P64k Meningococcal Protein as Immunological ... - Wiley Online Library

Scand. J. Immunol. 52, 113±116, 2000

P64k Meningococcal Protein as Immunological Carrier for Weak Immunogens Â LEZ,* A. ALVAREZ,* E. CABALLERO,* L. VIN Ä A,² G. GUILLEÂ N* & R. SILVA* S. GONZA *Center for Genetic Engineering and Biotechnology, Havana, Cuba (Received 21 February 2000; Accepted in revised form 5 June 2000)

GonzaÂlez S, Alvarez A, Caballero E, VinÄa L, GuilleÂn G, Silva R. P64k Meningococcal Protein as Immunological Carrier for Weak Immunogens. Scand J Immunol 2000:52:113±116 Previously, the P64k meningococcal protein, an antigen of 64 kDa expressed in Escherichia coli, has been extensively characterized. We have successfully conjugated several synthetic peptides and meningococcal group C polysaccharide to P64k. In three out of four model peptides, the murine humoral immune response against the homologous peptide, evaluated after three doses of conjugate, was higher in the animals immunized with the coupled peptide than in those that received free peptide. The fourth and largest was immunogenic by itself. Similarly, the antigroup C polysaccharide levels reached by conjugated polysaccharide were signi®cantly higher than those produced against unconjugated polysaccharide. As a carrier for one of the peptides, P64k was compared with bovine serum albumin (BSA) and tetanus toxoid (TT), being able to induce slightly higher or similar antipeptide antibody levels than these well-establish protein carriers. Our results suggest that recombinant P64k protein could be a readily available immunological carrier, as ef®cient as other commonly used large carrier molecules. Dr S. GonzaÂlez, DivisioÂn de Vacunas, Centro de IngenieriÂa GeneÂtica y BiotecnologiÂa, Apdo 6162, C. Habana 10600, Cuba. E-mail: [email protected]

INTRODUCTION Frequently, there is a need of conjugating synthetic peptides and microbial polysaccharides to protein carriers, to increase their immunogenicity [1] and/or to turn them in T-cell dependent antigens [2]. Large proteins, like bacterial toxoids, BSA and keyhole limpet hemocyanin, that contain suf®cient reactive groups are widely used for chemical conjugation of peptides and polysaccharides. The high molecular weight meningococcal protein P64k has been expressed as a soluble antigen in Escherichia coli, accounting for 20% of the total cell proteins [3]. Pure recombinant protein, that has been extensively characterized [4,5], was used to immunize mice, rabbits and monkeys. The protein was found to be immunogenic in all these species [6]. Moreover, the antigen seems to elicit antibodies in humans who suffered meningococcal disease [3]. As a result of all these features we decided to evaluate it as a carrier for weak immunogens and to compare its performance with the one shown by known protein carriers, like BSA and tetanus toxoid (TT).

² Present address: Center of Molecular Immunology, PO Box 16040, Havana 11600, Cuba.

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MATERIALS AND METHODS Immunogens. Recombinant P64k protein was obtained as described earlier [3]. BSA (Fraction V Powder) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). TT was kindly donated by the Division of Formulations of our centre. Four peptides were synthesized at the Peptide Synthesis Unit of our institute. The peptide sequences are the following: SP1 from human immunode®ciency virus (HIV) 1 gp120 protein, RQSTPIGLGQALYTT; SP2 from HIV 1 p24 protein, IRQGP KEPFRDYVDRFYK; SP3 from HCV core protein, PKPQRKTKRN TNRRPQDVKFPGGGQIVGGVY; and SP4 from HCV NS4 protein, SGRPAVIPDREVLYQEFDEMEECASHLPYIEQGMQLAEQFKQKA LGL. Meningococcal serogroup C polysaccharide (Men C) was supplied by the Instituto Finlay, Havana, Cuba. Conjugation. The peptides were conjugated to protein carriers by the glutaraldehyde method, as previously described [7]. After coupling, peptide-protein conjugates were examined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) [8] and protein concentration was determined using the Lowry's method [9]. Additionally, the conjugates were analyzed by Immunoblot [10], using monoclonal or polyclonal antibodies that recognized each particular peptide. SP1 was conjugated either to BSA, TT or P64k. SP2 to SP4 were coupled to P64k protein only. Men C was conjugated to P64k via adipic acid dihydrazide, by using the carbodiimide method, as previously described [11]. The polysaccharide content in the samples was determined using the method reported by Svennerholm [12].

114 S. GonzaÂlez et al. Immunizations. In all experiments 5 to 10 female BALB/c mice (8± 10-week-old) per group were immunized. In a ®rst experiment, three doses (20 mg each) of either SP1-BSA, SP1-P64k, BSA, P64k, or free peptide, emulsi®ed with Freund's Adjuvant were subcutaneously (s.c.) administered, at two-week intervals, to mice divided in ®ve experimental groups. In a second experiment, mice received three doses (10 mg each) of SP1-TT, SP1-P64k or free peptide, absorbed to 400 mg of aluminium hydroxide (Superfos Biosector, Vedbaek, Denmark). The doses were given s.c. at two-week intervals. A third immunization schedule was performed with the same antigen amount, adjuvant, route, and time intervals. Mice were injected with three doses of SP2, SP3 or SP4, respectively, either free or coupled to P64k. In a fourth experiment, the animals were s.c. immunized with three doses of either Men C (2,5 mg), P64k (3 mg) or Men C-P64k (2,5 mg Men C and 3 mg P64k/dose) absorbed to 100 mg of aluminium hydroxide. In all cases, the immunizations were given in a total volume of 100 ml on days 0, 14 and 28. Serum samples were obtained from blood extracted from mice at days 0, 14, 28, 35 and 42. ELISA. Antibody levels in sera were determined by Enzyme Linked Immunosorbent Assay (ELISA). To detect antipeptide antibodies, 96well plates (High Binding, Costar, USA) were coated with 100 ml/well of SP1 (20 mg/ml), SP2 (10 mg/ml), SP3 (0.8 mg/ml) or SP4 (1.6 mg/ml) in carbonate buffer (0.05 M Na2CO3, pH 9.6). Skim-milk powder (3%) was used as a blocking reagent. Plates were processed as published elsewhere [13]. Anti-Men C antibodies were measured as previously described [14]. All sera were analyzed in duplicate. Serum antipeptide and antipolysaccharide antibody levels were expressed as their absorbance (492 nm) values in ELISA and used for statistical analysis. Moreover, pooled sera from each group were titrated by serial dilution, being the cut-off values calculated as twice the mean absorbance of preimmune serum. Statistical methods. The signi®cance of differences between data in the second and third immunization experiment was determined with the Student's t-test. Conversely, in the ®rst experiment, the signi®cance of differences between antibody levels was analyzed by using a split plot design (simple) with mice as plots, protein carrier as the between subjects factor (factor A) and time point as within blocks factor (factor B). In the last immunization routine, Newman±Keuls multiple comparison test was used to determined differences between the data. A P-value of < 0.05 was considered statistically signi®cant. In the ®gures, bars represent the mean of antibody levels 6 the standard deviation for each experimental group. Reciprocal antipeptide or antipolysaccharide titers of pooled sera corresponding to each experimental group are shown on the bars.

the mice. Figure 1(A) re¯ects the time course of antipeptide antibody levels observed in the groups that received SP1 coupled to the two protein carriers. As it can be seen, both conjugates were immunogenic in mice. The level of anti-SP1 antibodies was slightly higher (with statistical signi®cance) in the group immunized with SP1-P64k than in the group injected with SP1-BSA. The antibody levels remained negligible, after three doses of antigen, for the animals immunized either with carrier protein or free peptide (data not shown). In a second experiment, the carrier capacity previously found for P64k was demonstrated by comparing it with that showed by TT. The conjugate SP1-TT migrated in SDS-PAGE like the previously described conjugates, but the upper zone contained most of the molecules. Again, the peptide substitution rates were estimated to be similar for both conjugates by Immunoblot. Mice were immunized with SP1 either conjugated to P64k, to TT or

RESULTS P64k as a carrier, compared to BSA and TT In a parallel coupling reaction, the peptide SP1 (15 mer) was linked ®rst to P64k and BSA. Both conjugates migrated in SDSPAGE as a smear of multiple bands, which are mainly concentrated in two zones (data not shown). The upper zone included bands of molecular weight higher than 116 kDa and the lower zone was composed by molecules weighing between 97 kDa and 66 kDa, respectively. A peptide-speci®c Immunoblot suggested similar peptide substitution rates for both conjugates. Protein carriers, conjugates, and free peptide were employed to immunize

Fig. 1. Anti-SP1 antibody levels. (A) Mice (n 5) were subcutaneously (s.c.) immunized with three doses (20 mg/dose) of SP1-P64k or SP1-BSA. (B) Mice (n 10) were s.c. immunized with three doses (10 mg/dose) of SP1-P64k or SP1-TT. Anti-peptide antibody levels in sera are expressed as their absorbance (492 nm) values in ELISA (Serum dilution: 1: 250). Reciprocal antipeptide titers of pooled sera corresponding to each experimental group are shown on the bars.

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P64k as a Carrier

Fig. 2. Anti-peptide antibody levels. Mice (n 8) were s.c. immunized with three doses (10 mg/dose) of free SP2, SP3, or SP4, or the same amount of the respective P64k conjugate. Antibody levels in sera were measured against the homologous peptide and are expressed as their absorbance (492 nm) values in ELISA (Serum dilution 1 : 100). Reciprocal antipeptide titers of pooled sera corresponding to each experimental group are shown on the bars.

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Fig. 3. Anti-meningococcal group C polysaccharide (Men C) antibodies. Mice (n 7) were immunized with three doses of Men C (2,5 mg/dose), P64k (3 mg/dose), or Men C-P64k (2,5 mg Men C/dose). Anti-Men C antibody levels in sera are expressed as their absorbance (492 nm) values in ELISA (Serum dilution 1 : 100). Reciprocal antiMen C titers of pooled sera corresponding to each experimental group are shown on the bars.

DISCUSSION free, using aluminium hydroxide as an adjuvant. Figure 1(B) shows the time course of anti-SP1 antibody levels for the two experimental groups that received conjugates. Both conjugates elicited similar antipeptide antibodies after the second dose (P < 0.05), that remained true after a third one. The uncoupled peptide was not immunogenic in mice, even after three injections (data not shown). P64k as a carrier for peptides of variable length To further study the carrier ability of the recombinant meningococcal protein, we chemically coupled peptides 18 amino acid residues (aa) (SP2), 31 aa (SP3) and 47 aa long (SP4), respectively, to P64k and injected mice either with the conjugates or free homologous peptide. Two weeks after the second dose, there was a signi®cant statistical difference between the group that received the conjugate and the group immunized with free peptide (data not shown). Fifteen days after the third dose, the same difference was observed for SP2 and SP3 (Fig. 2), whereas similar levels of antipeptide antibodies were found for SP4 free or coupled to P64k.

P64k as a carrier for Men C polysaccharide Men C polysaccharide conjugated to P64k elicited antipolysaccharide antibodies in mice after immunization (Fig. 3). Even after the ®rst dose, the conjugate was immunogenic. The antibody levels continued to increase after the second dose and remained stable after the third one. There was no signi®cant statistical difference between data obtained after the second and third dose of conjugate.

In the present study, we have coupled P64k to four peptides and Men C polysaccharide and administered three doses of conjugates to mice. After two doses of peptide coupled to P64k, most of the mice seroconverted; having the expected response, considering its high molecular weight and observed immunogenicity. Even after three doses of antigen, three of the uncoupled peptides failed to elicit a signi®cant antibody response. Only SP4 (47 mer), induced antibody levels similar to those produced by the conjugated peptide, that could be expected owing to its length. Most probably it contains both T-cell and B-cell epitopes, having itself the ability to induce a humoral immune response after repeated immunizations. Owing to the encouraging results achieved in the ®rst immunization experiment, we decided to half the antigen amount in the second and third one, obtaining positive results as well. It is worth noting that P64k can exert its carrier function absorbed on aluminium hydroxide, one of the few adjuvants widely used in human vaccines. The antibody levels against the meningococcal group C polysaccharide were signi®cantly increased after its conjugation to P64k. Similar results were obtained by Costantino and coworkers [15], who coupled Men C to CRM 197 and injected the conjugate in mice and rabbits. However, MenC±P64k could prime the host for a T-cell memory response to the pathogen, by employing a carrier protein derived from the same bacterium. BSA, as a carrier, is widely used at the laboratory scale because of its availability and reduced cost. However, its mammalian origin, has been reported to reduce its ef®ciency for that purpose [16]. The foreignness of meningococcal P64k can contribute to its performance in this respect. Frequently, the haptens have been coupled to TT or cholera toxoid in human vaccines, because these proteins are readily available and have been used in humans without side-effects [17]. Nonetheless,


116 S. GonzaÂlez et al. owing to the successful development of new conjugate vaccines and the limited availability of protein carriers, concern is increasing regarding the epitope overload and suppression that can take place when the same molecule is used in several vaccines [18,19]. The meningococcal protein employed by us can circumvent these drawbacks of known carriers. Recently, a Phase I clinical trial conducted in healthy human volunteers showed that P64k is safe and immunogenic after a three-dose immunization protocol [20]. The recombinant P64k meningococcal protein could be used in future conjugate vaccines. The T-cell epitopes present in this protein are under investigation.

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ACKNOWLEDGMENTS

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We are grateful to Ms Dagmara Pichardo for her excellent work in the animal care and immunization. We also acknowledge the help of Dr Carlos Duarte for providing us with peptide SP1 and helpful advice. Dr Lidia I. Novoa kindly provided us with peptides SP3 and SP4.

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