FEMS Immunology and Medical Microbiology 43 (2005) 133–140 www.fems-microbiology.org
Memory immune response generated in Cercopithecusaethiops against meningococcal polysaccharide serogroup C conjugate vaccine ´ lvarez, L. Canaa´n, S. Gonza´lez, T. Carmenate *, M. Guirola, A. A E. Caballero, T. Mene´ndez, G. Guille´n Centro de Ingenierı´a Gene´tica y Biotecnologı´a, Avenue 135, P.O. Box 6162, Havana 10600, Cuba Received 24 March 2004; received in revised form 4 June 2004; accepted 1 July 2004 First published online 2 August 2004
Abstract The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Conjugate vaccines; Memory response; Neisseria meningitidis
1. Introduction The development of conjugate vaccines for the control of bacterial diseases has been an important field of investigation for researchers associated with human health. Several steps have been accomplished towards the elimination of bacterial meningitis. The first one was the implementation in several countries of the conjugate vaccine against Haemophilus influenzae type b disease [1,2]. The second one was the development and subsequent licensure of the conjugate vaccine against Streptococcus pneumoniae [3]. Finally, the development of a conjugate vaccine against the meningococcal sero*
Corresponding author. Tel.: +53 7 2718008; fax: +53 7 214764. E-mail addresses:
[email protected],
[email protected] (T. Carmenate).
groups A and C opened the doors for the elimination the remaining bacterial meningitis cases [4]. Although the vaccine against serogroup A was abandoned, several vaccines against serogroup C have been licensed and introduced in England with a great impact in public health [5,6]. Plain polysaccharide vaccines do not generate a memory immune response against the pathogen and are not effective in young children, who are the principal riskgroup of the disease [7]. It is well established that conjugation of bacterial polysaccharides with carrier proteins overcomes those problems [8]. Specifically, the conjugate vaccines against meningococcal disease caused by serogroup C have been proven to induce immunological memory in infants [9]. For all the developed and licensed conjugate vaccines only a few proteins have been used as carriers, mainly
0928-8244/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2004.07.003
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tetanus toxoid and a mutant of the diphtheria toxin polypeptide (CRM 197). The characterization of new carrier proteins seems to be a compulsory step for the achievement of a proper immune response against all the polysaccharide antigens needed for childhood vaccination. In this work, we study the memory response generated by a polysaccharide C conjugate with the recombinant protein P64k. This protein is an outer membrane associated antigen isolated from Neisseria meningitidis that has been extensively characterized by physicochemical and immunological methods [10]. The P64k protein was assayed in a Phase I clinical trial and its safety and immunogenicity in human volunteers were demonstrated [11]. This protein has been used as a carrier for peptides with satisfactory results and also a preliminary assay where it was employed as carrier for the polysaccharide C has been published [12].
2. Materials and methods 2.1. Materials Native meningococcal serogroup C polysaccharide (CCPS) was obtained from meningococcal strain (C11 ATCC). The molecular size of the native polysaccharide was P 60,000 Da and the degree of O-acetylation was 3.1 lmol mg 1. This material was donated by ‘‘Instituto Finlay’’, Habana, Cuba. The recombinant P64k protein used for conjugation was produced at the ‘‘Centro de Ingenierı´a Gene´tica y Biotecnologı´a’’, Havana, Cuba. Outer membrane vesicles preparation (OMV) was obtained from the meningococcal strain CU385/83, using the standard extraction method with LiCl2 [13]. Human sera from healthy Cuban volunteers previously immunized with the polysaccharide vaccine A + C from Pasteur Vaccines, France were available in our laboratory. Volunteers were immunized once and serum samples were taken on days 0 and 30. 2.2. CCPS–P64k conjugate The conjugate used for immunization was obtained by amination reductive method [14]. Briefly, 50 mg of native CCPS was oxidized by incubation with 20 mg of NaIO4 previously dissolved in 5 ml of acetate buffer (5%), pH 5.5. The oxidation reaction proceeded during 30 min in the dark at room temperature (rt) and was stopped by addition of ethylene glycol. Oxidized polysaccharide was dialyzed against double distilled water using a membrane (MWCO: 12–14 000 Spectra/Por, Inc., USA) and dried by lyophilization. Purified, oxidized polysaccharide (10 mg) was coupled to the P64k protein (6.6 mg) by reductive amination with sodium cyanoborohydride. The conjugation reaction was moni-
tored by gel filtration chromatography with a Bio-Gel A0.5 column (2.6 · 100 cm, BioRad). The reaction was considered completed when the magnitude of void volume-peak remained constant. The conjugate named CCPS–P64k, was treated with sodium borohydride and incubated at rt for 60 min to reduce any remaining aldehyde, it was dialyzed against sterile water at 4 C and lyophilized to dryness. The final protein: polysaccharide ratio was 10:1 (w:w). The conjugate was assayed by NMR for determination of the acetylation level and a high degree of acetylation was founded in the carbon 7 (data not shown). 2.3. Immunization To evaluate the immunological response against the conjugate, three groups of three African Green Monkeys (Cercopithecus aethiops) each, were immunized intramuscularly. The first group was immunized with 5 lg of CCPS–P64k conjugate, the second group was immunized with 25 lg of CCPS mixed with 5 lg of OMV from the strain CU385/83 and the third one with 5 lg of plain CCPS. Two doses were administered 30 days apart and a booster dose was injected on day 390. Blood extractions were performed on days 0, 30, 60, 270, 390 and 420. 2.4. ELISAs For evaluation of IgG titers against native CCPS three different procedures were used. Traditional protocol. Microtiter plates (High Binding, Costar) were activated with 2 lg ml 1 of poly-L -lysine in carbonate buffer, pH 9.6, at 37 C for 2 h. Plates were washed three times with double distilled water. Then, they were coated with 1 lg ml 1 CCPS diluted in PBS, pH 7.6, at 4 C overnight. After that, plates were washed three times with PBS with 0.05% (v:v) Tween 20 (TPBS). Plates were blocked with 1% skim milk in TPBS. Twofold serum dilutions were prepared in T-PBS and incubated at 37 C for 2 h in the plates. After three washes with T-PBS the plates were incubated with antimonkey IgG-HRP or with anti-human IgG-HRP at rt for 2 h. Finally plates were washed as indicated and the substrate H2O2 (3%) and o-phenylenediamine (0.5 mg ml 1) in citrate buffer 0.1 M, pH 5, was used to develop the chromogenic reaction that was chemically stopped after 15 min. The optical density (OD) was read at 492 nm, in a plate reader [15]. Standard ELISA. The standardized ELISA protocol described by Gheesling et al. [16] was performed with some modifications. Briefly, 5 lg ml 1 of methylated human albumin and 5 lg ml 1 of CCPS in carbonate buffer were used as coating antigen, the plates were incubated overnight at 4 C, and then plates were washed with PBS containing 0.1% of Brij 35 detergent.
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After that, plates were blocked with PBS containing 1% of BSA and 0.1% of Brij 35 for 1 h at 37 C. The serum samples were twofold diluted and incubated in the plates overnight at 4 C in the same buffer. The rest of the experiment was performed as described above. Modified protocol. Finally, sera were assayed in ELISA using derivatized CCPS as coating antigen as described by Granoff et al. [17]. The ELISA was performed with some modifications; specifically the potassium thiocyanate was not added to the incubating buffer. Briefly, native CCPS was derivatized with adipic acid dihydrazide (ADH) by the carbodiimide method [18]. For the ELISA assay, microtiter plates were coated with 100 ll of 1 lg ml 1 derivatized CCPS for 1 h at 37 C. Following the incubation period, the plates were washed three times with T-PBS and blocked with 1% of BSA in PBS for 1 h at 37 C. The serum samples were twofold diluted and incubated in the plates overnight at 4 C in the same buffer. The rest was performed as described above. Titers were determined as the reciprocal dilution yielding twofold OD values of preimmune sera. When human sera were evaluated, a curve of the standard serum CDC 1992 was included and the concentration in lg ml 1 was determined for each serum. 2.5. Avidity index We determined the avidity index (AI) of serum using potassium thiocyanate. Plates were coated with 100 ll of 1 lg ml 1 derivatized CCPS and blocked as described above. Two dilution curves were prepared for each serum, either in T-PBS or in T-PBS containing 0.1 M KSCN. The working concentration of the chaotropic agent was previously selected for properly removing the low avidity antibody without removing the medium or high avidity antigen–antibody complex. Serum dilutions were incubated in the plates at 37 C for 1 h. Bound antibodies were detected incubating with antimonkey IgG-HRP at rt for 1 h. To evaluate the avidity we use an AI expressed as the percentage of antibodies that remained bound to the antigen in the presence of KSCN [19]. 2.6. Bactericidal assay Bactericidal assays were performed in microtiter plates as described by Høiby et al. [20]. Briefly, bacteria of the strain C11 ATCC, were grown overnight in brain– heart infusion (BHI) agar at 37 C in a candle jar. To prepare the inoculum for the test, bacteria were passed to a second plate of BHI agar and allowed to grow for 4 h in the same atmosphere and temperature conditions. Twofold dilutions of the sera, previously inactivated for 30 min at 56 C, were tested with an inoculum of 80–100 colony forming units (cfu) per well, in the presence of 25% of complement. As complement sources, we used
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either a normal human serum or pooled baby rabbit sera, both lacking detectable intrinsic bactericidal activity. The reaction was incubated at 37 C for 60 min as standardized by Maslanka et al. [21]. Titers were determined as the reciprocal of the final serum dilution yielding P 50% killing with respect the control. Simultaneously, the expression of the CCPS after these growth conditions, was confirmed by recognition with the anti capsular monoclonal antibody 4-2-C, from the NIBSC, UK. 2.7. Functional activity of CCPS–P64k conjugates in the infant rat passive protection assay Functional activity was determined as described previously [22], with some modifications. Briefly, rat pups aged five or six days were injected intraperitoneally (i.p) with 100 ll of sera previously diluted 1:10 and ferric dextran. One hour later, infant rats were challenged with 5 · 106 cfu of meningococcal strain C11 ATCC. Six rats were used for each pooled sera, and the mean of the cfu recovered from blood were analyzed. The development of bacteremia was assessed by culturing samples of blood taken 4 h after challenge. The animals were considered protected when the cfu recovered were statistically different from the negative control. 2.8. Statistical analysis For purpose of analysis, the serum titers were logarithmically transformed, and the titers measured by different methods were compared. The significance of differences in antibody levels was assessed by using the Kruskal–Wallis non-parametric analysis followed by DunnÕs test for multiple comparisons. The difference in antibody levels before and after KSCN treatment was determined by Bonferroni pairwise comparison t test. The significance of differences in cfu in the protection studies were assessed by using a single way ANOVA followed by DunnetÕs test for comparison of groups versus control. (Instat version 2.0 software; Graphpad Software, Inc., San Diego, CA). To determine the relationship between IgG titers and the bactericidal titer, Pearson correlation coefficient was calculated.
3. Results 3.1. Comparison of the ELISA procedures In order to compare the performance of the different ELISA procedures in our laboratory, we evaluated 11 paired human sera from healthy volunteers immunized with the polysaccharide vaccine A + C using three ELISA protocols. The standard serum CDC1992 was included in each plate and the antibody concentration
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Standard procedure 60
50
50 IgG µg/mL
IgG µg/mL
Traditional procedure 60
40 30 20
30 20 10
10
0
0 2 (a)
40
6
7
8
9 16 22 volunteers
23 28
32
33
2
6
7
8
9 16 22 volunteers
23
28
32
33
(b)
23
28
32
33
Modified procedure 60
IgG µg/mL
50 40 30 20 10 0 2
6
7
8
(c)
9 16 22 volunteers
Fig. 1. Antibody levels against CCPS detected in the paired human volunteers sera using the three ELISA protocols. (a) Traditional protocol using poly-L -Lys as the activating molecule. (b) ELISA using methylated human albumin mixed with plain CCPS as coating antigen. (c) Modified ELISA using ADH-derivatized CCPS as coating antigen. White columns: preimmune sera. Black columns: immune sera. The modified protocol (panel (c)) was the only method detecting statistical differences between the pre and post vaccinated sera (p = 0.0113).
(lg ml 1) was assigned for each serum. Fig. 1 shows the results obtained. The antibody titers measured by the standard and traditional protocols were higher than those measured by the modified ELISA, especially for the prevaccinated sera. Neither the traditional nor the standard protocols were able to detect the difference in the antibody levels when the preimmune and the immune sera were compared, on the other hand, the modified protocol detected statistical differences between the pre and post vaccinated sera (p = 0.0113). The sera were also assayed for the bactericidal activity against the strain C11 ATCC (Table 1). The bactericidal titers only correlated with the antibody titers measured by the modified ELISA procedure (r = 0.85). 3.2. Antibody response in African Green Monkeys
Table 1 Serum bactericidal titers to N. meningitidis strain C11 ATCC of human volunteers vaccinated with the polysaccharide vaccine A + C from Pasteur vaccines, France, titers were determined before and one month after vaccination Volunteer
2 6 7 8 9 16 22 23 28 32 33
Bactericidal titers Pre vaccination
Post vaccination
– – – – – – – – 1:32 1:128 –
– – 1:256 – 1:256 1:256 – – 1:32 1:256 1:512
The dashes represent no detectable bactericidal activity.
To evaluate the memory immune response generated by the CCPS–P64k conjugate three groups of African Green monkeys were immunized. Two doses were administered one month apart and a year later animals were immunized again for measurement of the booster effect. The antibody response was measured using the same ELISA protocols described above. Fig. 2 shows the kinetics of the antibody response and in all cases the group immunized with the CCPS–P64k conjugate
showed the highest antibody levels. However, there were some differences between the ELISA protocols used. In the case of the traditional protocol, the antibody titers measured for the group immunized with the conjugate were higher than the titers of the other two groups during the entire schedule. This was statistically different in
T. Carmenate et al. / FEMS Immunology and Medical Microbiology 43 (2005) 133–140
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1200
900 800
1000
700 800
500
titer
titer
600 400
600 400
300 200
200
100 0 (a)
30 CCPS-P64k
0 60 270 CCPS + OMV
390 CCPS
420 t (days)
(b)
30 CCPS-P64k
60 270 CCPS + OMV
390 CCPS
420 t (days)
3000 2500
titer
2000 1500 1000 500 0 (c)
30 CCPS-P64k
60 270 CCPS + OMV
390 CCPS
420 t (days)
Fig. 2. Kinetics of the antibody response against native CCPS elicited in African Green Monkeys and measured by three ELISA protocols. (a) Traditional protocol using poly-L -Lys as the activating molecule. (b) Standard ELISA using methylated human albumin mixed with plain CCPS as coating antigen. (c) Modified ELISA using ADH-derivatized CCPS as coating antigen. Arrows indicate the immunization dates. Titers were determined as the reciprocal dilution yielding twofold OD values of preimmune sera.
all the cases with the exception of the extraction after the booster dose. Unexpectedly, after the booster dose only a slightly increment of the antibody titer was observed and this increment did not reach the level of the antibody titer measured after the second dose. The other two ELISA protocols showed a different performance compared with the traditional protocol. The antibody levels were very similar for the three groups after the first and the second doses but after the booster dose a high increment of the titers was observed especially for the group immunized with the conjugate. A threefold increment was measured with the standard protocol and a sixfold increment with the modified protocol. In both cases the antibody titers after the booster dose were statistically different from the titers after the second dose. The antibody titers of the group immunized with the CCPS–P64k were statistically different from the group immunized with plain polysaccharide. In spite of the protocol used, after the booster dose the group immunized with the CCPS–P64k conjugate showed the highest antibody titer followed by the group immunized with the CCPS mixed with OMV. The group immunized with plain polysaccharide developed the poorest response.
3.3. Avidity index To further analyze the maturation of the immune response after the booster dose, the AI of the elicited antibodies was evaluated by comparing the titer measured with and without KSCN added to the antibody diluting buffer during the ELISA procedure. We considered more than 50% as a high AI. The group immunized with the CCPS–P64k conjugate showed high avidity indexes after both the second or the third dose (65.17% and 79.27%, respectively), the group immunized with the CCPS mixed with OMV showed an increment of the AI only after the booster dose (69.74%), while the group immunized with plain CCPS did not show any increment in the AI remaining at 14.83% after the booster dose. 3.4. Bactericidal response The bactericidal activity of the elicited antibodies was determined using either rabbit or human serum as a complement source (Table 2). In general, the bactericidal titers measured with rabbit complement were higher than those measured with human complement.
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Table 2 Mean bactericidal titers of monkey sera to N. meningitidis strain C11 ATCC, determined using baby rabbit or human sera as a complement sources Rabbit complement
1st dose 2nd dose 3rd dose
Human complement
CCPS–P64k
CCPS + OMV
Plain CCPS
CCPS–P64k
CCPS + OMV
Plain CCPS
1:1706 1:2730 1:4437
1:256 1:1024 1:2048
1:160 1:186 1:202
