Division of Bacteriology, National Institute for Biological Standards and Control, Hertfordshire United Kingdom
We assessed a rat model to evaluate the immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines and the effect on Hib immunogenicity of combining 2 Hib vaccines (Hibtetanus toxoid [TT]A and Hib-TT-B) with diphtheria-TTacellular pertussis (DTaP)3 or DTaP5/inactivated poliovirus (IPV) vaccines. Rats were immunized subcutaneously with Hib alone or with Hib and DTaPbased vaccines; antiHib capsular polysaccharide IgG, poly-ribosyl-ribitol-phosphate (PRP), IgG subclass, and cellular immune responses were evaluated. Results showed a significant reduction in the antibody response to PRP when Hib-TT-A was administered in combination with DTaP3 and showed changes in the anti-PRP IgG subclass distribution between the separate and combination groups. However, combining Hib-TT-B with DTaP5/IPV did not reduce the anti-PRP antibody response. These results suggest that the model can predict the effect of combined administration of Hib and DTaP vaccines on Hib immunogenicity and would be suitable for preclinical studies of mechanisms of interference in Hib/DTaP vaccines.
Combination vaccines have been introduced with the aim of simplifying the immunization of children against multiple diseases. Many of these vaccines are based on Haemophilus influenzae type b (Hib) conjugate vaccine and DTwP vaccine (diphtheria-tetanus toxoids [DT]whole-cell pertussis antigens [wP], which are inactivated suspension of Bordetella pertussis), or DTaP vaccine, which contains purified acellular pertussis antigens (aPs). However, because many countries are changing from wP to aP vaccines because of the reduced reactogenicity of the latter, it is more likely that the new combinations will be based on DTaP vaccines. Several Hib-DTaP combination vaccines are now available that differ in the number and amount of aPs, the type of adjuvant, and the presence of other antigens, such as inactivated poliovirus (IPV) and hepatitis B virus.
Results of trials on the effect of combination vaccines on Hib immunogenicity after the primary series of infant immunizations are conflictingmost studies that have used Hib combined with DTwP reported no significant reduction in antipoly-ribosyl-ribitol-phosphate (PRP) antibody levels [1, 2]; a number of clinical trials with a Hib/DTaP3 combined vaccine that contained pertussis toxoid (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) showed significant reduction in anti-PRP antibody levels, especially in preterm infants [37], compared with Hib and DTaP3 vaccines administered separately. Other clinical trials that used a different Hib-DTaP5/IPV combination vaccine containing 5 pertussis componentsPT, FHA, PRN, and fimbriae (Fims 2 and 3) and inactivated (Vero types 1, 2, and 3) IPV vaccinesreported no significant effect on Hib immunogenicity [810]. Although the clinical importance of this reduction in Hib immunogenicity is not clear, it might have implications for protection against Hib disease, as was seen with the increase in the incidence of Hib disease in the United Kingdom after the use of a Hib/DTaP combination vaccine [11]. This could be particularly serious in disadvantaged groups, such as premature or immunosuppressed infants, or in populations at high risk for Hib disease, such as Alaska Natives [12].
The mechanism by which mixing the vaccines reduced Hib antibody response is not known, but several possibilities have been considered, including the incompatibility of Hib vaccines with aluminium adjuvant [13, 14], carrier-induced epitopic suppression [15], the increased load of tetanus toxoid (TT) from Hib-TT and DTaP, and chemical, physical, or immunological interactions between components of the vaccines that might alter the conformation or presentation of specific components.
The lack of a suitable animal model has hampered basic research studies of the immunological mechanisms of interference between Hib and components of these vaccines and has limited the development of quality-control tests. Animal models that have been used to study Hib immunogenicity include mice, rats, guinea pigs, and rabbits [16, 17]. Miceregardless of breed, age, or method of injectionare poor responders to Hib conjugate vaccines, and the response in guinea pigs is highly variable between animals. The response in rats is less variable between individual animals, making this animal the most suitable model for Hib immunogenicity studies [17]. In the present study, we have investigated the utility of the rat model to evaluate the immunogenicity of Hib vaccines and to analyze the characteristics of the immune response to Hib vaccine after separate or combined administration with 2 different DTaP-based vaccines.
MATERIALS AND METHODS
Vaccines.
Hib capsular polysaccharide (PRP), TT, and 3 different commercial Hib conjugate vaccines were used: Hib-TT-A, Hib-TT-B, and Hib-CRM (table 1). In the first 2, PRP is conjugated to TT, and the last formulation uses CRM197 (a nontoxic cross-reacting mutant protein of diphtheria toxin from Corynebacterium diphtheriae) as the carrier protein. DTaP3 vaccine containing DT and aPs (PT, FHA, and PRN) adsorbed to aluminium hydroxide was obtained from the manufacturer of Hib-TT-A. DTaP5/IPV and DTaP5/IPV/Hib vaccines containing aPs (PT, FHA, PRN, and Fims 2 and 3) and IPV vaccine were obtained from the manufacturer of Hib-TT-B (table 1). The lyophilized Hib-TT vaccines were reconstituted to the appropriate dose with sterile saline. The preparation of the Hib-TT-A/DTaP3 combination vaccines was done by the reconstitution of lyophilized Hib with the liquid DTaP3. Vaccines were injected into rats within 1 h of preparation. For the spleen-cell proliferation assay, TT from the same manufacturer as that of Hib-TT-A was used.
Immunization of rats.
Female, 46-week-old Sprague-Dawley rats, in groups of 58, were injected subcutaneously with different doses of Hib vaccines or with Hib and DTaP3 or DTaP5/IPV concomitantly, either on different sides or as a single injection, on days 0, 28, and 42. Control rats were injected with saline. Blood was obtained on days 0, 28, 42, and 49. Serum samples were stored frozen at -20°C until testing. Animal studies were conducted according to the UK Home Office regulations and were approved by local ethics committee.
Detection of antiPRP IgG and IgG subclasses by ELISA.
Plastic microtiter plates (Nunc Maxisorb) were coated with 1 g/mL Hib capsular oligosaccharide conjugated to human serum albumin (HbO-HA), provided by Wyeth [18], in PBS for 90 min at 37°C and were stored at 4°C overnight. The next day, plates were blocked with PBS that contained 1% bovine serum albumin, and 11 serial 2-fold dilutions of immune rat serum samples were distributed in the wells (100 L/well); then, the plates were incubated for 90 min at room temperature, followed by incubation with biotin-conjugated goat antirat IgG (Southern Biotechnology) for 2 h and streptavidinhorseradish peroxidase (Serotec) for 1 h. The optical density at 492 nm was measured by use of an automated reader (Labsystems Multiscan MS) and Genesis software (version 3.05; Thermo Life Sciences). Results are presented as the titer being equal to the reciprocal of the serum dilution giving an OD of 0.5. A reference serum was used in each assay, to allow correction for assay variability within the same experiment. For quantification of the various IgG subclasses, immune rat serum samples were incubated, at subsaturating dilutions, in HbO-HAcoated wells, and bound antibodies were detected by biotin-conjugated mouse antirat isotype-specific monoclonal antibodies (Pharmingen). The relative concentrations of the antibody isotypes were determined from the standard curves of absorbance of serially diluted rat IgG subclass myeloma proteins (Pharmingen) by use of the same ELISA, except that the wells were coated with goat antirat IgG (Pharmingen) instead of HbO-HA.
The specificity of the rat anti-PRP assay was checked by inhibition ELISA performed by preincubation of the serum samples with PRP, and we found 94% inhibition of the ELISA response with 7.5 g/mL PRP. Repeated analysis of a positive control serum sample showed inter- and intra-assay coefficients of variation of 14% and 16%, respectively.
Spleen-cell proliferation and cytokine production.
To determine the proliferative response and the cytokine profile induced by Hib conjugate vaccine, spleens were removed 2 weeks after the second immunization, and single-cell suspensions were prepared. Aliquots of 4 × 105 cells were cultured for 4 days in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 50 mmol/L 2-mercaptoethanol, 100 U/mL penicillin, and 100 g/mL streptomycin, in the presence of 10 g/mL TT and in a total volume of 200 L. Before the termination of the incubation period, each well was pulsed with 0.25 Ci of 3H-thymidine (Amersham), and plates were incubated for a further 6 h and then harvested on glass filters by use of an automated cell-harvesting apparatus (Skatron). 3H-thymidine incorporation was measured in a -scintillation counter. Results were expressed as the stimulation index (SI; counts per minute of cells cultured with antigen/counts per minute of cells cultured alone). Bulk cultures were set up in parallel for cytokine analysis. Levels of interleukin (IL)4 and interferon (IFN) in the supernates of stimulated cells were measured by standard sandwich ELISA, according to the manufacturer's instructions (Pharmingen). Values for cytokines were expressed by reference to a standard curve constructed by assaying serial dilutions of the respective rat cytokine. The assay cutoff was 4 and 16 pg/mL, respectively, for IL-4 and IFN-. Antigen-specific cytokine secretion was obtained by subtracting the cytokine content of the supernates from that of splenocytes cultured alone.
RESULTS
Immunogenicity of Hib Conjugate Vaccines in the Rat Model
Immunization of rats with one-fifth of a single human dose (SHD; 0.5 mL) of 3 different Hib vaccines induced a significant (P < .05) primary antibody response to PRP (table 2). A second immunization 4 weeks later increased the response for all vaccines, with the increase being significant (P < .05) only for Hib-TT-A, and a third immunization at 6 weeks induced a further, but not significant, increase (P > .05) in the antibody response to all vaccines tested. Unconjugated PRP induced a very weak primary antibody response that was not boostable.
Titration of the Hib dose was done for the 3 Hib vaccines, and the response was evaluated after the boost. All 3 vaccines resulted in a dose-dependent anti-PRP response within a range of the doses tested. However, the optimum dose varied for the 3 vaccines and was 0.2, 0.04, and 0.02 SHD, respectively, for Hib-CRM, Hib-TT-A, and Hib-TT-B (figure 1). A further increase in the Hib dose resulted in a clear reduction in the anti-PRP response.
Immunogenicity of Hib-TT-A and Hib-TT-B in Combination Vaccines
The effect of the combined administration of Hib and DTaP vaccines on Hib immunogenicity was investigated in rats immunized with Hib-TT-A and DTaP3 or with Hib-TT-B and DTaP5/IPV vaccines. Rats immunized with Hib-TT-A and DTaP3 but not with Hib-TT-B and DTaP5/IPV mounted a significantly lower anti-PRP response in the combined vaccine group than in the separate vaccine group (P < .05, experiments 1 and 2; P < .01, experiment 3) (table 3). The reduction in the anti-PRP response ranged from 3- to 16-fold in experiments that used Hib-TT-A combined with DTaP3. Similar results were obtained when a range of different amounts of 2 vaccines, Hib-TT-A/DTaP3 and Hib-TT-B/DTaP5/IPV, was used (table 4). Moreover, the magnitude of reduction in anti-PRP antibody level seems to be related to the number of doses administered, with the level of reduction increasing from 7-fold after a single dose to 17- and 23-fold, respectively, after 2 and 3 doses (table 5).
Characterization of the Hib Immune Response after Combined Versus Separate Administration of Hib-TT-A and DTaP3 Vaccines
To understand the causes and mechanisms of reduced Hib immunogenicity in Hib-TT-A/DTaP3 combination vaccines, further experiments were designed to analyze the characteristics of the humoral and cellular immune responses to Hib-TT-A in combined versus separate administration with DTaP3.
Anti-PRP IgG subclass distribution.
Analysis of the relative concentrations of the anti-PRP IgG subclasses showed that, although all 4 subclasses were represented in both the combined and separate vaccine groups (figure 2), there were clear and significant differences in the percentage of each subclass produced by the 2 groups. Although the IgG2b subclass was the most abundant subclass in the separate vaccine group (60% of total IgG), it was reduced to 25% (P < .05) in the combined vaccine group. In contrast, the percentage of IgG2a increased significantly, from 17% in the separate vaccine group to 39% in the combined vaccine group (P < .02). Similarly, the percentage of IgG1 was 14% in the separate vaccine group and 32% in the combined vaccine group, but this difference was not statistically significant (P > .05).
Cellular immune response to Hib-TT-A.
The cellular immune response induced by the carrier protein TT was evaluated in rats immunized with Hib-TT-A and DTaP3 vaccines administered separately or in combination, by measuring the proliferative response of spleen cells from immunized rats to recall antigen TT and by analysis of the cytokine profiles in the culture supernatants. Immune spleen cells proliferated vigorously in the presence of TT in both groups (table 6). However, the magnitude of the response was significantly diminished to 50% in the combination vaccine group (SI, 17), compared with the separate group (SI, 37) (P < .05).
In the same experiment, analysis of the cytokine patterns secreted by immune spleen cells from both groups showed that, although cells from the separate vaccine group secreted a substantial amount of IFN- (mean, 1320 pg/mL) and a very low level of IL-4 (mean, 5 pg/mL), spleen cells from the combined vaccine group secreted a significantly higher level of IL-4 (mean, 18 g/mL; P < .05) and little IFN-, with only 1 of 5 rats secreting a small amount of IFN- (50 pg/mL) (figure 3).
DISCUSSION
Combining Hib with DTaP vaccine reduces the number of injections and visits to the clinic and increases vaccine coverage and compliance. However, reduction of the anti-PRP response has been demonstrated after the primary series of vaccinations in infancy with Hib vaccine combined with DTaP3 [37] but not with other Hib vaccines combined with DTaP5/IPV [810]. The reason for the interference with the Hib response in some but not all combination vaccines is unclear, and investigation into mechanisms of reduced Hib immunogenicity in combined vaccines has been hampered by the lack of a suitable animal model. Previous models for Hib immunogenicity have included the mouse, rat, guinea pig, and rabbit. Of these models, the guinea pig and rat have been shown to be the most promising [16, 17], whereas mice, regardless of breed, age, or method of injection, were relatively poor responders to conventional Hib conjugate vaccines [16]. The response in rabbits is highly variable between individual animals (authors' unpublished data).
In the present study, we have shown that the immune response of adult rats immunized with Hib vaccines qualitatively resembled that of human infants: no response was detected in rats immunized with Hib polysaccharide (PRP) alone, whereas substantial responses were measured against the 3 Hib conjugate vaccines used, with evidence of an anamnestic memory response after the first and second boosts. Moreover, similar to the clinical trial findings [310], the anti-PRP response in our rat model was significantly reduced when Hib-TT-A was administered in combination with DTaP3 but not when Hib-TT-B was administered in combination with DTaP5/IPV, in comparison with the vaccines being administered at separate sites. In addition, the finding that the level of reduction of anti-PRP antibody response is related to the number of doses is in keeping with Daum et al.'s [7] finding that a significant decrease in the mean anti-PRP antibody response occurred in infants as the number of doses of Hib-DTaP combination vaccine increased. This suggests that similar mechanisms of interference might be operating in both species when Hib-TT-A and DTaP are used.
Immunization with protein-conjugated polysaccharide (PS) induces protein-specific CD4+ T helper cells, which are thought to provide help to PS-specific B cells, which act as antigen-presenting cells for the carrier protein through direct cell-cell contact and cytokine secretion, which results in B cell differentiation toward memory or plasma cells [19]. In the rat, CD4+ cells can be subdivided into 2 major subsets on the basis of their different cytokine-production patterns [20]. Th1 cells, which produce IL-2 and IFN-, can preferentially induce the synthesis of the IgG2b isotype [21, 22]. Conversely, Th2 cells produce IL-4 and mainly elicit the production of IgG1, IgG2a, and IgE. To the best of our knowledge, the present study is the first to show, in a rat model, that, in addition to the reduced total anti-PRP IgG response, combining Hib-TT-A with DTaP3 also modulated the IgG isotype of the anti-PRP response. Although the separate vaccine group showed a clear bias toward a Th1-mediated response (high IgG2b), this was substantially reduced in the combined vaccine group, which suggests a suppression of the Th1-mediated response to Hib. This modulation of the IgG subclasses is clearly the result of modulation of the T helper response induced by the carrier protein, as evidenced by the reduced proliferation of immune spleen cells in response to the carrier protein TT and by the suppression of IFN- production in the combined vaccine group. Possible causes for the reduction and modulation of Hib-TT-A response could include the adsorption of Hib-TT-A onto the adjuvant aluminum hydroxide Al(OH)3 after mixing with DTaP3. A previous study by Sturgess et al. [14] showed that the adsorption of Hib conjugate vaccines on Al(OH)3 induces a catalytic reaction between the phosphodiester bond of PRP and Al(OH)3 that results in hydrolysis of the PRP polymer into smaller chain lengths and liberation of PRP oligomers from the conjugate particle. Another possible cause could be the presence of free carrier protein in the Hib/DTaP mixture that could have resulted in competition for antigen capture and presentation between B cells with surface immunoglobulin specific for epitopes on the carrier protein and B cells specific for the PS. The presence of free TT might also prevent the binding of the conjugate to PS-specific B cells by the tetanus protein and suppression of the response to PS by clonal expansion of the number of carrier-specific B cells [15, 23].
It has been accepted that whole-cell pertussis vaccines have adjuvant activity for coinjected antigens, and this has been attributed to endotoxin, which enhances the production of IL-12 [24], and also to residual levels of active PT [25]. In contrast, adjuvant activity in acellular pertussis vaccines has been more difficult to demonstrate. Although some researchers have reported enhanced Th1 and Th2 responses to antigens coadministered with chemically detoxified PT [25], others have reported a suppressive/modulating effect of FHA on coadministered antigens [26].
Taking together the modulation of IgG subclass and the suppression of IFN- production in the combined vaccine group, this suggests that the reduced antibody response to Hib could be mediated by a suppression of the Th1 response to the carrier protein on which the PS-specific B cells are dependent for differentiation and Ig secretion. We are currently investigating the effect of individual pertussis components on the characteristics of the immune response to Hib-TT-A in combined versus separate administration.
Overall, our results in the rat model suggest that in the combined administration of Hib-TT-A and DTaP3, the Hib response was not only reduced but also modulated. This suppression/modulation of the Hib-TT-A response in the Hib-TT-A/DTaP combination vaccine could be the result of several interacting factors. The development of this rat model may facilitate a better understanding of the mutual interactions between the different components of a combined vaccine and of the mechanism(s) of interference with Hib immune response in Hib-DTaP combined vaccines.
Acknowledgments
We thank L. Frost, R. Huskisson, and A. Belton, for technical support; and S. Baker and G. Crossland, for excellent animal husbandry.
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