INFECTION AND IMMUNITY, Sept. 2005, p. 5835–5841 0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.9.5835–5841.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 9
Combined Conjugate Vaccines: Enhanced Immunogenicity with the N19 Polyepitope as a Carrier Protein Karin Baraldo,1 Elena Mori,1 Antonella Bartoloni,2 Francesco Norelli,2 Guido Grandi,1 Rino Rappuoli,1 Oretta Finco,1 and Giuseppe Del Giudice1* Research Center1 and Technology Development,2 Chiron Vaccines, via Fiorentina 1, 53100 Siena, Italy Received 25 March 2005/Returned for modification 11 April 2005/Accepted 12 May 2005
The N19 polyepitope, consisting of a sequential string of universal human CD4ⴙ-T-cell epitopes, was tested as a carrier protein in a formulation of combined glycoconjugate vaccines containing the capsular polysaccharides (PSs) of Neisseria meningitidis serogroups A, C, W-135, and Y. Good antibody responses to all four polysaccharides were induced by one single immunization of mice with N19-based conjugates. Two immunizations with N19 conjugates elicited anti-MenACWY antibody titers comparable to those induced after three doses of glycoconjugates containing CRM197 as carrier protein. Compared to cross-reacting material (CRM)based constructs, lower amounts of N19-MenACWY conjugates still induced high bactericidal titers to all four PSs. Moreover, N19-MenACWY-conjugated constructs induced faster and higher antibody avidity maturation against meningococcal C PS than CRM-based conjugates. Very importantly, N19-specific antibodies did not cross-react with the parent protein from which N19 epitopes were derived, e.g., tetanus toxoid and influenza virus hemagglutinin. Finally, T helper epitopes of the N19 carrier protein were effectively generated both in vivo (after immunization with the N19 itself) and in vitro (after restimulation of epitope-specific spleen cells). Taken together, these data show that the N19 polyepitope represents a strong and valid option for the generation of improved or new combined glycoconjugate vaccines. sponse has been attributed to carrier overload or carrier-mediated epitope suppression (13, 17, 35), resulting in the competition between carrier- and PS-specific B cells and consequently in a reduced antibody response to the PSs (6, 13, 14, 40). This argues for the need of alternative carrier molecules. Abrogation of suppression was obtained by replacing fulllength proteins with peptides containing T-helper-cell epitopes and lacking B-cell epitopes (1, 7, 15). The use of human universal epitopes, being able to bind most of the HLA class II molecules, would enable the whole population to respond to the immunization irrespective of their major histocompatibility complex makeup (2, 7, 16, 29, 30). Along these lines, novel polyepitope carrier proteins have been genetically engineered in our laboratories by assembling 6, 10, or 19 human universal T helper epitopes (referred to as N6, N10, and N19, respectively) (16). In a previous work, we reported that the N19 polyepitope conjugated to MenC PS exerts a stronger carrier effect than the conventional carrier protein CRM197 in terms of induction of anti-MenC serum antibody titers and of antibodies with bactericidal activity (5). Here, we report the results of experiments aimed at investigating the carrier effect of the N19 polyepitope in a combined conjugate vaccine containing capsular PSs of Neisseria meningitidis serogroups A, C, W-135, and Y (MenACWY). We examined the antibody response to the capsular PSs in terms of bactericidal activity and avidity. Moreover, we addressed the issue of the potential cross-reactivity of anticarrier antibodies with the parent proteins from which N19 epitopes derive, among them, TT and influenza hemagglutinin (HA). Finally, we investigated the generation in mice of the T-helper-cell epitopes present in N19.
The limited immune response of infants to most bacterial capsular polysaccharides (PSs) makes them a population at risk of infections with encapsulated bacteria such as Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, Neisseria meningitidis (Men), and others. Immunization with conjugate vaccines consisting of the capsular PS covalently linked to a protein carrier has resulted in a remarkable decline in the incidence of disease caused by those pathogens (28, 37, 46). The conjugation of a PS to carrier proteins generates T-celldependent antibody responses that lead to the production of protective anti-PS immunoglobulin G (IgG) and induction of immunologic memory even at a very young age (25, 27). Since protective immunity is mediated by antibodies to group-specific PSs and since many different serotypes of the same pathogen are associated with disease, the approach is to combine several conjugates in the same formulation in which each PS is individually coupled to a carrier molecule. Several combined conjugate vaccines have been developed, such as the heptavalent pneumococcal vaccine (8) and the tetravalent meningococcal combination vaccine (44), and others are under development. Most licensed conjugate vaccines utilize only a few carrier proteins, mainly tetanus toxoid (TT) and diphtheria antigens (DT and CRM197), which are also commonly used vaccines, and few others. The limited number of carriers implies an increasing number of conjugate vaccines using the same carrier, with the consequent risk of a reduced immunogenicity of individual conjugates when administered in multivalent formulations (8, 26). The observed impaired anti-PS antibody re-
* Corresponding author. Mailing address: Research Center, Chiron Vaccines, via Fiorentina 1, 53100 Siena, Italy. Phone: 39-0577-243261. Fax: 39-0577-243564. E-mail: [email protected]
BARALDO ET AL. MATERIALS AND METHODS
Preparation of N19-MenACWY conjugates. N19 is a recombinant polyepitope consisting of 19 human universal CD4⫹-T-cell epitopes derived from various microbial antigens (5, 16). N19 recombinant polyepitope was expressed in Escherichia coli and purified as previously described in detail (16). Meningococcal serogroup A, C, W-135, and Y PSs (MenA, MenC, MenW-135, MenY) and CRM197-based conjugates were prepared as already described (11, 12, 39). The same conjugation chemistry was used for the preparation of N19-MenACWY constructs (5). The saccharide content of MenC, MenW-135, and MenY conjugates was quantified by sialic acid determination (45), while that of MenA conjugate was quantified by mannosamine-1-phosphate chromatographic determination (41). The protein content was measured by a micro-bicinchoninic acid assay (Pierce, Rockford, IL). The sugar-to-protein ratio of N19-based conjugates ranged between 0.3 and 1.5, similar to that of cross-reacting material (CRM)based conjugates. Mouse immunizations. Groups of six female 7-week-old BALB/c mice (Charles River, Calco, Italy) were used in each experiment. Tetravalent formulations were prepared by mixing together in equivalent saccharide amounts N19-MenA, N19-MenC, N19-MenW, and N19-MenY (N19-MenACWY) or the equivalent CRM-based conjugates. Each group of mice was immunized subcutaneously three times with various dosages of tetravalent conjugate (from 2 to 0.074 g of each MenPS per dose) in the presence of 0.06 mg aluminum phosphate as adjuvant. Mice were immunized on days 0, 21, and 35 and bled at days ⫺1 (pre), 20 (post-1), 34 (post-2), and 45 (post-3). Individual serum samples were taken at each time point and kept frozen at ⫺20°C until use. Titration of antibodies specific for the meningococcal PS serogroup, carriers, and parent proteins. Titration of MenA-, MenC-, MenW-135-, and MenYspecific IgG antibodies was performed on individual sera from each mouse according to the assays already described (10). Briefly, enzyme-linked immunosorbent assay (ELISA) plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 5 g/ml of purified N. meningitidis serogroup A, C, W-135, or Y PSs in the presence of 5 g/ml methylated human serum albumin. Titration of antibodies to N19, CRM197, TT, HA (A/New Caledonia H1N1 and A/Panama H3N2 hemagglutinin), and DT (all from Chiron Vaccines, Siena, Italy) was performed on pooled sera as described previously (5). ELISA plates were coated overnight at 4°C with a phosphate-buffered saline solution containing 2 g/ml of N19, TT, HA, or CRM197 or 5 g/ml of DT protein. The titers of antigen-specific IgG were determined by using alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma Chemical Co., St Louis, MO). Titers were calculated by using the reference line method (22) and expressed as the logarithm of ELISA units (EU)/ml. Preimmunization values consistently gave optical density values below 0.1. Titration of serum meningococcal serogroup-specific bactericidal antibodies. Serum bactericidal antibody activity (SBA) was measured against N. meningitidis serogroup A (strain F8238), C (strain 11), W-135 (strain 240070), or Y (strain 240539) using baby rabbit serum as a source of complement (PelFreeze; Rogers, AR) (31). Titers were determined by calculating the dilution of test serum showing a 50% decrease in the number of CFU after 1 h of incubation compared with time zero. Avidity of MenA- and MenC-specific antibodies. The avidity of MenA- and MenC-specific IgG antibodies was determined by a modified ELISA on pooled sera, using 75 mM of ammonium thiocyanate (NH4SCN) as chaotropic agent as described previously (24). Assay validation included the assessment of antigen stability following incubation with 4 M NH4SCN (42). ELISA plates were coated overnight at 4°C with 5 g/ml of purified N. meningitidis PSs separately. Duplicate twofold dilutions of test and reference sera were prepared in a microplate. Serum samples in one of the duplicates were incubated 15 min at room temperature with 75 mM NH4SCN in serum dilution buffer, whereas dilution buffer was added to the other duplicate. After washing, the plates were incubated with alkaline-phosphatase-conjugated goat anti-mouse IgG antibodies (Sigma Chemical Co.) as described above. Results were expressed as the avidity index (AI), expressed as the percentage of antibodies that remained bound to the antigens after thiocyanate treatment. The AI was calculated as follows: AI ⫽ (titer with NH4SCN)/(titer without NH4SCN) ⫻ 100 (3). Statistical analysis. Student’s t test (two tails) was used to compare antibody titers between groups and at different times. A P value of ⬍0.05 was considered statistically significant. Lymphocyte proliferation assay. Spleens of mice immunized as described above with N19-MenACWY were collected and tested for their ability to proliferate following in vitro stimulation with synthetic peptides (Primm srl, Milan, Italy) reproducing the epitopes present in the N19 protein. A total of 5 ⫻ 105 cells/well were cultured in 200 l of RPMI (GIBCO) supplemented with 25 mM
INFECT. IMMUN. HEPES buffer, antibiotics, 0.5 M 2-mercaptoethanol, L-glutamine, sodium pyruvate, vitamins, and a cocktail of nonessential amino acids (1% of a 100⫻ stock; GIBCO) and 5% fetal calf serum (HyClone). Cells were cultured at 37°C in 5% CO2 in a flat-bottom 96-well cell culture cluster (Corning, NY) in the presence of various concentrations of the single peptides or with medium alone. On day 5, the cultures were pulsed with 1 Ci of [3H]thymidine/well. After 18 h, cultures were harvested and thymidine incorporation was measured by using a liquid scintillation counter (cpm value). The stimulation index (SI) was calculated as the ratio between the mean cpm of triplicate cultures stimulated with peptide and the mean cpm of triplicate cultures treated with medium alone. Alternatively, groups of three mice were immunized at the base of the tail with a 50-l volume containing 50 g of antigen emulsified 1:1 in complete Freund’s adjuvant (CFA). Then, 7 days later, inguinal lymph nodes (LN) were removed and LN cells (3 ⫻ 105 cells/well) were tested for their capacity to proliferate in the presence of the homologous antigens or of the N19 polyepitope (47) after a 6-day culture. Results are expressed as averages of SIs of groups of three mice.
RESULTS IgG antibody responses against capsular MenA, MenC, MenW-135, and MenY PS. Dose response studies were conducted to assess the strength of the carrier effect of N19 in a tetravalent conjugate vaccine compared to that of a CRMbased vaccine. Analysis of IgG antibody levels against the four MenPSs (Fig. 1) showed that conjugates based on N19, but not those containing CRM197, induced remarkable serum anti-PS IgG responses already after the first immunization in a dosedependent fashion. In particular, the antibody titers against MenA and MenW-135 induced by one injection of N19 conjugates were significantly higher than those induced by CRMMenACWY when given at the highest dosage (post-1 at [2 g] N19 versus CRM; P ⬍ 0.05). Nevertheless, one injection of N19 conjugates elicited antibody titers against MenC significantly higher than those with CRM conjugates at all dosages tested (post-1 at [0.074 to 2 g] N19 versus CRM; P ⬍ 0.05). After two immunizations, the antibody responses to N19MenACWY constructs, at whatever dosage, reached a plateau level and did not significantly increase after the third dose. It is noteworthy that, after the second immunization, the antibody titers against serogroups A and C induced by N19-based conjugates were significantly higher than those induced by CRMbased conjugates (IgG anti-MenA and anti-MenC, post-2 at [0.074 to 2 g] N19 versus CRM; P ⬍ 0.05). To reach titers against MenA and MenC comparable to those induced by N19 conjugates, CRM conjugates required three injections. The higher immunogenicity of N19-based conjugates was also evident in terms of the number of mice responding to immunization (Table 1). Indeed, after the first and the second doses, more animals responded to N19 conjugates than to CRM conjugates by producing detectable titers of antiMenACWY antibodies. The IgG antibody response to the conjugates was characterized predominantly by the IgG1 isotype (data not shown). Serogroup-specific bactericidal antibody activity. SBA activity has been shown to correlate with protection from meningococcal disease in a serogroup-specific fashion (21); vaccineinduced bactericidal antibodies are regarded as a surrogate of efficacy (4). N19 conjugates were highly effective in inducing bactericidal antibodies against the four meningococcal capsular PSs. Notably, the N19 carrier was significantly better than the CRM197 carrier in inducing MenC-specific serum bactericidal activity after two doses, at all dose regimens (data not shown). Moreover, by reducing the dosage of conjugates, those
VOL. 73, 2005
N19 POLYEPITOPE AS CARRIER PROTEIN
FIG. 1. Serum anti-MenACWY IgG antibody responses. Groups of six BALB/c mice were immunized three times subcutaneously with increasing amounts (0.074, 0.22, 0.67, 2 g/dose of each MenPS) of N19-MenACWY (closed symbols) or CRM-MenACWY (open symbols) in the presence of 0.06 mg of aluminum phosphate as adjuvant. Serum anti-MenA-, anti-MenC-, anti-MenW-135-, and anti-MenY-specific IgG antibody titers were measured after each immunization (post-1, -2, and -3). Each point represents the mean antibody titer of each group at each time point.
based on the carrier N19 were stronger in maintaining their ability to induce high levels of SBA antibodies, in particular, against MenC and MenW-135, even at the lowest dosage. As shown in Fig. 2, N19 conjugates at the lowest dosage (0.074 g) induced better SBA titers than CRM conjugates against all four serogroups. Again, N19-based conjugates required only two injections to induce anti-MenC and W-135 SBA titers remarkably higher than those induced by three injections of CRM conjugates. Antibody avidity maturation. Antibody avidity has been also considered as a measure of the protective functional activity of antimeningococcal PS antibodies (19, 20). We then asked
TABLE 1. Percentage of mice responding to the immunizations with glycoconjugates containing N19 or CRM197 as carrier proteina % of responder mice to MenPS PS (g/dose)
Post-1 2 0.67 0.22 0.074
83 67 17 0
0 17 0 0
100 100 83 83
Post-2 2 0.67 0.22 0.074
100 100 100 100
67 50 83 33
Post-3 2 0.67 0.22 0.074
100 100 100 83
83 83 100 100
0 0 0 0
83 67 50 0
0 17 0 17
100 100 50 67
33 17 33 17
100 100 100 100
100 83 100 67
100 100 100 100
83 83 100 83
100 100 100 100
100 83 100 100
100 100 100 100
100 83 100 100
100 100 100 100
83 67 100 67
100 100 100 100
100 83 100 100
Groups of six BALB/c mice were immunized three times with either N19MenACWY or CRM-MenACWY as described in Materials and Methods. After each immunization (Post-1, Post-2, Post-3), single sera were tested by ELISA to determine the titers of IgG antibodies specific for each of the four PSs. Mice were considered responders when the log10 of the IgG ELISA unit was ⬎0.3.
whether the better carrier effect of N19 reflected also the capability of this polyepitope to induce serogroup-specific antibodies with a higher avidity than those induced by CRMbased conjugates. We followed the avidity maturation over time and doses of immunization. Remarkably, after the first dose of the 2-g regimen of N19 conjugates, the anti-MenC antibody avidity was already high (Fig. 3) and after the second dose, the antibodies present had a high avidity index. Lower avidity maturation profiles were found with CRM conjugates at 2- to 0.074-g regimen and with N19 conjugates at a 0.67- to 0.074-g regimen. Antibody responses to carriers and parent proteins. It has been postulated in humans that the anticarrier antibodies in serum can interfere with the immune response to PSs following immunization with glycoconjugates containing that carrier (13). Since there is an overload of carrier protein in combination vaccines, we analyzed the antibody response to the N19 polyepitope and to the CRM197 protein, as well as to their parent proteins (Fig. 4). As expected by the nature of CRM197 and in agreement with data from others (33, 38), we found that CRM-based conjugates induce the production of anti-CRM
FIG. 2. Bactericidal activity against N. meningitidis serogroups A, C, W-135, and Y in pooled serum from mice after two (post-2) and three (post-3) immunizations with 0.074 g of each MenPS per dose (N19-MenACWY or CRM-MenACWY). Titers are expressed as reciprocal values of the highest serum dilution giving at least 50% bacterial killing.
BARALDO ET AL.
FIG. 3. Avidity maturation of anti-MenC antibodies in mice after immunization with N19-MenACWY or CRM-MenACWY. Avidity index of specific IgG antibodies was measured on pooled serum by a modified ELISA as described in detail in Materials and Methods. AI corresponds to the percentage of bound antibodies after elution with 75 mM NH4SCN of each group at each time point.
antibodies and that these antibodies recognize DT also (Fig. 4, left panel). On the contrary, although N19 at the dosages given induced antibodies against itself, these antibodies did not recognize any of the tested parent proteins from which N19 epitopes were derived (16), e.g., TT and influenza HA (Fig. 4, right panel). N19 epitope-driven T-cell proliferative responses. The epitopes assembled within the N19 polyepitope were selected since they were known to be universal CD4⫹-T-cell epitopes in humans. In previous work, we showed that these epitopes were recognized by specific human T-cell clones (16). Here, we asked whether N19 epitopes were indeed generated also in the mouse after immunization. To answer this question, two approaches were undertaken. In the first one, mice were immunized with N19-MenACWY conjugates as in the experiments reported above and spleen cells were stimulated in vitro with synthetic peptides reproducing the epitopes contained in the N19 protein. Figure 5A shows that, in addition to the N19 polyepitope itself, various TT
FIG. 4. Antibody responses to carrier proteins and to their parent proteins in pooled sera obtained after the third immunization. Since mice were immunized with an equivalent amount of each MenPS in either conjugate, the final amount of carrier differs in the groups receiving CRM-MenACWY and those receiving N19-MenACWY due to a different sugar-to-protein ratio in the four constructs. Each point represents the antibody titer of each group after three immunizations as described before.
FIG. 5. N19 epitope-driven T-cell proliferative responses. (A) Spleen cells from mice immunized subcutaneously with N19MenACWY were stimulated in vitro in the presence of the indicated antigens (N19 polyepitope or synthetic peptides), and proliferation was measured as described in Materials and Methods. Results are expressed as SI ⫽ (cpm experimental/cpm background unstimulated) obtained at concentrations of peptides ranging from 10 to 30 g/ml. (B) Stimulation indices to HA, HBsAg, P23TT peptides, and N19 protein as determined in proliferation assay. Groups of three mice were immunized at the base of the tail with a 50-l volume containing 50 g of P23TT, HBsAg, or HA peptides emulsified 1:1 in CFA. Seven days later, draining LN were removed and LN cells tested for their capacity to proliferate in presence of the homologous peptide or N19 at concentrations ranging from 10 to 30 g/ml. Each column represents the mean of triplicate cultures of LN cells from three mice.
epitopes (e.g., P32TT, P30TT, P23TT), the HA, and the HBsAg peptides induced the proliferation of spleen cells, showing that T-cell precursors specific for these epitopes had been generated in vivo in mice following immunization with N19MenACWY conjugates. No proliferative responses were detected after in vitro stimulation with the P21TT and P2TT peptides. In the second approach, mice were immunized at the base of the tail with single synthetic peptides reproducing some of the epitopes contained in the N19 polyepitope; then, cells collected from draining LN were stimulated in vitro either with the homologous peptide or with the N19 polyepitope. As shown in Fig. 5B, proliferative responses of LN cells were detectable not only after restimulation in vitro with the homologous peptides but also and importantly with the N19 protein. These results clearly demonstrate that mouse antigen-presenting cells in vitro were able to process the N19 protein and to generate the correct epitopes which, in turn, were able to reactivate the specific T cells, which had been primed by the in vivo immunization with the single peptide. DISCUSSION In searching for novel carriers for conjugate vaccines, the N19 polyepitope was conceived based on the rational assembling of various universal human T-cell epitopes able to inter-
VOL. 73, 2005
act with the vast majority of HLA-DR molecules and to induce T-cell help for antibody production to the conjugated PS in the vast majority of the human population (16). The enhanced immunogenicity and protective efficacy of N19 polyepitopebased monovalent conjugate vaccines previously reported (5, 16) prompted us to investigate the strength of the carrier effect of the N19 polyepitope in multivalent vaccines. In the present study, we have shown the higher immunogenicity of N19-based conjugates compared to those based on the conventional carrier CRM197, in a combined tetravalent meningococcal vaccine formulation. Indeed, in agreement with previous data with the monovalent MenC conjugate (5), the use of the N19 polyepitope carrier induced a faster and stronger immune response to the four conjugated meningococcal capsular PSs already after one single administration; the plateau of antibody levels was reached after the second dose. In contrast, CRM-based conjugates consistently required a third injection to reach titers comparable to those obtained with N19 conjugates. This demonstrates the feasibility of combining several N19-based conjugates together in order to broaden the coverage of the vaccination to multiple meningococcal serogroups. The stronger helper effect of the N19 polyepitope was also reflected by the ability of this carrier, compared to that of the CRM197 protein, to induce higher titers of bactericidal antibodies, which are known to mediate protection against meningococci (23). It is noteworthy that, unlike the CRM conjugates, higher bactericidal antibody titers were induced even at the lowest dosages of N19 conjugates. This was particularly evident for N19-based MenC and MenW-135 conjugates, which induced bactericidal antibody titers remarkably higher after two doses than those induced by three doses of CRM conjugates. All these data suggest that the use of N19 as a carrier protein could allow the induction of effective protective immunity faster and with a lower amount of vaccine than conventional conjugates. It has been hypothesized that preexisting immunity against carrier proteins can negatively affect the immune response to conjugated vaccines containing the same carrier protein (40, 43). The analysis of the immune response to the carrier after immunization with a conjugate is thus of critical importance to understanding the outcome of the immune response to the PS moiety of the conjugate. As amply expected from the structure of CRM197, which differs from the native DT at the level of only 1 amino acid (18), immunization with CRM-based conjugates induces a strong antibody response against DT. These data are in full agreement with data obtained from children immunized with CRM197-based conjugated vaccines (33, 34) and from adults immunized with CRM197 to boost anti-DT immunity primed at infancy (32, 36). We have previously shown that immunization with the monovalent N19-MenC conjugate does not induce significant anti-N19 antibody levels (5). In the work reported here, we found that the concomitant administration of four N19-based meningococcal conjugate vaccines induces anti-N19 antibody titers higher than those after immunization with monovalent N19-MenC conjugate. Nevertheless, these antibodies did not cross-react with the native proteins from which the epitopes contained in the N19 protein were derived. This negative finding is particularly relevant for TT, since more than 50% of the N19 sequence is composed of epitopes derived from TT. The
N19 POLYEPITOPE AS CARRIER PROTEIN
lack of interference between N19 and TT immunity was further stressed by experiments in vivo in which the pretreatment of mice with high doses of TT in Freund’s adjuvant did not affect the anti-PS antibody response after immunization with the four N19-based meningococcal conjugates (data not shown). Taken together, these data demonstrate that N19 exerts a strong helper effect for multiple conjugate vaccines without inducing an immune response to itself that may interfere with those directed to the native proteins. The enhanced helper effect of the N19 polyepitope compared to that of conventional carrier proteins, such as CRM197, is also demonstrated by the avidity indices of the anti-PS antibodies induced. Indeed, anti-PS antibodies induced by N19-based conjugates exhibit avidity indices higher than those of serum antibodies induced by CRM-based conjugates. It is known that anti-PS antibodies with high avidity have better bactericidal activity than antibodies of low avidity, such as those induced by vaccines consisting of plain PSs (9). This would suggest that the kinetics and strength of avidity maturation can be driven by the intrinsic characteristics of the carrier protein present in the glycoconjugate and that, probably through the string of sequential T helper epitopes present in its sequence, N19 would be better suited than a conventional carrier protein to induce anti-PS antibodies with higher avidity. Also, the high number of universal CD4⫹ epitopes present in the N19 sequence may account for the enhanced helper effect to the conjugated PS moiety observed. We had previously shown that some epitopes present in N19 were correctly generated, since this polyepitope was able to induce the proliferation of human T-cell clones specific for some of the TT epitopes present in the N19 sequence (16). In this paper, we have shown that most of the epitopes represented in the N19 sequence are indeed generated also in the mouse. The fact that synthetic peptides reproducing some N19 epitopes did not induce detectable proliferative responses can be explained by the fact that CD4⫹-T-cell precursors are not generated to each epitope at the same frequency. The fact that these epitopes are indeed generated in the mouse was formally demonstrated by the observation that priming in vivo with single peptides generated specific CD4⫹ cells which proliferated when restimulated in vitro with the N19 polyepitope, demonstrating that the epitope(s) was correctly generated and presented by the murine antigen-presenting cells. In conclusion, we have shown that the N19 polyepitope behaves as a potent carrier protein for combined conjugated vaccines, inducing potent protective bactericidal antibody responses and fast and strong avidity maturation. The intrinsic nature of the N19 polyepitope, exquisitely consisting of universal T-cell epitopes, renders it suitable for the generation of improved or new combined conjugate vaccines with a very low risk of unwanted effects due to carrier overload or carrier epitope suppression.
ACKNOWLEDGMENTS We thank Giampietro Corradin, University of Lausanne, Lausanne, Switzerland, for insightful suggestions with the use of synthetic peptides; Francesca Meini and Cristiana Balocchi for expert performance of the bactericidal assay; Marco Tortoli for technical support with mice; and Giorgio Corsi for preparation of the figures.
BARALDO ET AL.
REFERENCES 1. Alexander, J., M. F. del Guercio, B. Frame, A. Maewal, A. Sette, M. H. Nahm, and M. J. Newman. 2004. Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE). Vaccine 22:2362–2367. 2. Alexander, J., M. F. del Guercio, A. Maewal, L. Qiao, J. Fikes, R. W. Chesnut, J. Paulson, D. R. Bundle, S. DeFrees, and A. Sette. 2000. Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. J. Immunol. 164:1625–1633. 3. Anttila, M., J. Eskola, H. Ahman, and H. Kayhty. 1999. Differences in the avidity of antibodies evoked by four different pneumococcal conjugate vaccines in early childhood. Vaccine 17:1970–1977. 4. Balmer, P., and R. Borrow. 2004. Serologic correlates of protection for evaluating the response to meningococcal vaccines. Expert Rev. Vaccines 3:77–87. 5. Baraldo, K., E. Mori, A. Bartoloni, R. Petracca, A. Giannozzi, F. Norelli, R. Rappuoli, G. Grandi, and G. Del Giudice. 2004. N19 polyepitope as a carrier for enhanced immunogenicity and protective efficacy of meningococcal conjugate vaccines. Infect. Immun. 72:4884–4887. 6. Bergquist, C., T. Lagergard, and J. Holmgren. 1997. Anticarrier immunity suppresses the antibody response to polysaccharide antigens after intranasal immunization with the polysaccharide-protein conjugate. Infect. Immun. 65:1579–1583. 7. Bixler, G. S., Jr., R. Eby, K. M. Dermody, R. M. Woods, R. C. Seid, and S. Pillai. 1989. Synthetic peptide representing a T-cell epitope of CRM197 substitutes as carrier molecule in a Haemophilus influenzae type B (Hib) conjugate vaccine. Adv. Exp. Med. Biol. 251:175–180. 8. Bogaert, D., P. W. Hermans, P. V. Adrian, H. C. Rumke, and R. de Groot. 2004. Pneumococcal vaccines: an update on current strategies. Vaccine 22: 2209–2220. 9. Burrage, M., A. Robinson, R. Borrow, N. Andrews, J. Southern, J. Findlow, S. Martin, C. Thornton, D. Goldblatt, M. Corbel, D. Sesardic, K. Cartwight, P. Richmond, and E. Miller. 2002. Effect of vaccination with carrier protein on response to meningococcal C conjugate vaccines and value of different immunoassays as predictors of protection. Infect. Immun. 70:4946–4954. 10. Carlone, G. M., C. E. Frasch, G. R. Siber, S. Quataert, L. L. Gheesling, S. H. Turner, B. D. Plikaytis, L. O. Helsel, W. E. DeWitt, and W. F. Bibb. 1992. Multicenter comparison of levels of antibody to the Neisseria meningitidis group A capsular polysaccharide measured by using an enzyme-linked immunosorbent assay. J. Clin. Microbiol. 30:154–159. 11. Costantino, P., F. Norelli, A. Giannozzi, S. D’Ascenzi, A. Bartoloni, S. Kaur, D. Tang, R. Seid, S. Viti, R. Paffetti, M. Bigio, C. Pennatini, G. Averani, V. Guarnieri, E. Gallo, N. Ravenscroft, C. Lazzeroni, R. Rappuoli, and C. Ceccarini. 1999. Size fractionation of bacterial capsular polysaccharides for their use in conjugate vaccines. Vaccine 17:1251–1263. 12. Costantino, P., S. Viti, A. Podda, M. A. Velmonte, L. Nencioni, and R. Rappuoli. 1992. Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691–698. 13. Dagan, R., J. Eskola, C. Leclerc, and O. Leroy. 1998. Reduced response to multiple vaccines sharing common protein epitopes that are administered simultaneously to infants. Infect. Immun. 66:2093–2098. 14. Di John, D., S. S. Wasserman, J. R. Torres, M. J. Cortesia, J. Murillo, G. A. Losonsky, D. A. Herrington, D. Sturcher, and M. M. Levine. 1989. Effect of priming with carrier on response to conjugate vaccine. Lancet 2:1415–1418. 15. Etlinger, H. M., D. Gillessen, H. W. Lahm, H. Matile, H. J. Schonfeld, and A. Trzeciak. 1990. Use of prior vaccinations for the development of new vaccines. Science 249:423–425. 16. Falugi, F., R. Petracca, M. Mariani, E. Luzzi, S. Mancianti, V. Carinci, M. L. Melli, O. Finco, A. Wack, A. Di Tommaso, M. T. De Magistris, P. Costantino, G. Del Giudice, S. Abrignani, R. Rappuoli, and G. Grandi. 2001. Rationally designed strings of promiscuous CD4(⫹) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines. Eur. J. Immunol. 31:3816–3824. 17. Fattom, A., Y. H. Cho, C. Chu, S. Fuller, L. Fries, and R. Naso. 1999. Epitopic overload at the site of injection may result in suppression of the immune response to combined capsular polysaccharide conjugate vaccines. Vaccine 17:126–133. 18. Giannini, G., R. Rappuoli, and G. Ratti. 1984. The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 12:4063–4069. 19. Goldblatt, D., R. Borrow, and E. Miller. 2002. Natural and vaccine-induced immunity and immunologic memory to Neisseria meningitidis serogroup C in young adults. J. Infect. Dis. 185:397–400. 20. Goldblatt, D., A. R. Vaz, and E. Miller. 1998. Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate vaccines following infant immunization. J. Infect. Dis. 177:1112–1115. 21. Goldschneider, I., E. C. Gotschlich, and M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129:1307–1326. 22. Grabowska, K., X. Wang, A. Jacobsson, and J. Dillner. 2002. Evaluation of
28. 29. 30. 31.
cost-precision rations of different strategies for ELISA measurement of serum antibody levels. J. Immunol. Methods 271:1–15. Granoff, D. M., and S. L. Harris. 2004. Protective activity of group C anticapsular antibodies elicited in two-year-olds by an investigational quadrivalent Neisseria meningitidis-diphtheria toxoid conjugate vaccine. Pediatr. Infect. Dis. J. 23:490–497. Granoff, D. M., S. E. Maslanka, G. M. Carlone, B. D. Plikaytis, G. F. Santos, A. Mokatrin, and H. V. Raff. 1998. A modified enzyme-linked immunosorbent assay for measurement of antibody responses to meningococcal C polysaccharide that correlate with bactericidal responses. Clin. Diagn. Lab. Immunol. 5:479–485. Guttormsen, H.-K., A. H. Sharpe, A. K. Chandraker, A. K. Brigtsen, M. H. Sayegh, and D. L. Kasper. 1999. Cognate stimulatory B-cell–T-cell interactions are critical for T-cell help recruited by glycoconjugate vaccines. Infect. Immun. 67:6375–6384. Insel, R. A. 1995. Potential alterations in immunogenicity by combining or simultaneously administering vaccine components. Ann. N. Y. Acad. Sci. 754:35–47. Kamboj, K. K., C. L. King, N. S. Greenspan, H. L. Kirchner, and J. R. Schreiber. 2001. Immunization with Haemophilus influenzae type b-CRM(197) conjugate vaccine elicits a mixed Th1 and Th2 CD(4⫹) T cell cytokine response that correlates with the isotype of antipolysaccharide antibody. J. Infect. Dis. 184:931–935. Kelly, D. F., E. R. Moxon, and A. J. Pollard. 2004. Haemophilus influenzae type b conjugate vaccines. Immunology 113:163–174. Lockhart, S. 2003. Conjugate vaccines. Expert Rev. Vaccines 2:633–648. Lussow, A. R., M. T. Aguado, G. Del Giudice, and P. H. Lambert. 1990. Towards vaccine optimisation. Immunol. Lett. 25:255–263. Maslanka, S. E., L. L. Gheesling, D. E. Libutti, K. B. J. Donaldson, H. S. Harakeh, J. K. Dykes, F. F. Arhin, S. J. N. Devi, C. E. Frasch, J. C. Huang, P. Kriz-Kuzemenska, R. D. Lemmon, M. Lorange, C. C. A. M. Peeters, S. Quataert, J. Y. Tai, G. M. Carlone, and the Multilaboratory Study Group. 1997. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. Clin. Diagn. Lab. Immunol. 4:156–167. McNeela, E. A., I. Jabbal-Gill, L. Illum, M. Pizza, R. Rappuoli, A. Podda, D. J. Lewis, and K. H. Mills. 2004. Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan. Vaccine 22:909–914. McVernon, J., J. MacLennan, E. Clutterbuck, J. Buttery, and E. R. Moxon. 2003. Effect of infant immunisation with meningococcus serogroup C-CRM(197) conjugate vaccine on diphtheria immunity and reactogenicity in pre-school aged children. Vaccine 21:2573–2579. Olander, R. M., T. Wuorimaa, H. Kayhty, O. Leroy, R. Dagan, and J. Eskola. 2001. Booster response to the tetanus and diphtheria toxoid carriers of 11-valent pneumococcal conjugate vaccine in adults and toddlers. Vaccine 20:336–341. Peeters, C. C., A. M. Tenbergen-Meekes, J. T. Poolman, M. Beurret, B. J. Zegers, and G. T. Rijkers. 1991. Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines. Infect. Immun. 59:3504–3510. Podda, A., N. Vescia, D. Donati, I. Marsili, G. Volpini, L. Nencioni, I. Mastroeni, R. Rappuoli, and G. M. Fara. 1991. A phase-I clinical trial of a new antitetanus/antidiphtheria vaccine for adults. Ann. Ig. 3:79–84. [In Italian.] Poolman, J. T. 2004. Pneumococcal vaccine development. Expert Rev. Vaccines 3:597–604. Porro, M., M. Saletti, L. Nencioni, L. Tagliaferri, and I. Marsili. 1980. Immunogenic correlation between cross-reacting material (CRM197) produced by a mutant of Corynebacterium diphtheriae and diphtheria toxoid. J. Infect. Dis. 142:716–724. Ravenscroft, N., G. Averani, A. Bartoloni, S. Berti, M. Bigio, V. Carinci, P. Costantino, S. D’Ascenzi, A. Giannozzi, F. Norelli, C. Pennatini, D. Proietti, C. Ceccarini, and P. Cescutti. 1999. Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines. Vaccine 17:2802–2816. Renjifo, X., S. Wolf, P. P. Pastoret, H. Bazin, J. Urbain, O. Leo, and M. Moser. 1998. Carrier-induced, hapten-specific suppression: a problem of antigen presentation? J. Immunol. 161:702–706. Ricci, S., A. Bardotti, S. D’Ascenzi, and N. Ravenscroft. 2001. Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection. Vaccine 19:1989–1997. Schallert, N., M. Pihlgren, J. Kovarik, C. Roduit, C. Tougne, P. Bozzotti, G. Del Giudice, C. A. Siegrist, and P. H. Lambert. 2002. Generation of adultlike antibody avidity profiles after early-life immunization with protein vaccines. Eur. J. Immunol. 32:752–760. Schutze, M. P., C. Leclerc, M. Jolivet, F. Audibert, and L. Chedid. 1985.
VOL. 73, 2005 Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J. Immunol. 135:2319–2322. 44. Snape, M. D., and A. J. Pollard. 2005. Meningococcal polysaccharide-protein conjugate vaccines. Lancet Infect. Dis. 5:21–30. 45. Svennerholm, L. 1957. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta 24:604–611.
Editor: D. L. Burns
N19 POLYEPITOPE AS CARRIER PROTEIN
46. Trotter, C. L., N. J. Andrews, E. B. Kaczmarski, E. Miller, and M. E. Ramsay. 2004. Effectiveness of meningococcal serogroup C conjugate vaccine 4 years after introduction. Lancet 364:365–367. 47. Valmori, D., A. Pessi, E. Bianchi, and G. Corradin. 1992. Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. J. Immunol. 149:717–721.