a proper standard (due to its hemiacetal form in solution) for estimation of aldehydes generated by periodate oxidation of PRP. In this work, BCA assay was used to establish relative values of degree of activation for different sized PRP using glucose as a standard. Moreover, relative molecular sizes of PRP reported in this work were determined from calibration curve plotted by running known molecular weight Pullulan standards on HPSEC but not calculated from the reducing end groups generated by periodate treatment. It was observed that highly derivatized TT-H tends to precipitate in reaction mixture which led to lower yield of TT. To counter this effect, the reaction mixture was observed during incubation and reaction was quenched if precipitation was observed. The precipitation is most likely due to change in isoelectric point of TT, which is normally between 6.2 and 6.5. The isoelectric point may increase due to loading of hydrazide on the TT molecule which may have led to precipitation of protein at a lower pH [45]. HPSEC profile and SDS-PAGE analysis indicates the coupling of PRP and TT. The bivalency of PRP generated with periodate chemistry may give rise to cross-linked species, which will result in a population of diverse molecular weight conjugates as is evident from the HPSEC chromatogram and SDS-PAGE. The purification of PRP–TT conjugate by ammonium sulfate precipitation would remove unconjugated PRP and high molecular weight peak represents conjugate only. Further, no free TT was observed after analyzing conjugates on HPSEC and SDS-PAGE. The degree of

activation of protein was found to be inversely correlated with free PS content, i.e. greater the number of hydrazide moieties incorporated into TT-H, the lower the free PS in conjugate. Increasing the number of reactive sites on TT should improve reactivity and more reactive sites would increase the probability of an interaction with the terminal aldehyde of activated PRP; thus decreasing the free PS. The free PS content was below set limit of 10% with a maximum of 9.5%. The difference observed on the free saccharide content could have been due to different behaviour of the conjugates, depending on the size and purification method employed to remove unconjugated PRP. Further, the conjugation yield was quite satisfactory i.e. up to 19% for the 10 kDa, up to 21% for the 50 kDa and up to 25% for the 100 kDa PRP–TT conjugates. This can be explained by the fact that the activated TT carries fixed number of reactive hydrazide groups for conjugating high as well as low molecular size PRP. Hence lesser number of moles of PRP could be conjugated on TT when PRP chain length is shorter which leads to lower PS to protein ratio and yield in comparison of long chain PRP. However, experimental conditions can further be optimized to increase the conjugation 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 all the immunogenicity studies conducted, the 10 kDa PRP–TT conjugates gave rise to comparable or better antibody titres after three injections as compared to both the higher molecular weight inhouse conjugates (50 and 100 kDa PRP–TT) when tested at 1 ␮g dose level (Fig. 1A) after third dose. This may be explained by extent of glycosylation in various conjugates. Within a similar range of polysaccharide to protein ratio, the lower size PRP conjugates would have higher degree of glycosylation on a molar basis which might potentially influence the antibody response of glycoconjugate vaccine. The dose ranging study on 10 kDa PRP–TT conjugate showed that the highest response was achieved with 1 ␮g dose which 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 dose suggesting 1 ␮g dose to be preferred for animal test model. This type of reduction in antibody titres with higher doses as compared to optimal dose has also been reported earlier for PRP–TT [35]. Also the 10 kDa PRP–TT conjugates gave rise to an equivalent or better antibody titres as compared to both the licensed vaccines (Table 4, Fig. 2C). The differences in 10 kDa PRP–TT conjugates and licensed vaccine-1 immune responses in terms of IgG GMCs were not statistically significant by t-test comparisons, however in 5 out of 6 studies, the former was more immunogenic (1.5–4 fold higher IgG GMCs) than the later after third dose. Similarly, the differences between the 10 kDa PRP–TT versus 50 or 100 kDa PRP–TT conjugate immune responses were not statistically different. The lack of statistical difference may be due to the inherent very high variation in the animal to animal responses as observed by other authors also [35]. There was no defined trend observed for post 1 (day 28) and post 2 (day 42) IgG antibody responses for 10 kDa conjugate and licensed vaccine (data not shown). Interestingly, in three of the




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four studies, an equivalent or better response for 10 kDa PRP–TT was observed at lower dose level of 0.5 ␮g in comparison to 1 ␮g of the licensed vaccine-1 (data not shown). The data was further boosted by comparison of the lead 10 kDa PRP–TT candidate with another comparator (licensed vaccine-2) in two studies and both of them showed a higher response to the former with statistically significant difference in one study. Theoretical considerations and limited experimental evidence suggest that saccharide fragments of shorter chain length, as opposed to high-molecular-weight saccharides, may be better 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 to longer chain length (average 27 kDa) oligosaccharide–TT conjugates. However presence of conformational epitopes may be an important determinant of the optimal size of the derivatized oligosaccharides used in conjugate vaccines. The conjugation yields in this study were found marginally higher for high kDa RPP–TT conjugates in comparison to 10 kDa conjugates. In conclusion, we report here the highly immunogenic PRP–TT conjugates generated from shorter chain PRP as compared to their high molecular weight counterparts from the same laboratory and also to the licensed vaccines. We have optimized methods to prepare more immunogenic low molecular weight PRP–TT conjugate in a reproducible manner. The higher reactivity of hydrazide groups as compared to the lysine ␧-amino group resulted in a reduced conjugation time and an optimal yield. The conjugates thus produced are significantly immunogenic in rats. Hallmark of this research is the development of highly immunogenic PRP–TT conjugates using short chain PRP which forms the basis for further development of oligosaccharide conjugates as successful vaccine candidates alone or in combination formats. Acknowledgements Authors thank Dr. Davinder Gill and Dr. Zimra Israel for their able guidance and Sandeep Sharma, Madhu Madan for their invaluable technical help. Conflict of interest statement: The authors declare no conflict of interest. References [1] Frasch C. Regulatory perspectives in vaccine licensure. In: Ellis RW, Granoff DM, editors. Development and clinical uses of Haemophilus influenzae type b conjugate vaccines. New York: Marcel Dekker; 1994. p. 435–53. [2] Peltola H, Makela PH, Kayhty H, Jousimies H, Herva E, Hallstrom K, et al. Clinical efficacy of meningococcus group A capsular polysaccharide vaccine in children three months to five years of age. N Engl J Med 1977;297:686–91. [3] Makela PH, Peltola H, Kayhty H, Jousimies H, Pettay O, Ruoslahti E, et al. Polysaccharide vaccines of group A Neisseria meningitidis and Haemophilus influenzae type b: a field trial in Finland. J Infect Dis 1977;136:S43–50. [4] Peltola H, Kayhty H, Sivonen A, Makela PH. Haemophilus influenzae type b capsular polysaccharide vaccine in children: a double-blind filed study of 100,000 vaccinees 3 months to 5 years of age in Finland. Pediatrics 1977;60:730–7. [5] Avery OT, Goebel WF. Chemo-immunological studies on conjugated carbohydrate-proteins: II. Immunological specificity of synthetic sugar-protein antigens. J Exp Med 1929;50:533–50. [6] Guttormsen HK, Sharpe AH, Chandraker AK, Brigtsen AK, Sayegh MH, Kasper DL. Cognate stimulatory B-cell-T-cell interactions are critical for T-cell help recruited by glycoconjugate vaccines. Infect Immun 1999;67:6375–84. [7] Guttormsen HK, Wetzler LM, Finberg RW, Kasper DL. Immunogenic memory induced by a glycoconjugate vaccine in a murine adoptive lymphocyte transfer model. Infect Immun 1998;66:2026–32. [8] Avci FY, Kasper DL. How bacterial carbohydrates influence the adaptive immune system. Annu Rev Immunol 2010;28:107–30. [9] Schneerson R, Barrera O, Sutton A, Robbins JB. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J Exp Med 1980;152:361–76. [10] Eskola J, Peltola H, Takala AK, Kayhty H, Hakulinen M, Karanko V, et al. Efficacy of Haemophilus influenzae type b polysaccharide-diphtheria toxoid conjugate vaccine in infancy. N Engl J Med 1987;317:717–22.

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Efficacy and safety of a Haemophilus influenzae type b capsular polysaccharide-tetanus protein conjugate vaccine.

Evaluating the Haemophilus influenzae type b conjugate vaccine PRP-D.

Immunogenicity of Haemophilus influenzae type b polysaccharide-tetanus protein conjugate vaccine in children with sickle hemoglobinopathy or malignancies, and after systemic Haemophilus influenzae type b infection.

Haemophilus influenzae type B conjugate vaccines.

Response to a Haemophilus influenzae type b diphtheria CRM197 conjugate vaccine in children with a defect of antibody production to Haemophilus influenzae type b polysaccharide.

Hepatitis B surface antigen conjugate vaccine.

Clinical experience with PedvaxHIB, a conjugate vaccine of Haemophilus influenzae type b polysaccharide--Neisseria meningitidis outer membrane protein.

Antibody responses to four Haemophilus influenzae type b conjugate vaccines.

Efficacy of Haemophilus influenzae type B conjugate vaccine in Oxford region.

Immunogenicity of Haemophilus influenzae type b conjugate vaccine in children with sickle cell disease.

Haemophilus b disease after vaccination with Haemophilus b polysaccharide or conjugate vaccine.

Immunogenicity of Haemophilus influenzae type b conjugate vaccine in allogeneic bone marrow recipients.

Response to Haemophilus influenzae type B conjugate vaccine in children undergoing splenectomy.

Mucosal antibodies to Haemophilus influenzae type b capsular polysaccharide.

Cost-effectiveness of Haemophilus influenzae type b vaccine in Vietnam.

Influence of prevaccination immunity on the human B-lymphocyte response to a Haemophilus influenzae type b conjugate vaccine.

Characterization of an ampicillin-resistant Haemophilus influenzae type B.

Group B Streptococcus type II polysaccharide-tetanus toxoid conjugate vaccine.

The efficacy in Navajo infants of a conjugate vaccine consisting of Haemophilus influenzae type b polysaccharide and Neisseria meningitidis outer-membrane protein complex.

Haemophilus influenzae type b pneumonia.

Haemophilus influenzae type B endocarditis and meningitis.

Type b and non-type b Haemophilus influenzae endocarditis.

Limited efficacy of a Haemophilus influenzae type b conjugate vaccine in Alaska Native infants. The Alaska H. influenzae Vaccine Study Group.

Statement on Haemophilus influenzae type b conjugate vaccines for use in infants and children.