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Mutual Interactions Between DTaP-IPV and Haemophilus influenzaeType b (Hib)-conjugated Vaccines in Laboratory Animal Models
Biologicals (1999) 27, 227–240 Article No. biol.1999.0180, available online at http://www.idealibrary.com on
Mutual Interactions Between DTaP-IPV and Haemophilus influenzae Type b (Hib)-conjugated Vaccines in Laboratory Animal Models 1
Homayoun Shams1*† and Iver Heron2‡ Bacterial Vaccines Department, Statens Serum Institut, Copenhagen, Denmark 2 Amvax Inc., Indian Creeck Court, Beltsville, Maryland, U.S.A.
Abstract. Potency and/or immunogenicity of three different Haemophilus influenzae type b-conjugated vaccines (Hib) and a DTaP-IPV vaccine alone, and their mutual interactions in DTaP-IPV-Hib combination was tested. In a mouse model, only combination of Act–Hib, in which tetanus toxoid (TT) was as active as non-conjugated TT, significantly increased the immunogenicity and potency of TT component of DTaP-IPV vaccine. Also, only combination of Hib-TITER, in which CRM197 was used as the carrier with DTaP-IPV, increased the potency of diphtheria toxoid (DT) component of DTaP-IPV vaccine significantly. It shows that the additive effect of tested Hib vaccines on immunogenicity and/or potency of TT and DT was mostly due to the existence of TT and CRM197, respectively, as the carrier in the mentioned Hib vaccines. No difference was shown in inoculation of DTaP-IPV and Hib conjugated vaccines in the same syringe or at separate sites. DTaP-IPV had dual effects on anti-Hib capsular polysaccharide (HibCP) responses to Hib vaccines in the mouse model. This duality was probably related to the carrier B-cell epitopes activity of Hib conjugated vaccines. The immunogenicity of TT component of Act-Hib and Amvax Hib-TT in the guinea pig model was shown and combination of mentioned Hib vaccines with DTaP-IPV, remarkably increased anti-TT antibody responses to the TT component of DTaP-IPV vaccine. These confirmed our results in the mouse model. Using two different protocols to evaluate the guinea pig model for induction of anti-HibCP immunity showed that a ‘‘long interval’’ protocol does not have any advantage over the ‘‘short interval’’ protocol. Also, combination of DTaP-IPV with Hib vaccines did not have any noticeable effect on anti-HibCP antibodies in the guinea pig model. Taken together, our observations in laboratory animal models may facilitate a better understanding of the mutual interactions between the different antigen components of a combined vaccine such as DTaP-IPV© 1999 The International Association for Biologicals Hib vaccine.
Introduction Combined vaccines which protect against two or more diseases make vaccination plans more practical and economical. This is especially important in developing countries due to the large number of pathogens which threaten the health of infants.1 The need to administer many vaccines to infants and children has stimulated e#orts to determine *To whom correspondence should be addressed. †Current address: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, P.O. Box 830905, Lincoln, NE 68583–0905, U.S.A. ‡Deceased. 1045–1056/99/030227+14$30.00/0
their compatibility for incorporation as a combined vaccine into a single injection. The trend is toward combining more and more of the newly developed conjugated vaccines with other combined vaccines. For example, the DTaP-IPV constitutes a complex mixture, the diphtheria toxoid (DT) – tetanus toxoid (TT) acellular pertussis (aP) and three serotypes (types 1, 2 and 3) of inactivated polio vaccine (IPV). Addition of another antigen such as a Haemophilus influenzae type b (Hib)-conjugated vaccine further increases this complexity. The current concept is that combination of the DTaP-IPV with a Hibconjugated vaccine can provide a unique opportunity for broadening protection against more 1999 The International Association for Biologicals
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Table 1. Vaccines Vaccines
Act-Hib* Hib-TT Hib-TITER† DTaP-IPV‡
Manufacturer
Pasteur Merieux Amvax Lederle-Praxis Statens Serum Institut/Amvax
Polysaccharide
Carrier
size§
g/HD
nature¶
g/HD
Large Small Small N/A
10 10 10 N/A
Tetanus Toxoid Tetanus Toxoid CRM197 N/A
24 28·5 25 N/A
* Batches No.H1239 and J0812. † Batches No.S815 and L0284B4. ‡ Each dose is 0·5 ml and contains 1 mg Aluminium, 25 Lf diphtheria toxoid, 7 Lf tetanus toxoid, 40/8/32 DU type 1,2,3 of poliovirus, and 40 g pertussis toxoid (PT) is the major component of acellular pertussis vaccine (aP). § Native Hib-CP was considered as large size and reduced size oligosaccharides, containing 15–30 repeat units were considered as small size. ¶ CRM197 is a mutant of Corynebacterium diphtheriae toxin.
diseases without discomfort of additional injections. Four Hib-conjugate vaccines have been licensed for sale in the United States, but there is no welldefined and universally accepted method for testing immunogenicity of Hib-conjugate vaccines in laboratory animals.2 Also, the U.S. Food and Drug Administration (FDA) and European Pharmacopoeia (EP) requirements for potency tests of DT and TT containing vaccines are not alike. Therefore, further experiments to determine the mutual interactions between DTaP-IPV vaccine and Hibconjugated vaccines, and to clarify the role of di#erent factors [e.g. di#erent carriers, size of Hib capsular polysaccharides (HibCP)] on immunogenicity of Hib-conjugated vaccines in laboratory animal models are required. In this study, we used three di#erent Hibconjugated vaccines, Act-Hib (large size HibCP and TT as the carrier), Amvax Hib-TT (small size HibCP and TT as the carrier) and Hib-TITER (small size HibCP and CRM197 as the carrier; CRM197 or ‘‘cross-reacting material’’ is a non-toxic mutant of diphtheria toxin and serologically cross-reacts with diphtheria antitoxin) and a DTaP-IPV vaccine to investigate: (1) the e#ect of Hib-conjugated vaccines on immunogenicity and/or potency of di#erent components of a DTaP-IPV vaccine; (2) the role of di#erent factors in the immunogenicity of Hibconjugated vaccines; (3) the e#ect of the DTaP-IPV vaccine on immunogenicity of di#erent Hibconjugated vaccines, in a mouse and in a guinea pig model. DTP-Hib vaccine was evaluated in a mouse model3,4 and Gupta et al. proposed the guinea pig model for evaluation of DTaP-Hib vaccines.5 To our
knowledge, this is the first study in which DTaPIPV-Hib vaccines have been evaluated in both the mouse and the guinea pig model.
Materials and methods Vaccines
The following vaccines were used in this study: Act-Hib (Institute Pasteur Merieux Lyon, France); Hib-TT conjugate (Amvax Inc., Beltsville, MD, U.S.A.); Hib-TITER (Lederle-Praxis Biologicals, Pearl River, U.S.A.); and a paediatric diphtheria– tetanus–acellular pertussis (aP)-inactivated polio vaccine (DTaP-IPV, Statens Serum Institut, Copenhagen, Denmark / Amvax, Inc. Beltsville, MD, U.S.A.). The volume of each single human dose (HD) was 0·5 ml. The details of each vaccine component are shown in Table 1. Animals
Outbred, female, CF1, 16–18 g mice; and white, female, 250–300 g Hartley strain guinea pigs were used as animal models. All the animals were obtained from Hvidesteen Laboratory Animal Farms, Statens Serum Institut, Denmark. Immunization in the mouse model
Two di#erent immunization schedules were carried out: a single shot programme to measure the potency of DT and TT, and a multiple shot programme to measure the immunogenicity and kinetics of antibody responses to HibCP, DT, TT, and acellular pertussis (aP).
DTaP-IPV & Hib vaccines in animal models
Single shot immunization programme. This pro-
gramme was chosen according to the o#icial oneinjection protocol in the mouse model6,7 to measure the relative potency of DT and of TT component of di#erent preparations. Methods were selected on the basis of the World Health Organization (WHO) and EP requirements as follows: (A) Diphtheria Vero potency assay. The method has been described elsewhere,6–9 briefly, groups of mice (16–20 mice/dose) were immunized once subcutaneously with at least three di#erent doses of either the international standard toxoid for adsorbed diphtheria vaccine (DIXA), Hib-TITER, DTaP-IPV, combination of DTaP-IPV+Act-Hib (either injected in separate sites or mixed in the same syringe), combination of DTaP-IPV+Amvax Hib-TT (mixed in the same syringe and four di#erent dilution groups for this), or with the combination of DTaP-IPV+HibTITER (either injected in separate sites or mixed in the same syringe, and four di#erent dilution groups for this). Doses for DIXA were 8, 4, and 2 IU/mouse, and doses for test preparations were 1/20, 1/40, 1/80 and in a few cases 1/160 HD. Four weeks later, immunized mice were bled and sera were collected. The concentration of neutralizing anti-diphtheriatoxin antibodies of sera was measured by Vero cell assay.6,9,10 (B) Tetanus lethal potency assay. This assay has been 6–8,10–12
briefly groups of 16–20 described elsewhere, mice/dose were immunized once subcutaneously with three di#erent doses of either tetanus standard toxoid, DTaP-IPV, Act-Hib, Amvax Hib-TT, combination of Act-Hib+DTaP-IPV (either injected in separate sites or mixed in the same syringe), combination of Amvax Hib-TT+DTaP-IPV (mixed in the same syringe, and four di#erent dilution group for this), or combination of Hib-TITER+DTaP-IPV (either injected in separate sites or mixed in the same syringe). Doses for tetanus standard toxoid were 3·425, 1·713 and 0·856 IU/mouse, and the test vaccines dilutions were 1/40, 1/80, 1/160 and in a few cases 1/320 HD. Four weeks later, immunized mice were challenged with median paralytic dose (PD50) tetanus toxin, and lethal or paralytic e#ects were recorded within the next 4 days. Multiple-shot immunization programme. In order to investigate the mutual interactions of Hibconjugated vaccines and DTaP-IPV in the mouse model, the mice (16–20 mice/dose) that had been bled to analyse the potency of diphtheria toxoid along with other two groups which had been immu-
229
nized with either Act-Hib or Amvax Hib-TT were immunized for second time (at the same day after bleeding for diphtheria potency) with the same doses and components as they had been immunized in the first shot (described in the diphtheria potency assay). Two weeks after second injection, second bleedings and at the same day third immunization were done. Two weeks, 4 weeks, 8 weeks and 12 weeks after third immunization, third, fourth, fifth and sixth bleedings were performed. In the Tables 2 and Table 3, results are only shown for the first three bleedings. Immunization in the guinea pig model
In this model, groups of six animals were immunized subcutaneously with 1/2 HD of either DTaPIPV, Act-Hib, DTaP-IPV+Act-Hib (either injected in separate sites or mixed in the same syringe), Amvax Hib-TT, DTaP-IPV+Amvax Hib-TT (either injected in separate sites or mixed in the same syringe), Hib-TITER, or combination of DTaPIPV+Hib-TITER (either injected in separate sites or mixed in the same syringe). Guinea pigs were immunized twice in the two di#erent immunization schedule (Table 4). Measurement of anti-diphtheria antibodies by Vero cell assay
Details of the assay have been described elsewhere.9 Briefly, a two-fold dilution series of each of the sera and diphtheria standard antitoxin (an international WHO standard for diphtheria antitoxin was used which is a hyperimmune horse antiserum containing 10 IU/ml, and was used at 1:100 dilution) were made in Nunc flat-bottomed Micro Well plates and a quantity of 50 l was left in each well. Then, 20 l diphtheria toxin containing 10 3 Lf/ml was added to each well and incubated for 2 h at 20C, and then into each well, 150 l Vero cell suspension (6104 African Green Monkey Kidney Cells/ml) was added. Plates were incubated for 5 days at 37C, 5% CO2 with 90% relative humidity. Complete Dulbecco’s modified Eagle medium containing 10 m Hepes, 100 IU/ml penicillin, 100 g/ml streptomycin and 10% fetal calf serum (FCS) was used. The concentration of neutralizing antidiphtheria toxin antibodies in each serum was calculated in comparison with the standard for diphtheria antitoxin, using a computerized Vero program from the Bacterial Vaccines Department, Statens Serum Institut, Denmark. This program is based on a regression analysis calculation. Data are shown in the scale of International Unit (IU).
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Table 2. Geometric mean of anti-tetanus antibodies (IU) in the mice immunized with di#erent vaccines in three di#erent set of experiments (95% confidence limits) Dose*
4 wks post 1st immunization
2 wks post 2nd immunization
2 wks post 3rd immunization
1/20 1/40 1/80
0·08(0·05–0·12) 0·06(0·04–0·10) 0·02(0·01–0·03)
1·88(1·42–2·50) 1·09(0·72–1·65) 0·54(0·33–0·87)
1·89(1·53–2·32) 1·43(1·18–1·72) 1·37(1·04–1·82)
0·12(0·06–0·22) 0·04(0·02–0·12) 0·03(0·01–0·07)
1·69(1·10–2·60) 0·57(0·24–1·33) 0·26(0·08–0·95)
2·15(1·63–2·84) 1·10(0·62–1·96) 0·27(0·10–0·73)
0·14(0·10–0·20) 0·11(0·06–0·19) 0·02(0·01–0·04) syringe) 0·16(0·11–0·24) 0·12(0·08–0·19) 0·05(0·03–0·09)
3·06(2·31–4·04) 2·25(1·53–3·30) 0·93(0·58–1·49)
2·66(2·12–3·34) 2·42(1·81–3·24) 1·32(0·90–1·94)
3·86(2·88–5·19) 2·45(1·75–3·44) 1·38(0·97–1·99)
2·86(2·02–4·05) 2·03(1·41–2·92) 1·61(1·17–2·21)
0·08(0·05–0·14) 0·06(0·04–0·10) 0·02(0·01–0·03)
2·22(1·56–3·16) 1·25(0·93–1·69) 0·65(0·30–1·42)
1·84(1·27–2·68) 0·76(0·54–1·06) 0·71(0·52–0·97)
1/20 † 1/40 † 1/80 † DTaP-IPV+Amvax Hib-TT (mixed in the same syringe) 1/20 0·22(0·15–0·33) 1/40 0·05(0·03–0·10) 1/80 0·03(0·01–0·06) 1/160 †
0·01(0·002–0·01) † †
0·06(0·01–0·30) 0·01(0·002–0·02) 0·01(0·002–0·01)
4·06(2·87–5·74) 2·11(0·99–4·49) 0·85(0·36–2·00) 0·25(0·12–0·54)
2·67(1·90–3·75) 2·71(1·99–3·69) 1·21(0·86–1·71) 0·85(0·60–1·20)
Panel C: DTaP-IPV
1·79(1·31–2·43) 1·36(0·97–1·90) 0·40(0·22–0·74)
1·58(1·23–2·03) 1·38(1·08–1·75) 0·85(0·63–1·15)
1·36(1·03–1·79) 0·86(0·55–1·35) 0·44(0·25–0·76) 0·07(0·03–0·17)
1·33(1·07–1·65) 1·25(1·00–1·57) 0·63(0·45–0·87) 0·53(0·35–0·79)
1·58(1·17–2·14) 1·05(0·75–1·48) 0·45(0·20–0·99) 0·08(0·04–0·16)
1·15(0·89–1·49) 0·90(0·70–1·15) 0·78(0·51–1·19) 0·51(0·35–0·72)
Vaccines Panel A: DTaP-IPV Act-Hib
1/20 1/40 1/80 DTaP-IPV+Act-Hib (separate sites) 1/20 1/40 1/80 DTaP-IPV+Act-Hib (mixed in the same 1/20 1/40 1/80 Panel B: DTaP-IPV Amvax Hib-TT
1/20 1/40 1/80
1/20 0·04(0·02–0·06) 1/40 0·03(0·01–0·06) 1/80 0·01(0·004–0·01) DTaP-IPV+Hib-TITER (separate sites) 1/20 0·06(0·04–0·10) 1/40 0·02(0·01–0·03) 1/80 0·01(0·01–0·02) 1/160 † DTaP-IPV+Hib-TITER (mixed in the same syringe) 1/20 0·05(0·03–0·08) 1/40 0·01(0·01–0·03) 1/80 † 1/160 0·01(0·004–0·02) * Human Dose. † Anti-tetanus antibody levels were less than 0·01 IU.
Measurement of anti-tetanus antibodies by ELISA
Sera from animals were tested for anti-tetanus antibodies using an enzyme-linked immunosorbent assay (ELISA) as described previously.13,14 Briefly,
coating the plastic microtitre plates (Nunc Intermed, Roskilde, Denmark) with purified tetanus toxoid (750 Lf/ml) diluted in carbonate bu#er (1:10 000, overnight), was followed by blocking bu#er, and then di#erent dilutions of sera were
DTaP-IPV & Hib vaccines in animal models
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Table 3. Geometric mean of anti-diphtheria antibodies (IU) in the mice immunized with di#erent vaccines in three di#erent set of experiments (95% confidence limits) Vaccines
Dose*
4 wks post 1st immunization
Panel A DTaP-IPV
1/20 0·03(0·02–0·04) 1/40 0·01(0·01–0·02) 1/80 0·01(0·01–0·02) 0·13(0·06–0·29) DTaP-IPV+Act-Hib (separate sites) 1/20 0·02(0·01–0·02) 1/40 0·01(0·01–0·01) 1/80 0·01(0·01–0·01) DTaP-IPV+Act-Hib (mixed in the same syringe) 1/20 0·02(0·02–0·03) 1/40 0·01(0·01–0·02) 1/80 0·01(0·01–0·01) Panel B DTaP-IPV 1/20 0·02(0·01–0·03) 1/40 0·02(0·01–0·03) 1/80 0·004(0·003–0·006) DTaP-IPV+Amvax Hib-TT (mixed in the same syringe) 1/20 0·02(0·01–0·03) 1/40 0·01(0·01–0·02) 1/80 0·01(0·01–0·01) 1/160 † Panel C DTaP-IPV 1/20 0·01(0·004–0·01) 1/40 0·004(0·003–0·006) 1/80 0·002(0·001–0·003) Hib-TITER 1/20 † 1/40 † 1/80 † DTaP-IPV+Hib-TITER (separate sites) 1/20 0·01(0·01–0·02) 1/40 0·01(0·004–0·01) 1/80 † 1/160 † DTaP-IPV+Hib-TITER (mixed in the same syringe) 1/20 0·02(0·01–0·03) 1/40 0·01(0·004–0·01) 1/80 † 1/160 †
2 wks post 2nd immunization
2 wks post 3rd immunization
0·86(0·51–1·47) 0·32(0·16–0·64) 0·23(0·13–0·42)
0·62(0·42–0·92) 0·32(0·20–0·53)
0·62(0·39–0·97) 0·19(0·08–0·45) 0·17(0·08–0·36)
0·51(0·34–0·74) 0·24(0·13–0·42) 0·27(0·17–0·42)
1·16(0·73–1·85) 0·42(0·23–0·78) 0·17(0·08–0·37)
0·76(0·54–1·11) 0·41(0·25–0·67) 0·26(0·13–0·51)
1·15(0·75–1·76) 0·82(0·55–1·23) 0·15(0·06–0·43)
1·65(1·01–2·69) 0·41(0·21–0·81) 0·21(0·11–0·40)
0·41(0·16–1·03) 0·52(0·26–1·04) 0·17(0·07–0·42) 0·04(0·01–0·11)
0·48(0·29–0·83) 0·35(0·18–0·69) 0·22(0·10–0·48) 0·13(0·04–0·45)
0·73(0·47–1·13) 0·37(0·17–0·80) 0·10(0·05–0·18)
0·68(0·43–1·10) 0·36(0·18–0·75) 0·23(0·11–0·48)
† 0·01(0·003–0·01) 0·01(0·004–0·01)
0·01(0·01–0·3) 0·02(0·01–0·05) 0·02(0·01–0·04)
0·94(0·37–2·38) 0·30(0·13–0·70) 0·34(0·19–0·61) 0·03(0·01–0·08)
0·79(0·41–1·53) 0·74(0·43–1·28) 0·55(0·32–0·94) 0·21(0·13–0·33)
1·04(0·63–1·70) 0·44(0·26–0·75) 0·09(0·03–0·28) 0·01(0·004–0·02)
1·08(0·69–1·69) 0·60(0·43–0·83) 0·49(0·26–0·95) 0·09(0·06–0·14)
*Human dose. †Anti-diphtheria antibody levels were less than 0·002 IU.
made. After overnight incubation at 4C, plates were washed and peroxidase labelled rabbit-antimouse immunoglobulin antibodies (DAKO) were used. After washing, plates were developed by substrate. Addition of 1 H2SO4 after 30 min stopped the chromogenic reaction. OD values were measured with Immunoreader NJ 2000 at 490 nm. Calculation was performed using parallel line assay against the home reference serum (MuTe91). The
results are shown on the scale of International Unit (IU). Measurement of anti-HibCP antibodies by ELISA
Plates were coated with 50 l of HibCP conjugated to human serum albumin (2 g/ml HibCPHSA, Amvax, Inc.; Beltsville, MD, U.S.A.) and incubated overnight at 4C. Coated plates were washed and blocked with 100 l rabbit normal
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Table 4. Immunization and bleeding schedule of guinea pigs Immunization on the week of 1-Short interval protocol 2-Long interval protocol
0 0
serum(1%). For control, sera of the animals immunized with DTaP-IPV were pooled and used as the negative control. By direct ELISA. Di#erent dilutions of the murine sera were made and the rest of the technique was as described previously. The titre of antibody was determined by endpoint titration after subtraction of values for the negative control. The last dilution at which the OD value exceeded that observed in negative control was taken as the titre. By competitive ELISA. To recognize individual responders in the guinea pig model, a competitive ELISA was employed. On the coated plates, 25 l of di#erent dilutions of non-conjugated HibCP with four-fold serial dilutions (seven dilutions starting at a concentration of 100 g/ml) and 25 l dilution bu#er in the control well were applied. Then the constant amount of each serum (25 l) was added to eight wells of each column (from A to H). Plates were incubated at 4C overnight and washed and stained as described earlier. Sera that showed _1·000 OD value (after subtraction of background) and were inhibited by _0·02 g HibCP were recognized as responders. Results are expressed as the amounts of non-conjugated HibCP that have been able to inhibit the 50% OD value (after subtraction of background). Statistical analysis
The potency of the TT component of the test vaccines was calculated by the probit analysis, and for the potency of DT and CRM197 the parallel line assay was used. The results are shown in the term of relative potency in comparison with the relevant standard.6 Comparison of potency results was done by calculation of 95% confidence limits. The di#erences in values outside of the 95% confidence limits were considered significant. Student’s t-test was used in the analysis for di#erences in antibody levels between and within groups. The significance level chosen for the tests was 5%.
4 6
Bleedings on the week of 4 6
6 8
— 12
Results Effect of Hib-conjugated vaccines on immunogenicity of TT in the mouse model
Table 2 shows the kinetics of antibody responses to the TT component of Hib-conjugated vaccines, and the e#ect of Hib-conjugated vaccines on kinetics of antibody responses to the TT component of DTaP-IPV vaccine. Act-Hib alone produced anti-TT antibodies as much as the TT component of DTaP-IPV vaccine, and the combination of Act-Hib and DTaP-IPV increased anti-TT antibodies. The overall results show that combination of Act-Hib and DTaP-IPV, either injected in separate sites or mixed in the same syringe, had an additive e#ect on anti-TT antibodies, and there were no significant di#erences in anti-TT antibody responses between injection of DTaP-IPV+Act-Hib in separate sites and the mixture of DTaP-IPV+Act-Hib in the same syringe. Amvax Hib-TT alone produced no noticeable anti-TT antibodies on its own. Combination of Hib-TITER with DTaP-IPV did not significantly a#ect antibody responses to the TT component of DTaP-IPV. Effect of Hib-conjugated vaccines on immunogenicity of DT in the mouse model
Table 3 gives the results of the e#ect of Hibconjugated vaccines on kinetics of antibody response to the DT component of the DTaP-IPV vaccine. In the Act-Hib groups, no significant di#erences in the anti-DT antibody responses was observed. Anti-DT antibodies in the mice immunized with mixture of DTaP-IPV+Amvax Hib-TT were lower, specially after third injection with the highest dose (1/20HD) than anti-DT antibodies produced by DTaP-IPV alone, while, the e#ect of Amvax Hib-TT on immunogenicity of the DT component of DTaP-IPV in the lower doses (1/40HD and 1/80HD) was only minor. Hib-TITER alone did not show any anti-DT activity on its own, and anti-DT antibody levels in the mice immunized with DTaPIPV+Hib-TITER mostly were not significantly
DTaP-IPV & Hib vaccines in animal models
233
Table 5. Results of tetanus lethal potency assay and diphtheria Vero potency assay. The relative potency of the TT and DT component of vaccines was calculated by comparing the results from the test vaccines with the results from either the tetanus standard toxoid or the international standard toxoid for adsorbed diphtheria vaccine (DIXA), respectively. Using this standard calculation, the relative potency of DTaP-IPV was then set at 100%. The relative potency of the other preparations was calculated using the relative potency of the TT and DT component of DTaP-IPV set at 100% Vaccines DTaP-IPV Act-Hib DTaP-IPV+Act-Hib (separate sites) DTaP-IPV+Act-Hib (mixed in the same syringe) 1/2DTaP-IPV+1/2Act-Hib (separate sites) 1/2DTaP-IPV+1/2Act-Hib (mixed in the same syringe) Amvax Hib-TT DTaP-IPV+Amvax Hib-TT (mixed in the same syringe) Hib-TITER DTaP-IPV+Hib-TITER (separate sites) DTaP-IPV+Hib-TITER (mixed in the same syringe) 1/2DTaP-IPV+1/2Hib-TITER (separate sites) 1/2DTaP-IPV+1/2Hib-TITER (mixed in the same syringe)
Tetanus potency*
Diphtheria Potency*
100% 86% 558% N/A 70·2% 104% 0% 75·7% N/D 95·2% 128% N/D N/D
100% N/D 39% 58·4% N/D N/D N/D 109% 0% 171%† 166%† 82·3% 77·9%
* Results have been compared with DTaP-IPV. † Significantly di#erent from DTaP-IPV. N/A: Due to unacceptable linearity of dose response curve, calculation of potency was not applicable. N/D: Not done.
di#erent from the mice immunized with DTaP-IPV alone. Effect of Hib-conjugated vaccines on the tetanus lethal potency assay and diphtheria Vero potency assay in the mouse model
The results of the potency assays are shown in Table 5. It is clear that the TT component of Act-Hib was as active as the TT component of the DTaP-IPV vaccine. Thus, combination of DTaP-IPV+Act-Hib, injected in separate sites, augmented the potency of combination more than five-fold. Since the responses to the mixture of DTaP-IPV+Act-Hib in the same syringe had approached the plateau phase of the dose response, it was impossible to calculate the potency of this mixture. The use of 1/2DTaP-IPV +1/2Act-Hib, either injected in separate sites or mixed in the same syringe, adjusted the potency of the combinations to 70·2% and 104%, respectively, which were not significantly di#erent from DTaPIPV alone. Amvax Hib-TT alone did not show any TT potency on its own and a mixture of DTaPIPV+Amvax Hib-TT did not significantly a#ect the potency of TT. Combination of DTaP-IPV +HibTITER did not display any significant e#ect on potency of TT. In the combination groups, the
groups which had been immunized with mixture of DTaP-IPV+Hib-conjugated vaccines in the same syringe did not show di#erent TT potency from the groups which had been immunized with DTaPIPV+Hib-conjugated vaccines injected in the separate sites. Combination of DTaP-IPV+Act-Hib diminished the relative potency of the DT component of DTaPIPV. Mixture of DTaP-IPV+Amvax Hib-TT in the same syringe did not have any notable e#ect on potency of the DT component of DTaP-IPV vaccine. Hib-TITER did not show any DT potency on its own, but combination of Hib-TITER with DTaP-IPV elevated significantly the immunogenic potential of the DT component of DTaP-IPV vaccine. Immunization with combination of 1/2DTaP-IPV+1/2HibTITER resulted in modulation of DT potency. Di#erences of DT potency between two combination groups, injected in separate sites and mixed in the same syringe were minor. Effect of Hib-conjugated vaccines on immunogenicity of PT in the mouse model
The e#ect of Hib-conjugated vaccines on immunogenicity of the pertussis toxoid, the main component of the aP vaccine, was studied. Anti-PT antibody
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responses were una#ected when DTaP-IPV was administered with all three tested Hib-conjugated vaccines (data not shown). Immunogenicity of Hib-conjugated vaccines alone and in combination with DTaP-IPV in the mouse model
Figure 1 shows anti-HibCP antibodies in pooled sera of the mice immunized with either Hib vaccines or combination of Hib vaccines with DTaP-IPV. Non-dose dependent response to HibCP was shown in all immunized groups. Act-Hib alone produced pronounced anti-HibCP antibodies, while combination of Act-Hib with DTaP-IPV in general decreased anti-HibCP titre, and mixture of Act-Hib with DTaP-IPV in a same syringe showed obviously lower titre of anti-HibCP antibodies than Act-Hib alone. In the mice which had been immunized with mixture of Hib-TITER with DTaP-IPV in the same syringe, the levels of antibody were considerably higher than in both the Hib-TITER alone and separate sites combined group. The levels of anti-HibCP antibodies in the mice immunized with mixture of Amvax Hib-TT with DTaP-IPV in the same syringe were markedly higher than the mice which had been immunized with Amvax Hib-TT alone. The overall view was that Act-Hib alone produced higher anti-HibCP antibodies than combination of Act-Hib+DTaPIPV, while mixture of either Hib-TITER or Amvax Hib-TT with DTaP-IPV in the same syringe had a positive additive e#ect on antibody responses to HibCP. Effect of Hib-conjugated vaccines on immunogenicity of TT, DT, aP, and IPV in the guinea pig model
Act-Hib and Amvax Hib-TT alone showed pronounced anti-TT antibodies in the guinea pig model (Table 6). But only in the short interval protocol, combination of either Act-Hib or Amvax Hib-TT with DTaP-IPV increased significantly the anti-TT antibody responses to the TT component of DTaPIPV. This feature was shown both after first and second immunization. None of Hib-conjugated vaccines showed remarkable e#ect on immunogenicity of DT (Table 6), aP, and IPV components of the DTaP-IPV vaccine in the guinea pig model (results for aP and IPV are not shown). Immunogenicity of Hib-conjugated vaccines alone and in combination with the DTaP-IPV in the guinea pig model
Table 7 shows the capability of the guinea pig model to produce anti-HibCP antibodies using two
di#erent protocols. In the ‘‘short interval’’ protocol, four weeks after first immunization, only one animal showed detectable anti-HibCP antibodies, and the rest of animals responded after second injection. Boosting e#ect was seen in all immunized groups. There was no considerable di#erence between the groups of guinea pigs which had been immunized with either Hib-conjugated vaccines alone or combination of Hib-conjugated vaccines with DTaP-IPV. In the ‘‘long interval’’ protocol, five of the immunized animals died under anaesthesia during the first bleeding, one in the Act-Hib+DTaP-IPV (MS) group, three in the Amvax Hib-TT group, and one in the Amvax Hib-TT+DTaP-IPV (SS) group. It can be seen that only two animals, one in the Act-Hib+DTaP-IPV(MS) group and one in the HibTITER+DTaP-IPV (SS) group, had detectable antiHibCP antibodies after first immunization. Two weeks after boosting, anti-HibCP antibodies were detected in most of the groups, and the maximum responses were reached six weeks after second injection. In the ‘‘long interval’’ protocol, a boosting e#ect was shown in all groups (except Amvax Hib-TT alone) and the number of responders in a few groups increased over the time from two weeks post second injection to six weeks post second injection. Discussion The magnitude of the antibody responses of immunized mice to the TT component of di#erent combination groups was variable. It was demonstrated that in the mouse model the immunogenic potential of the TT component of Act-Hib in the multiple shot program was as great as that of the TT component of DTaP-IPV vaccine. Therefore, combination of DTaP-IPV+Act-Hib, either injected in separate sites or mixed in the same syringe, induced, as expected, higher anti-TT antibodies than DTaP-IPV alone. Similar results were obtained in the tetanus lethal potency assay for Act-Hib alone and for combination of DTaP-IPV+Act-Hib. However, the TT component of Amvax Hib-TT did not show any immunogenic potential on its own, either in the multiple shot program nor in the lethal potency assay. Also in the lethal potency assay, combination of Amvax Hib-TT with DTaP-IPV did not increase the potency of the TT component of DTaP-IPV, attributable to occupancy of the tetanus toxoid B-cell epitopes by small size HibCP.15 However, in the multiple-shot programme, when DTaP-IPV was
0
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DTaP-IPV & Hib vaccines in animal models 235
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Figure 1. Anti HibCP antibodies in the pool sera of the mice immunized with di#erent preparations. Results are shown as titre in comparison with negative control serum. ( ), 1/20 HD; ( ), 1/40 HI ( ), 1/80 HD.
SS: separate sites. MS: mixed in the same syringe. IU: International Unit. * : antibodies of the pooled sera. N/D: not done.
DTaP-IPV Act-Hib Act-Hib+DTaP-IPV(SS) Act-Hib+DTaP-IPV(MS) Amvax Hib-TT Amvax Hib-TT+DTaP-IPV(SS) Amvax Hib-TT+DTaP-IPV(MS) Hib-TITER Hib-TITER+DTaP-IPV(SS) Hib-TITER+DTaP-IPV(MS)
2·67(1·82–3·92) 1·45(0·60–3·52) 9·71(7·73–12·2) 12·2(9·98–14·8) 2·19(1·22–3·94) 4·54(3·25–6·34) 5·99(4·35–8·27) N/D N/D N/D
Anti-TT 0·07(0·04–0·12) N/D 0·09(0·04–0·21) 0·05(0·04–0·07) N/D 0·06(0·04–0·12) 0·64(0·02–0·06) N/D N/D N/D
Anti-DT
4 wks post 1st immunization
15·6(11·5–21·1) 25·6(13·0–15·3) 44·1(30·7–63·4) 38·7(26·3–56·9) 23·7(13·1–43·0) 52·0(38·7–70·0) 33·8(19·3–59·2) N/D N/D N/D
Anti-TT 0·69(0·36–1·3) N/D 1·4(0·57–1·9) 0·72(0·50–1·0) N/D 1·4(0·21–2·3) 0·56(0·41–0·78) N/D N/D N/D
Anti-DT
2 wks post 2nd immunization
Short interval protocol
5·7(3·5–3·7) 1·7(0·43–7·1) 7·5(7·2–7·7) 6·1(5·1–7·3) 0·46(0·02–8·9) 5·3(4·5–6·2) 5·7(3·9–8·3) N/D 7·4(6·9–7·9) 3·5(2·8–4·4)
Anti-TT 1·51(0·09–2·58) N/D 1·06(0·47–2·38) 1·43(0·57–3·60) N/D 1·64(0·31–3·31) 1·45(0·73–2·69) 0·02(·002–0·26) 3·01(1·67–5·44) 2·04(1·26–3·29)
Anti-DT
6 wks post 1st immunization
12·49 14·48 15·47 7·14 14·62 21·82 14·49 N/D 8·18 10·63
Anti-TT*
11·9(6·44–22·0) N/D 6·19(2·27–16·8) 3·70(1·59–2·61) N/D 7·21(4·32–12·0) 5·54(4·36–7·17) 0·06(0·03–0·11) 4·24(2·65–6·73) 5·171*
Anti-DT
2 wks post 2nd immunization
Long interval protocol
Table 6. Geometric mean of anti-TT and anti-DT antibodies IU (95% confidence limits) in the guinea pig model
1·65 5·68 6·31 4·30 4·36 8·74 1·63 N/D 1·53 2·90
Anti-TT*
0·62(0·36–1·07) N/D 0·39(0·14–1·11) 0·99(0·20–1·79) N/D 0·77(0·31–1·91) 0·47(0·20–1·09) 0·01(0·01–0·20) 1·91(0·72–5·10) 0·16(0·10–0·28)
Anti-DT
6 wks post 2
236 H. Shams and I. Heron
DTaP-IPV & Hib vaccines in animal models
237
Table 7. Immunogenicity of HibCP-conjugated vaccines in the guinea pig model. Results are presented as responder(s)/total number of animals in each group(range of g HibCP to inhibit 50% OD value) Short interval Vaccines Act-Hib Act-Hib+DTaP-IPV(SS) Act-Hib+DTaP-IPV(MS) Amvax Hib-TT Amvax Hib-TT+DTaP-IPV(SS) Amvax Hib-TT+DTaP-IPV(MS) Hib-TITER Hib-TITER+DTaP-IPV(SS) Hib-TITER+DTaP-IPV(MS)
Long interval
4 wks post 1st immun.
2 wks post 2nd immun.
6 wks post 1st immun.
2 wks post 2nd immun.
6 wks post 2nd immun.
0/6 0/6 1/6(0·19) 0/6 0/6 0/6 N/D N/D N/D
3/6(0·53–0·62) 3/6(0·25–0·81) 2/6(0·06–0·21) 2/6(0·62–2·36) 3/6(0·14–1·17) 3/6(0·04–2·08) N/D N/D N/D
0/6 0/6 1/6(0·87) 0/3 0/5 0/6 0/6 1/6(0·56) 0/6
3/6(0·08–0·59) 4/6(0·05–0·90) 3/5(0·05–0·19) 0/3 1/5(0·03) 0/6 1/6(0·23) 2/6(0·02–0·39) 2/6(0·02–0·19)
4/6(0·02–0·86) 4/6(0·06–1·00) 3/5(0·21–2·57) 0/3 1/5(0·59) 3/6(0·19–1·35) 3/6(0·17–0·37) 2/6(0·02–0·59) 3/6(0·08–1·23)
SS: separate sites. MS: mixed in the same syringe. N/D: Not done.
combined with Amvax Hib-TT, an increase in antibody responses after boosting was observed. This might be related to intact T-cell epitopes of the TT component of Amvax Hib-TT.15 Furthermore, combination of Hib-TITER with DTaP-IPV, either injected in separate sites or mixed in the same syringe produced no significant change in the kinetics of serum anti-TT antibody levels as well as in the potency of TT, demonstrating no change in the total and in the neutralizing antibody responses to TT. In the guinea pig model, the immunogenicity of the TT component of Act-Hib and Amvax Hib-TT was confirmed. In the ‘‘short interval’’ protocol, primary and secondary responses to combination of either Act-Hib or Amvax Hib-TT with DTaP-IPV were significantly higher than DTaP-IPV alone. This verifies the e#ect of the TT component of Act-Hib and Amvax Hib-TT on immunogenicity of the TT component of DTaP-IPV in the guinea pig model. However, contrary to our results in the mouse model, Amvax Hib-TT induced pronounced anti-TT antibody response in the guinea pigs after the first immunization. This may be attributed to the high vaccine dose used in guinea pigs (1/2 HD) and uncharacterized animal species e#ects.12 Taken together, these experiments show that in the two animal species used the HibCP component of Hib-conjugated vaccines did not have any deleterious e#ect on potency and immunogenicity of TT component of DTaP-IPV on its own, and the additive e#ect of Hib-conjugated vaccines on potency and/or immunogenicity of TT component of
DTaP-IPV was mainly due to existence of TT as the carrier in the Hib-conjugated vaccines. Combination of Act-Hib with DTaP-IPV in the mouse model using the single shot program reduced the potency of the DT component of DTaP-IPV vaccine to 39–58%, which however, was not statistically significant because of the wide 95% confidence limits, demonstrating that changes were within the experimental variability of the assay; and in the multiple shot programme, combination of Act-Hib with DTaP-IPV did not considerably a#ect the serum antibody responses to the DT component of DTaP-IPV vaccine. In the mouse model using the single shot programme, mixing of Amvax Hib-TT with DTaP-IPV in the same syringe did not influence the potency of the DT component of DTaP-IPV. However, in the multiple shot programme, serum antibody responses to DT after third injection with 1/20HD of mixture of DTaP-IPV+Amvax Hib-TT were lower than DTaP-IPV alone, but at lower doses (1/40HD and 1/80HD) di#erences were negligible. In the mouse model, combining of DTaP-IPV with Hib-TITER, either injected in separate sites or mixed in the same syringe, increased significantly the potency of DT. This positive e#ect of Hib-TITER on the potency of DT component of DTaP-IPV might be partially due to e#ect of adjuvant [Al(OH)3] of DTaP-IPV on intact B-cell epitopes of CRM197 component of Hib-TITER.16 However, in the multiple shot programme, serum antibody responses to DT in the combination of DTaP-IPV with Hib-TITER were not significantly di#erent from DTaP-IPV alone.
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Remarkable di#erences were shown in anti-DT antibody levels between two protocols in the guinea pig model. Anti-DT antibody levels in the animals immunized in ‘‘long interval’’ protocol were much higher than those immunized in the ‘‘short interval’’ protocol. This indicates the importance of interval on anti-DT antibody responses in the guinea pig model and notifies that in quality control tests of DT that are performed in the guinea pig model, the defined interval between immunization and bleeding must be regarded carefully. The use of the guinea pig model to study the e#ect of Hib-conjugated vaccines on immunogenicity of DT revealed that in the ‘‘short interval’’ protocol, combination of either Act-Hib or Amvax Hib-TT with DTaP-IPV had no significant influence on the immunogenicity of DT component of DTaP-IPV. This di#ers from the findings of Siber et al.2 that shows that combination of Hib-TT vaccines with DTP has been resulted in significant decrease in anti-DT antibody responses in the guinea pig model, probably attributable to di#erent doses used. Our results and those of others2–4 show inconsistency regarding the e#ect of HibCP on immunogenicity and potency of DT, and imply that more studies should be carried out. In the current studies, pooled sera from mice and guinea pigs immunized with either DTaP-IPV alone or a combination of DTaP-IPV with Hib-conjugated vaccines were tested for antibody responses to pertussis toxoid (PT), the main component of the acellular pertussis (aP) vaccine. In general, we showed that Hib-conjugated vaccines did not reduce antibody responses to the PT in either used animal models. Our current work and that of others3,4 also indicate that Hib-conjugated vaccines have no negative e#ect on the kinetics of antibody responses to PT, or on potency of whole cell pertussis vaccine, respectively. However, in a human clinical trial, co-administration of Act-Hib with DTP suppressed the level of anti-PT antibodies,17 demonstrating that the results in animal model(s) can not predict the consequence of combining vaccine components for human immune response. Combination with DTaP-IPV showed various e#ects on immunogenicity of Hib-conjugated vaccines in the mouse model. A decrease in anti-HibCP antibody levels was observed when Act-Hib, in which TT was as active as non-conjugated TT, was combined with DTaP-IPV vaccine. Mixture of Amvax Hib-TT with DTaP-IPV in a same syringe showed higher levels of anti-HibCP antibodies than Amvax Hib-TT alone. Likewise, combination
of Hib-TITER with DTaP-IPV produced higher serum antibodies to HibCP than Hib-TITER alone. Moreover, the ine#iciency of the TT component of Amvax Hib-TT, and of the CRM197 component of Hib-TITER in induction of anti-TT and anti-DT antibodies, respectively, has already been demonstrated.15 In summary, our results in the mouse model and those of others in chicken and human18,19 demonstrate that potentiation of the antibody responses to carrier, either due to large dose of carrier (existence of active B cell epitopes on the carrier and combination with non-conjugated carrier) or existence of anti-carrier antibodies prior to administration of Hib-conjugated vaccines will result in suppression of serum antibody responses to HibCP. These observations are in agreement with carrier specific B- and T-cell clonal dominance hypothesis20–22 which suggest that inactivation of carrier B-cell epitopes and consequently prevention of clonal dominance of carrier-specific B cells leads to prevention of epitopic suppression.22 It is reasonable that during secondary immune response, memory B cells interact with the carrier epitopes more e#iciently than other non-immune antigen presenting cells.20 Our results in the mouse system seem in accordance with this and confirm that removing the carrier B-cell-epitopes elevates the likelihood for HibCP epitopes of Hib-conjugated vaccines to interact with HibCP-specific B cells, and thereby, presentation of carrier T cell epitopes by HibCP-specific B cells will result in anamnestic anti-HibCP antibody responses. Also, the same specific B cell precursors are activated either by a polysaccharide in a thymus-independent way or by conjugated-polysaccharide in a thymus-dependent way,23 and only carrier-specific T cells modify the result of immune responses. Thus, conjugation to a protein does not increase the chance of polysaccharides for primary interactions with B cells. The latter is a possible explanation for non-dose dependency for anti-HibCP antibody responses in our experiments and those of others.2 Vogel et al. have reported a modulatory e#ect of whole-cell Bordetella pertussis on carrier-induced epitopic suppression.24 So, the discrepancy between our results and those of Redhead et al.,3,4 who reported the increase in anti-HibCP antibodies in simultaneous injection of Act-Hib and DTP vaccines in a mouse model might be attributable to whole cell pertussis component of DTP vaccine. Lyng and Heron12 found that potency results depend on di#erent factors including animal
DTaP-IPV & Hib vaccines in animal models
species. In the current study, we found that concurrent immunization with Act-Hib and DTaP-IPV in the mouse model resulted in reduction of antiHibCP antibody responses, while co-administration of Act-Hib with DTaP-IPV did not have a remarkable e#ect on anti-HibCP responses in the guinea pig model. Selected dose in the guinea pig model and in the mouse model, on a weight to weight basis was almost the same, and the levels of anti-carrier antibodies (anti-TT) in the International Unit basis in the guinea pig model was much higher than in the mouse model. This is tempting to speculate that the repertoire of the murine immune system in response to the Hib-conjugated vaccines is relatively restricted, and therefore, induction of suppression with the Hib-conjugated vaccines in the mouse model is easier than in the guinea pig model. In the current study, anti-HibCP responses of the guinea pig model in two di#erent protocols (‘‘short interval’’ and ‘‘long interval’’ protocols) were compared. Immunization of guinea pigs with either Act-Hib alone or combination of Act-Hib with DTaP-IPV did not show any striking di#erence in anti-HibCP responses between the two protocols. In the Amvax Hib-TT groups (Amvax Hib-TT alone and combination of Amvax Hib-TT with DTaP-IPV), animals which had been immunized in the ‘‘short interval’’ protocol responded even better than the groups which had been immunized in ‘‘long interval’’ protocol. These demonstrate that the ‘‘long interval’’ protocol does not have any advantage over the ‘‘short interval’’ protocol for induction of anti-HibCP antibodies in the guinea pig model. Additional studies are required to clarify the factors that determine di#erential immune responses as a consequence of mutual interactions between components of combined vaccines in humans and laboratory animals. Acknowledgements
Authors wish to thank Henrick Aggerbeck, Gert Albert Hansen, Max Kristiansen, and Gitte Stawski for worthwhile arguments; Adam Gottschau for statistical analysis and interpretation of results; and Halina L. Pedersen and Vita Skov for their excellent technical assistance.
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polyribosylribitol phosphate (PRP) conjugated vaccines in the mouse model. APMIS 1998; 106: 526–534. Anderson P, Pichichero ME, Insel RA. Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197. J Clin Invest 1985; 26: 52–59. Clemens JD, Ferreccio C, Levine MM et al. Impact of Haemophilus influenzae type b polysaccharidetetanus protein conjugate vaccine on responses to concurrently administered diphtheria-tetanuspertussis vaccine. JAMA 1992; 267: 673–678. Barington T, Gyhrs A, Kristensen K et al. Opposite e#ect of actively and passively acquired immunity to the carrier on responses of human infants to a Haemophilus influenzae type b conjugate vaccine. Infect Immun 1994; 62: 9–14. Sarvas H, Ma¨ kela¨ O, Toivanen P et al. E#ect of carrier preimmunization on the anti-hapten response in the chicken. Scand J Immunol 1974; 3: 455–460.
20. Schutze MP, Deriaud E, Przewlock G et al. Carrierinduced epitopic suppression is initiated through clonal dominance. J Immunol 1989; 142: 2635–2640. 21. Schutze MP, Leclerc C, Lolivert M et al. Carrierinduced epitopic suppression, a major issue for future synthetic vaccines. J Immunol 1985; 135: 2319–2322. 22. Schutze M, Leclerc C, Vogel FR et al. Epitopic suppression in synthetic vaccine models: Analysis of the e#ector mechanisms. Cell Immunol 1987; 104: 79–90. 23. Willers J, Weiler E, Kolb C. Stimulation of the same B-cell population by thymus-independent dextran and by thymus-dependent oligosaccharide-carrier. Scand J Immunol 1995; 42: 345–352. 24. Vogel FR, Leclerc C, Schutze MP et al. ‘‘Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muranyl peptide’’. Cell Immunol 1987; 107: 40–51.
Received for publication 22 December 1998; accepted 3 June 1999
Mutual Interactions Between DTaP-IPV and Haemophilus influenzae Type b (Hib)-conjugated Vaccines in Laboratory Animal Models 1
Homayoun Shams1*† and Iver Heron2‡ Bacterial Vaccines Department, Statens Serum Institut, Copenhagen, Denmark 2 Amvax Inc., Indian Creeck Court, Beltsville, Maryland, U.S.A.
Abstract. Potency and/or immunogenicity of three different Haemophilus influenzae type b-conjugated vaccines (Hib) and a DTaP-IPV vaccine alone, and their mutual interactions in DTaP-IPV-Hib combination was tested. In a mouse model, only combination of Act–Hib, in which tetanus toxoid (TT) was as active as non-conjugated TT, significantly increased the immunogenicity and potency of TT component of DTaP-IPV vaccine. Also, only combination of Hib-TITER, in which CRM197 was used as the carrier with DTaP-IPV, increased the potency of diphtheria toxoid (DT) component of DTaP-IPV vaccine significantly. It shows that the additive effect of tested Hib vaccines on immunogenicity and/or potency of TT and DT was mostly due to the existence of TT and CRM197, respectively, as the carrier in the mentioned Hib vaccines. No difference was shown in inoculation of DTaP-IPV and Hib conjugated vaccines in the same syringe or at separate sites. DTaP-IPV had dual effects on anti-Hib capsular polysaccharide (HibCP) responses to Hib vaccines in the mouse model. This duality was probably related to the carrier B-cell epitopes activity of Hib conjugated vaccines. The immunogenicity of TT component of Act-Hib and Amvax Hib-TT in the guinea pig model was shown and combination of mentioned Hib vaccines with DTaP-IPV, remarkably increased anti-TT antibody responses to the TT component of DTaP-IPV vaccine. These confirmed our results in the mouse model. Using two different protocols to evaluate the guinea pig model for induction of anti-HibCP immunity showed that a ‘‘long interval’’ protocol does not have any advantage over the ‘‘short interval’’ protocol. Also, combination of DTaP-IPV with Hib vaccines did not have any noticeable effect on anti-HibCP antibodies in the guinea pig model. Taken together, our observations in laboratory animal models may facilitate a better understanding of the mutual interactions between the different antigen components of a combined vaccine such as DTaP-IPV© 1999 The International Association for Biologicals Hib vaccine.
Introduction Combined vaccines which protect against two or more diseases make vaccination plans more practical and economical. This is especially important in developing countries due to the large number of pathogens which threaten the health of infants.1 The need to administer many vaccines to infants and children has stimulated e#orts to determine *To whom correspondence should be addressed. †Current address: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, P.O. Box 830905, Lincoln, NE 68583–0905, U.S.A. ‡Deceased. 1045–1056/99/030227+14$30.00/0
their compatibility for incorporation as a combined vaccine into a single injection. The trend is toward combining more and more of the newly developed conjugated vaccines with other combined vaccines. For example, the DTaP-IPV constitutes a complex mixture, the diphtheria toxoid (DT) – tetanus toxoid (TT) acellular pertussis (aP) and three serotypes (types 1, 2 and 3) of inactivated polio vaccine (IPV). Addition of another antigen such as a Haemophilus influenzae type b (Hib)-conjugated vaccine further increases this complexity. The current concept is that combination of the DTaP-IPV with a Hibconjugated vaccine can provide a unique opportunity for broadening protection against more 1999 The International Association for Biologicals
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Table 1. Vaccines Vaccines
Act-Hib* Hib-TT Hib-TITER† DTaP-IPV‡
Manufacturer
Pasteur Merieux Amvax Lederle-Praxis Statens Serum Institut/Amvax
Polysaccharide
Carrier
size§
g/HD
nature¶
g/HD
Large Small Small N/A
10 10 10 N/A
Tetanus Toxoid Tetanus Toxoid CRM197 N/A
24 28·5 25 N/A
* Batches No.H1239 and J0812. † Batches No.S815 and L0284B4. ‡ Each dose is 0·5 ml and contains 1 mg Aluminium, 25 Lf diphtheria toxoid, 7 Lf tetanus toxoid, 40/8/32 DU type 1,2,3 of poliovirus, and 40 g pertussis toxoid (PT) is the major component of acellular pertussis vaccine (aP). § Native Hib-CP was considered as large size and reduced size oligosaccharides, containing 15–30 repeat units were considered as small size. ¶ CRM197 is a mutant of Corynebacterium diphtheriae toxin.
diseases without discomfort of additional injections. Four Hib-conjugate vaccines have been licensed for sale in the United States, but there is no welldefined and universally accepted method for testing immunogenicity of Hib-conjugate vaccines in laboratory animals.2 Also, the U.S. Food and Drug Administration (FDA) and European Pharmacopoeia (EP) requirements for potency tests of DT and TT containing vaccines are not alike. Therefore, further experiments to determine the mutual interactions between DTaP-IPV vaccine and Hibconjugated vaccines, and to clarify the role of di#erent factors [e.g. di#erent carriers, size of Hib capsular polysaccharides (HibCP)] on immunogenicity of Hib-conjugated vaccines in laboratory animal models are required. In this study, we used three di#erent Hibconjugated vaccines, Act-Hib (large size HibCP and TT as the carrier), Amvax Hib-TT (small size HibCP and TT as the carrier) and Hib-TITER (small size HibCP and CRM197 as the carrier; CRM197 or ‘‘cross-reacting material’’ is a non-toxic mutant of diphtheria toxin and serologically cross-reacts with diphtheria antitoxin) and a DTaP-IPV vaccine to investigate: (1) the e#ect of Hib-conjugated vaccines on immunogenicity and/or potency of di#erent components of a DTaP-IPV vaccine; (2) the role of di#erent factors in the immunogenicity of Hibconjugated vaccines; (3) the e#ect of the DTaP-IPV vaccine on immunogenicity of di#erent Hibconjugated vaccines, in a mouse and in a guinea pig model. DTP-Hib vaccine was evaluated in a mouse model3,4 and Gupta et al. proposed the guinea pig model for evaluation of DTaP-Hib vaccines.5 To our
knowledge, this is the first study in which DTaPIPV-Hib vaccines have been evaluated in both the mouse and the guinea pig model.
Materials and methods Vaccines
The following vaccines were used in this study: Act-Hib (Institute Pasteur Merieux Lyon, France); Hib-TT conjugate (Amvax Inc., Beltsville, MD, U.S.A.); Hib-TITER (Lederle-Praxis Biologicals, Pearl River, U.S.A.); and a paediatric diphtheria– tetanus–acellular pertussis (aP)-inactivated polio vaccine (DTaP-IPV, Statens Serum Institut, Copenhagen, Denmark / Amvax, Inc. Beltsville, MD, U.S.A.). The volume of each single human dose (HD) was 0·5 ml. The details of each vaccine component are shown in Table 1. Animals
Outbred, female, CF1, 16–18 g mice; and white, female, 250–300 g Hartley strain guinea pigs were used as animal models. All the animals were obtained from Hvidesteen Laboratory Animal Farms, Statens Serum Institut, Denmark. Immunization in the mouse model
Two di#erent immunization schedules were carried out: a single shot programme to measure the potency of DT and TT, and a multiple shot programme to measure the immunogenicity and kinetics of antibody responses to HibCP, DT, TT, and acellular pertussis (aP).
DTaP-IPV & Hib vaccines in animal models
Single shot immunization programme. This pro-
gramme was chosen according to the o#icial oneinjection protocol in the mouse model6,7 to measure the relative potency of DT and of TT component of di#erent preparations. Methods were selected on the basis of the World Health Organization (WHO) and EP requirements as follows: (A) Diphtheria Vero potency assay. The method has been described elsewhere,6–9 briefly, groups of mice (16–20 mice/dose) were immunized once subcutaneously with at least three di#erent doses of either the international standard toxoid for adsorbed diphtheria vaccine (DIXA), Hib-TITER, DTaP-IPV, combination of DTaP-IPV+Act-Hib (either injected in separate sites or mixed in the same syringe), combination of DTaP-IPV+Amvax Hib-TT (mixed in the same syringe and four di#erent dilution groups for this), or with the combination of DTaP-IPV+HibTITER (either injected in separate sites or mixed in the same syringe, and four di#erent dilution groups for this). Doses for DIXA were 8, 4, and 2 IU/mouse, and doses for test preparations were 1/20, 1/40, 1/80 and in a few cases 1/160 HD. Four weeks later, immunized mice were bled and sera were collected. The concentration of neutralizing anti-diphtheriatoxin antibodies of sera was measured by Vero cell assay.6,9,10 (B) Tetanus lethal potency assay. This assay has been 6–8,10–12
briefly groups of 16–20 described elsewhere, mice/dose were immunized once subcutaneously with three di#erent doses of either tetanus standard toxoid, DTaP-IPV, Act-Hib, Amvax Hib-TT, combination of Act-Hib+DTaP-IPV (either injected in separate sites or mixed in the same syringe), combination of Amvax Hib-TT+DTaP-IPV (mixed in the same syringe, and four di#erent dilution group for this), or combination of Hib-TITER+DTaP-IPV (either injected in separate sites or mixed in the same syringe). Doses for tetanus standard toxoid were 3·425, 1·713 and 0·856 IU/mouse, and the test vaccines dilutions were 1/40, 1/80, 1/160 and in a few cases 1/320 HD. Four weeks later, immunized mice were challenged with median paralytic dose (PD50) tetanus toxin, and lethal or paralytic e#ects were recorded within the next 4 days. Multiple-shot immunization programme. In order to investigate the mutual interactions of Hibconjugated vaccines and DTaP-IPV in the mouse model, the mice (16–20 mice/dose) that had been bled to analyse the potency of diphtheria toxoid along with other two groups which had been immu-
229
nized with either Act-Hib or Amvax Hib-TT were immunized for second time (at the same day after bleeding for diphtheria potency) with the same doses and components as they had been immunized in the first shot (described in the diphtheria potency assay). Two weeks after second injection, second bleedings and at the same day third immunization were done. Two weeks, 4 weeks, 8 weeks and 12 weeks after third immunization, third, fourth, fifth and sixth bleedings were performed. In the Tables 2 and Table 3, results are only shown for the first three bleedings. Immunization in the guinea pig model
In this model, groups of six animals were immunized subcutaneously with 1/2 HD of either DTaPIPV, Act-Hib, DTaP-IPV+Act-Hib (either injected in separate sites or mixed in the same syringe), Amvax Hib-TT, DTaP-IPV+Amvax Hib-TT (either injected in separate sites or mixed in the same syringe), Hib-TITER, or combination of DTaPIPV+Hib-TITER (either injected in separate sites or mixed in the same syringe). Guinea pigs were immunized twice in the two di#erent immunization schedule (Table 4). Measurement of anti-diphtheria antibodies by Vero cell assay
Details of the assay have been described elsewhere.9 Briefly, a two-fold dilution series of each of the sera and diphtheria standard antitoxin (an international WHO standard for diphtheria antitoxin was used which is a hyperimmune horse antiserum containing 10 IU/ml, and was used at 1:100 dilution) were made in Nunc flat-bottomed Micro Well plates and a quantity of 50 l was left in each well. Then, 20 l diphtheria toxin containing 10 3 Lf/ml was added to each well and incubated for 2 h at 20C, and then into each well, 150 l Vero cell suspension (6104 African Green Monkey Kidney Cells/ml) was added. Plates were incubated for 5 days at 37C, 5% CO2 with 90% relative humidity. Complete Dulbecco’s modified Eagle medium containing 10 m Hepes, 100 IU/ml penicillin, 100 g/ml streptomycin and 10% fetal calf serum (FCS) was used. The concentration of neutralizing antidiphtheria toxin antibodies in each serum was calculated in comparison with the standard for diphtheria antitoxin, using a computerized Vero program from the Bacterial Vaccines Department, Statens Serum Institut, Denmark. This program is based on a regression analysis calculation. Data are shown in the scale of International Unit (IU).
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Table 2. Geometric mean of anti-tetanus antibodies (IU) in the mice immunized with di#erent vaccines in three di#erent set of experiments (95% confidence limits) Dose*
4 wks post 1st immunization
2 wks post 2nd immunization
2 wks post 3rd immunization
1/20 1/40 1/80
0·08(0·05–0·12) 0·06(0·04–0·10) 0·02(0·01–0·03)
1·88(1·42–2·50) 1·09(0·72–1·65) 0·54(0·33–0·87)
1·89(1·53–2·32) 1·43(1·18–1·72) 1·37(1·04–1·82)
0·12(0·06–0·22) 0·04(0·02–0·12) 0·03(0·01–0·07)
1·69(1·10–2·60) 0·57(0·24–1·33) 0·26(0·08–0·95)
2·15(1·63–2·84) 1·10(0·62–1·96) 0·27(0·10–0·73)
0·14(0·10–0·20) 0·11(0·06–0·19) 0·02(0·01–0·04) syringe) 0·16(0·11–0·24) 0·12(0·08–0·19) 0·05(0·03–0·09)
3·06(2·31–4·04) 2·25(1·53–3·30) 0·93(0·58–1·49)
2·66(2·12–3·34) 2·42(1·81–3·24) 1·32(0·90–1·94)
3·86(2·88–5·19) 2·45(1·75–3·44) 1·38(0·97–1·99)
2·86(2·02–4·05) 2·03(1·41–2·92) 1·61(1·17–2·21)
0·08(0·05–0·14) 0·06(0·04–0·10) 0·02(0·01–0·03)
2·22(1·56–3·16) 1·25(0·93–1·69) 0·65(0·30–1·42)
1·84(1·27–2·68) 0·76(0·54–1·06) 0·71(0·52–0·97)
1/20 † 1/40 † 1/80 † DTaP-IPV+Amvax Hib-TT (mixed in the same syringe) 1/20 0·22(0·15–0·33) 1/40 0·05(0·03–0·10) 1/80 0·03(0·01–0·06) 1/160 †
0·01(0·002–0·01) † †
0·06(0·01–0·30) 0·01(0·002–0·02) 0·01(0·002–0·01)
4·06(2·87–5·74) 2·11(0·99–4·49) 0·85(0·36–2·00) 0·25(0·12–0·54)
2·67(1·90–3·75) 2·71(1·99–3·69) 1·21(0·86–1·71) 0·85(0·60–1·20)
Panel C: DTaP-IPV
1·79(1·31–2·43) 1·36(0·97–1·90) 0·40(0·22–0·74)
1·58(1·23–2·03) 1·38(1·08–1·75) 0·85(0·63–1·15)
1·36(1·03–1·79) 0·86(0·55–1·35) 0·44(0·25–0·76) 0·07(0·03–0·17)
1·33(1·07–1·65) 1·25(1·00–1·57) 0·63(0·45–0·87) 0·53(0·35–0·79)
1·58(1·17–2·14) 1·05(0·75–1·48) 0·45(0·20–0·99) 0·08(0·04–0·16)
1·15(0·89–1·49) 0·90(0·70–1·15) 0·78(0·51–1·19) 0·51(0·35–0·72)
Vaccines Panel A: DTaP-IPV Act-Hib
1/20 1/40 1/80 DTaP-IPV+Act-Hib (separate sites) 1/20 1/40 1/80 DTaP-IPV+Act-Hib (mixed in the same 1/20 1/40 1/80 Panel B: DTaP-IPV Amvax Hib-TT
1/20 1/40 1/80
1/20 0·04(0·02–0·06) 1/40 0·03(0·01–0·06) 1/80 0·01(0·004–0·01) DTaP-IPV+Hib-TITER (separate sites) 1/20 0·06(0·04–0·10) 1/40 0·02(0·01–0·03) 1/80 0·01(0·01–0·02) 1/160 † DTaP-IPV+Hib-TITER (mixed in the same syringe) 1/20 0·05(0·03–0·08) 1/40 0·01(0·01–0·03) 1/80 † 1/160 0·01(0·004–0·02) * Human Dose. † Anti-tetanus antibody levels were less than 0·01 IU.
Measurement of anti-tetanus antibodies by ELISA
Sera from animals were tested for anti-tetanus antibodies using an enzyme-linked immunosorbent assay (ELISA) as described previously.13,14 Briefly,
coating the plastic microtitre plates (Nunc Intermed, Roskilde, Denmark) with purified tetanus toxoid (750 Lf/ml) diluted in carbonate bu#er (1:10 000, overnight), was followed by blocking bu#er, and then di#erent dilutions of sera were
DTaP-IPV & Hib vaccines in animal models
231
Table 3. Geometric mean of anti-diphtheria antibodies (IU) in the mice immunized with di#erent vaccines in three di#erent set of experiments (95% confidence limits) Vaccines
Dose*
4 wks post 1st immunization
Panel A DTaP-IPV
1/20 0·03(0·02–0·04) 1/40 0·01(0·01–0·02) 1/80 0·01(0·01–0·02) 0·13(0·06–0·29) DTaP-IPV+Act-Hib (separate sites) 1/20 0·02(0·01–0·02) 1/40 0·01(0·01–0·01) 1/80 0·01(0·01–0·01) DTaP-IPV+Act-Hib (mixed in the same syringe) 1/20 0·02(0·02–0·03) 1/40 0·01(0·01–0·02) 1/80 0·01(0·01–0·01) Panel B DTaP-IPV 1/20 0·02(0·01–0·03) 1/40 0·02(0·01–0·03) 1/80 0·004(0·003–0·006) DTaP-IPV+Amvax Hib-TT (mixed in the same syringe) 1/20 0·02(0·01–0·03) 1/40 0·01(0·01–0·02) 1/80 0·01(0·01–0·01) 1/160 † Panel C DTaP-IPV 1/20 0·01(0·004–0·01) 1/40 0·004(0·003–0·006) 1/80 0·002(0·001–0·003) Hib-TITER 1/20 † 1/40 † 1/80 † DTaP-IPV+Hib-TITER (separate sites) 1/20 0·01(0·01–0·02) 1/40 0·01(0·004–0·01) 1/80 † 1/160 † DTaP-IPV+Hib-TITER (mixed in the same syringe) 1/20 0·02(0·01–0·03) 1/40 0·01(0·004–0·01) 1/80 † 1/160 †
2 wks post 2nd immunization
2 wks post 3rd immunization
0·86(0·51–1·47) 0·32(0·16–0·64) 0·23(0·13–0·42)
0·62(0·42–0·92) 0·32(0·20–0·53)
0·62(0·39–0·97) 0·19(0·08–0·45) 0·17(0·08–0·36)
0·51(0·34–0·74) 0·24(0·13–0·42) 0·27(0·17–0·42)
1·16(0·73–1·85) 0·42(0·23–0·78) 0·17(0·08–0·37)
0·76(0·54–1·11) 0·41(0·25–0·67) 0·26(0·13–0·51)
1·15(0·75–1·76) 0·82(0·55–1·23) 0·15(0·06–0·43)
1·65(1·01–2·69) 0·41(0·21–0·81) 0·21(0·11–0·40)
0·41(0·16–1·03) 0·52(0·26–1·04) 0·17(0·07–0·42) 0·04(0·01–0·11)
0·48(0·29–0·83) 0·35(0·18–0·69) 0·22(0·10–0·48) 0·13(0·04–0·45)
0·73(0·47–1·13) 0·37(0·17–0·80) 0·10(0·05–0·18)
0·68(0·43–1·10) 0·36(0·18–0·75) 0·23(0·11–0·48)
† 0·01(0·003–0·01) 0·01(0·004–0·01)
0·01(0·01–0·3) 0·02(0·01–0·05) 0·02(0·01–0·04)
0·94(0·37–2·38) 0·30(0·13–0·70) 0·34(0·19–0·61) 0·03(0·01–0·08)
0·79(0·41–1·53) 0·74(0·43–1·28) 0·55(0·32–0·94) 0·21(0·13–0·33)
1·04(0·63–1·70) 0·44(0·26–0·75) 0·09(0·03–0·28) 0·01(0·004–0·02)
1·08(0·69–1·69) 0·60(0·43–0·83) 0·49(0·26–0·95) 0·09(0·06–0·14)
*Human dose. †Anti-diphtheria antibody levels were less than 0·002 IU.
made. After overnight incubation at 4C, plates were washed and peroxidase labelled rabbit-antimouse immunoglobulin antibodies (DAKO) were used. After washing, plates were developed by substrate. Addition of 1 H2SO4 after 30 min stopped the chromogenic reaction. OD values were measured with Immunoreader NJ 2000 at 490 nm. Calculation was performed using parallel line assay against the home reference serum (MuTe91). The
results are shown on the scale of International Unit (IU). Measurement of anti-HibCP antibodies by ELISA
Plates were coated with 50 l of HibCP conjugated to human serum albumin (2 g/ml HibCPHSA, Amvax, Inc.; Beltsville, MD, U.S.A.) and incubated overnight at 4C. Coated plates were washed and blocked with 100 l rabbit normal
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Table 4. Immunization and bleeding schedule of guinea pigs Immunization on the week of 1-Short interval protocol 2-Long interval protocol
0 0
serum(1%). For control, sera of the animals immunized with DTaP-IPV were pooled and used as the negative control. By direct ELISA. Di#erent dilutions of the murine sera were made and the rest of the technique was as described previously. The titre of antibody was determined by endpoint titration after subtraction of values for the negative control. The last dilution at which the OD value exceeded that observed in negative control was taken as the titre. By competitive ELISA. To recognize individual responders in the guinea pig model, a competitive ELISA was employed. On the coated plates, 25 l of di#erent dilutions of non-conjugated HibCP with four-fold serial dilutions (seven dilutions starting at a concentration of 100 g/ml) and 25 l dilution bu#er in the control well were applied. Then the constant amount of each serum (25 l) was added to eight wells of each column (from A to H). Plates were incubated at 4C overnight and washed and stained as described earlier. Sera that showed _1·000 OD value (after subtraction of background) and were inhibited by _0·02 g HibCP were recognized as responders. Results are expressed as the amounts of non-conjugated HibCP that have been able to inhibit the 50% OD value (after subtraction of background). Statistical analysis
The potency of the TT component of the test vaccines was calculated by the probit analysis, and for the potency of DT and CRM197 the parallel line assay was used. The results are shown in the term of relative potency in comparison with the relevant standard.6 Comparison of potency results was done by calculation of 95% confidence limits. The di#erences in values outside of the 95% confidence limits were considered significant. Student’s t-test was used in the analysis for di#erences in antibody levels between and within groups. The significance level chosen for the tests was 5%.
4 6
Bleedings on the week of 4 6
6 8
— 12
Results Effect of Hib-conjugated vaccines on immunogenicity of TT in the mouse model
Table 2 shows the kinetics of antibody responses to the TT component of Hib-conjugated vaccines, and the e#ect of Hib-conjugated vaccines on kinetics of antibody responses to the TT component of DTaP-IPV vaccine. Act-Hib alone produced anti-TT antibodies as much as the TT component of DTaP-IPV vaccine, and the combination of Act-Hib and DTaP-IPV increased anti-TT antibodies. The overall results show that combination of Act-Hib and DTaP-IPV, either injected in separate sites or mixed in the same syringe, had an additive e#ect on anti-TT antibodies, and there were no significant di#erences in anti-TT antibody responses between injection of DTaP-IPV+Act-Hib in separate sites and the mixture of DTaP-IPV+Act-Hib in the same syringe. Amvax Hib-TT alone produced no noticeable anti-TT antibodies on its own. Combination of Hib-TITER with DTaP-IPV did not significantly a#ect antibody responses to the TT component of DTaP-IPV. Effect of Hib-conjugated vaccines on immunogenicity of DT in the mouse model
Table 3 gives the results of the e#ect of Hibconjugated vaccines on kinetics of antibody response to the DT component of the DTaP-IPV vaccine. In the Act-Hib groups, no significant di#erences in the anti-DT antibody responses was observed. Anti-DT antibodies in the mice immunized with mixture of DTaP-IPV+Amvax Hib-TT were lower, specially after third injection with the highest dose (1/20HD) than anti-DT antibodies produced by DTaP-IPV alone, while, the e#ect of Amvax Hib-TT on immunogenicity of the DT component of DTaP-IPV in the lower doses (1/40HD and 1/80HD) was only minor. Hib-TITER alone did not show any anti-DT activity on its own, and anti-DT antibody levels in the mice immunized with DTaPIPV+Hib-TITER mostly were not significantly
DTaP-IPV & Hib vaccines in animal models
233
Table 5. Results of tetanus lethal potency assay and diphtheria Vero potency assay. The relative potency of the TT and DT component of vaccines was calculated by comparing the results from the test vaccines with the results from either the tetanus standard toxoid or the international standard toxoid for adsorbed diphtheria vaccine (DIXA), respectively. Using this standard calculation, the relative potency of DTaP-IPV was then set at 100%. The relative potency of the other preparations was calculated using the relative potency of the TT and DT component of DTaP-IPV set at 100% Vaccines DTaP-IPV Act-Hib DTaP-IPV+Act-Hib (separate sites) DTaP-IPV+Act-Hib (mixed in the same syringe) 1/2DTaP-IPV+1/2Act-Hib (separate sites) 1/2DTaP-IPV+1/2Act-Hib (mixed in the same syringe) Amvax Hib-TT DTaP-IPV+Amvax Hib-TT (mixed in the same syringe) Hib-TITER DTaP-IPV+Hib-TITER (separate sites) DTaP-IPV+Hib-TITER (mixed in the same syringe) 1/2DTaP-IPV+1/2Hib-TITER (separate sites) 1/2DTaP-IPV+1/2Hib-TITER (mixed in the same syringe)
Tetanus potency*
Diphtheria Potency*
100% 86% 558% N/A 70·2% 104% 0% 75·7% N/D 95·2% 128% N/D N/D
100% N/D 39% 58·4% N/D N/D N/D 109% 0% 171%† 166%† 82·3% 77·9%
* Results have been compared with DTaP-IPV. † Significantly di#erent from DTaP-IPV. N/A: Due to unacceptable linearity of dose response curve, calculation of potency was not applicable. N/D: Not done.
di#erent from the mice immunized with DTaP-IPV alone. Effect of Hib-conjugated vaccines on the tetanus lethal potency assay and diphtheria Vero potency assay in the mouse model
The results of the potency assays are shown in Table 5. It is clear that the TT component of Act-Hib was as active as the TT component of the DTaP-IPV vaccine. Thus, combination of DTaP-IPV+Act-Hib, injected in separate sites, augmented the potency of combination more than five-fold. Since the responses to the mixture of DTaP-IPV+Act-Hib in the same syringe had approached the plateau phase of the dose response, it was impossible to calculate the potency of this mixture. The use of 1/2DTaP-IPV +1/2Act-Hib, either injected in separate sites or mixed in the same syringe, adjusted the potency of the combinations to 70·2% and 104%, respectively, which were not significantly di#erent from DTaPIPV alone. Amvax Hib-TT alone did not show any TT potency on its own and a mixture of DTaPIPV+Amvax Hib-TT did not significantly a#ect the potency of TT. Combination of DTaP-IPV +HibTITER did not display any significant e#ect on potency of TT. In the combination groups, the
groups which had been immunized with mixture of DTaP-IPV+Hib-conjugated vaccines in the same syringe did not show di#erent TT potency from the groups which had been immunized with DTaPIPV+Hib-conjugated vaccines injected in the separate sites. Combination of DTaP-IPV+Act-Hib diminished the relative potency of the DT component of DTaPIPV. Mixture of DTaP-IPV+Amvax Hib-TT in the same syringe did not have any notable e#ect on potency of the DT component of DTaP-IPV vaccine. Hib-TITER did not show any DT potency on its own, but combination of Hib-TITER with DTaP-IPV elevated significantly the immunogenic potential of the DT component of DTaP-IPV vaccine. Immunization with combination of 1/2DTaP-IPV+1/2HibTITER resulted in modulation of DT potency. Di#erences of DT potency between two combination groups, injected in separate sites and mixed in the same syringe were minor. Effect of Hib-conjugated vaccines on immunogenicity of PT in the mouse model
The e#ect of Hib-conjugated vaccines on immunogenicity of the pertussis toxoid, the main component of the aP vaccine, was studied. Anti-PT antibody
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responses were una#ected when DTaP-IPV was administered with all three tested Hib-conjugated vaccines (data not shown). Immunogenicity of Hib-conjugated vaccines alone and in combination with DTaP-IPV in the mouse model
Figure 1 shows anti-HibCP antibodies in pooled sera of the mice immunized with either Hib vaccines or combination of Hib vaccines with DTaP-IPV. Non-dose dependent response to HibCP was shown in all immunized groups. Act-Hib alone produced pronounced anti-HibCP antibodies, while combination of Act-Hib with DTaP-IPV in general decreased anti-HibCP titre, and mixture of Act-Hib with DTaP-IPV in a same syringe showed obviously lower titre of anti-HibCP antibodies than Act-Hib alone. In the mice which had been immunized with mixture of Hib-TITER with DTaP-IPV in the same syringe, the levels of antibody were considerably higher than in both the Hib-TITER alone and separate sites combined group. The levels of anti-HibCP antibodies in the mice immunized with mixture of Amvax Hib-TT with DTaP-IPV in the same syringe were markedly higher than the mice which had been immunized with Amvax Hib-TT alone. The overall view was that Act-Hib alone produced higher anti-HibCP antibodies than combination of Act-Hib+DTaPIPV, while mixture of either Hib-TITER or Amvax Hib-TT with DTaP-IPV in the same syringe had a positive additive e#ect on antibody responses to HibCP. Effect of Hib-conjugated vaccines on immunogenicity of TT, DT, aP, and IPV in the guinea pig model
Act-Hib and Amvax Hib-TT alone showed pronounced anti-TT antibodies in the guinea pig model (Table 6). But only in the short interval protocol, combination of either Act-Hib or Amvax Hib-TT with DTaP-IPV increased significantly the anti-TT antibody responses to the TT component of DTaPIPV. This feature was shown both after first and second immunization. None of Hib-conjugated vaccines showed remarkable e#ect on immunogenicity of DT (Table 6), aP, and IPV components of the DTaP-IPV vaccine in the guinea pig model (results for aP and IPV are not shown). Immunogenicity of Hib-conjugated vaccines alone and in combination with the DTaP-IPV in the guinea pig model
Table 7 shows the capability of the guinea pig model to produce anti-HibCP antibodies using two
di#erent protocols. In the ‘‘short interval’’ protocol, four weeks after first immunization, only one animal showed detectable anti-HibCP antibodies, and the rest of animals responded after second injection. Boosting e#ect was seen in all immunized groups. There was no considerable di#erence between the groups of guinea pigs which had been immunized with either Hib-conjugated vaccines alone or combination of Hib-conjugated vaccines with DTaP-IPV. In the ‘‘long interval’’ protocol, five of the immunized animals died under anaesthesia during the first bleeding, one in the Act-Hib+DTaP-IPV (MS) group, three in the Amvax Hib-TT group, and one in the Amvax Hib-TT+DTaP-IPV (SS) group. It can be seen that only two animals, one in the Act-Hib+DTaP-IPV(MS) group and one in the HibTITER+DTaP-IPV (SS) group, had detectable antiHibCP antibodies after first immunization. Two weeks after boosting, anti-HibCP antibodies were detected in most of the groups, and the maximum responses were reached six weeks after second injection. In the ‘‘long interval’’ protocol, a boosting e#ect was shown in all groups (except Amvax Hib-TT alone) and the number of responders in a few groups increased over the time from two weeks post second injection to six weeks post second injection. Discussion The magnitude of the antibody responses of immunized mice to the TT component of di#erent combination groups was variable. It was demonstrated that in the mouse model the immunogenic potential of the TT component of Act-Hib in the multiple shot program was as great as that of the TT component of DTaP-IPV vaccine. Therefore, combination of DTaP-IPV+Act-Hib, either injected in separate sites or mixed in the same syringe, induced, as expected, higher anti-TT antibodies than DTaP-IPV alone. Similar results were obtained in the tetanus lethal potency assay for Act-Hib alone and for combination of DTaP-IPV+Act-Hib. However, the TT component of Amvax Hib-TT did not show any immunogenic potential on its own, either in the multiple shot program nor in the lethal potency assay. Also in the lethal potency assay, combination of Amvax Hib-TT with DTaP-IPV did not increase the potency of the TT component of DTaP-IPV, attributable to occupancy of the tetanus toxoid B-cell epitopes by small size HibCP.15 However, in the multiple-shot programme, when DTaP-IPV was
0
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DTaP-IPV & Hib vaccines in animal models 235
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Figure 1. Anti HibCP antibodies in the pool sera of the mice immunized with di#erent preparations. Results are shown as titre in comparison with negative control serum. ( ), 1/20 HD; ( ), 1/40 HI ( ), 1/80 HD.
SS: separate sites. MS: mixed in the same syringe. IU: International Unit. * : antibodies of the pooled sera. N/D: not done.
DTaP-IPV Act-Hib Act-Hib+DTaP-IPV(SS) Act-Hib+DTaP-IPV(MS) Amvax Hib-TT Amvax Hib-TT+DTaP-IPV(SS) Amvax Hib-TT+DTaP-IPV(MS) Hib-TITER Hib-TITER+DTaP-IPV(SS) Hib-TITER+DTaP-IPV(MS)
2·67(1·82–3·92) 1·45(0·60–3·52) 9·71(7·73–12·2) 12·2(9·98–14·8) 2·19(1·22–3·94) 4·54(3·25–6·34) 5·99(4·35–8·27) N/D N/D N/D
Anti-TT 0·07(0·04–0·12) N/D 0·09(0·04–0·21) 0·05(0·04–0·07) N/D 0·06(0·04–0·12) 0·64(0·02–0·06) N/D N/D N/D
Anti-DT
4 wks post 1st immunization
15·6(11·5–21·1) 25·6(13·0–15·3) 44·1(30·7–63·4) 38·7(26·3–56·9) 23·7(13·1–43·0) 52·0(38·7–70·0) 33·8(19·3–59·2) N/D N/D N/D
Anti-TT 0·69(0·36–1·3) N/D 1·4(0·57–1·9) 0·72(0·50–1·0) N/D 1·4(0·21–2·3) 0·56(0·41–0·78) N/D N/D N/D
Anti-DT
2 wks post 2nd immunization
Short interval protocol
5·7(3·5–3·7) 1·7(0·43–7·1) 7·5(7·2–7·7) 6·1(5·1–7·3) 0·46(0·02–8·9) 5·3(4·5–6·2) 5·7(3·9–8·3) N/D 7·4(6·9–7·9) 3·5(2·8–4·4)
Anti-TT 1·51(0·09–2·58) N/D 1·06(0·47–2·38) 1·43(0·57–3·60) N/D 1·64(0·31–3·31) 1·45(0·73–2·69) 0·02(·002–0·26) 3·01(1·67–5·44) 2·04(1·26–3·29)
Anti-DT
6 wks post 1st immunization
12·49 14·48 15·47 7·14 14·62 21·82 14·49 N/D 8·18 10·63
Anti-TT*
11·9(6·44–22·0) N/D 6·19(2·27–16·8) 3·70(1·59–2·61) N/D 7·21(4·32–12·0) 5·54(4·36–7·17) 0·06(0·03–0·11) 4·24(2·65–6·73) 5·171*
Anti-DT
2 wks post 2nd immunization
Long interval protocol
Table 6. Geometric mean of anti-TT and anti-DT antibodies IU (95% confidence limits) in the guinea pig model
1·65 5·68 6·31 4·30 4·36 8·74 1·63 N/D 1·53 2·90
Anti-TT*
0·62(0·36–1·07) N/D 0·39(0·14–1·11) 0·99(0·20–1·79) N/D 0·77(0·31–1·91) 0·47(0·20–1·09) 0·01(0·01–0·20) 1·91(0·72–5·10) 0·16(0·10–0·28)
Anti-DT
6 wks post 2
236 H. Shams and I. Heron
DTaP-IPV & Hib vaccines in animal models
237
Table 7. Immunogenicity of HibCP-conjugated vaccines in the guinea pig model. Results are presented as responder(s)/total number of animals in each group(range of g HibCP to inhibit 50% OD value) Short interval Vaccines Act-Hib Act-Hib+DTaP-IPV(SS) Act-Hib+DTaP-IPV(MS) Amvax Hib-TT Amvax Hib-TT+DTaP-IPV(SS) Amvax Hib-TT+DTaP-IPV(MS) Hib-TITER Hib-TITER+DTaP-IPV(SS) Hib-TITER+DTaP-IPV(MS)
Long interval
4 wks post 1st immun.
2 wks post 2nd immun.
6 wks post 1st immun.
2 wks post 2nd immun.
6 wks post 2nd immun.
0/6 0/6 1/6(0·19) 0/6 0/6 0/6 N/D N/D N/D
3/6(0·53–0·62) 3/6(0·25–0·81) 2/6(0·06–0·21) 2/6(0·62–2·36) 3/6(0·14–1·17) 3/6(0·04–2·08) N/D N/D N/D
0/6 0/6 1/6(0·87) 0/3 0/5 0/6 0/6 1/6(0·56) 0/6
3/6(0·08–0·59) 4/6(0·05–0·90) 3/5(0·05–0·19) 0/3 1/5(0·03) 0/6 1/6(0·23) 2/6(0·02–0·39) 2/6(0·02–0·19)
4/6(0·02–0·86) 4/6(0·06–1·00) 3/5(0·21–2·57) 0/3 1/5(0·59) 3/6(0·19–1·35) 3/6(0·17–0·37) 2/6(0·02–0·59) 3/6(0·08–1·23)
SS: separate sites. MS: mixed in the same syringe. N/D: Not done.
combined with Amvax Hib-TT, an increase in antibody responses after boosting was observed. This might be related to intact T-cell epitopes of the TT component of Amvax Hib-TT.15 Furthermore, combination of Hib-TITER with DTaP-IPV, either injected in separate sites or mixed in the same syringe produced no significant change in the kinetics of serum anti-TT antibody levels as well as in the potency of TT, demonstrating no change in the total and in the neutralizing antibody responses to TT. In the guinea pig model, the immunogenicity of the TT component of Act-Hib and Amvax Hib-TT was confirmed. In the ‘‘short interval’’ protocol, primary and secondary responses to combination of either Act-Hib or Amvax Hib-TT with DTaP-IPV were significantly higher than DTaP-IPV alone. This verifies the e#ect of the TT component of Act-Hib and Amvax Hib-TT on immunogenicity of the TT component of DTaP-IPV in the guinea pig model. However, contrary to our results in the mouse model, Amvax Hib-TT induced pronounced anti-TT antibody response in the guinea pigs after the first immunization. This may be attributed to the high vaccine dose used in guinea pigs (1/2 HD) and uncharacterized animal species e#ects.12 Taken together, these experiments show that in the two animal species used the HibCP component of Hib-conjugated vaccines did not have any deleterious e#ect on potency and immunogenicity of TT component of DTaP-IPV on its own, and the additive e#ect of Hib-conjugated vaccines on potency and/or immunogenicity of TT component of
DTaP-IPV was mainly due to existence of TT as the carrier in the Hib-conjugated vaccines. Combination of Act-Hib with DTaP-IPV in the mouse model using the single shot program reduced the potency of the DT component of DTaP-IPV vaccine to 39–58%, which however, was not statistically significant because of the wide 95% confidence limits, demonstrating that changes were within the experimental variability of the assay; and in the multiple shot programme, combination of Act-Hib with DTaP-IPV did not considerably a#ect the serum antibody responses to the DT component of DTaP-IPV vaccine. In the mouse model using the single shot programme, mixing of Amvax Hib-TT with DTaP-IPV in the same syringe did not influence the potency of the DT component of DTaP-IPV. However, in the multiple shot programme, serum antibody responses to DT after third injection with 1/20HD of mixture of DTaP-IPV+Amvax Hib-TT were lower than DTaP-IPV alone, but at lower doses (1/40HD and 1/80HD) di#erences were negligible. In the mouse model, combining of DTaP-IPV with Hib-TITER, either injected in separate sites or mixed in the same syringe, increased significantly the potency of DT. This positive e#ect of Hib-TITER on the potency of DT component of DTaP-IPV might be partially due to e#ect of adjuvant [Al(OH)3] of DTaP-IPV on intact B-cell epitopes of CRM197 component of Hib-TITER.16 However, in the multiple shot programme, serum antibody responses to DT in the combination of DTaP-IPV with Hib-TITER were not significantly di#erent from DTaP-IPV alone.
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Remarkable di#erences were shown in anti-DT antibody levels between two protocols in the guinea pig model. Anti-DT antibody levels in the animals immunized in ‘‘long interval’’ protocol were much higher than those immunized in the ‘‘short interval’’ protocol. This indicates the importance of interval on anti-DT antibody responses in the guinea pig model and notifies that in quality control tests of DT that are performed in the guinea pig model, the defined interval between immunization and bleeding must be regarded carefully. The use of the guinea pig model to study the e#ect of Hib-conjugated vaccines on immunogenicity of DT revealed that in the ‘‘short interval’’ protocol, combination of either Act-Hib or Amvax Hib-TT with DTaP-IPV had no significant influence on the immunogenicity of DT component of DTaP-IPV. This di#ers from the findings of Siber et al.2 that shows that combination of Hib-TT vaccines with DTP has been resulted in significant decrease in anti-DT antibody responses in the guinea pig model, probably attributable to di#erent doses used. Our results and those of others2–4 show inconsistency regarding the e#ect of HibCP on immunogenicity and potency of DT, and imply that more studies should be carried out. In the current studies, pooled sera from mice and guinea pigs immunized with either DTaP-IPV alone or a combination of DTaP-IPV with Hib-conjugated vaccines were tested for antibody responses to pertussis toxoid (PT), the main component of the acellular pertussis (aP) vaccine. In general, we showed that Hib-conjugated vaccines did not reduce antibody responses to the PT in either used animal models. Our current work and that of others3,4 also indicate that Hib-conjugated vaccines have no negative e#ect on the kinetics of antibody responses to PT, or on potency of whole cell pertussis vaccine, respectively. However, in a human clinical trial, co-administration of Act-Hib with DTP suppressed the level of anti-PT antibodies,17 demonstrating that the results in animal model(s) can not predict the consequence of combining vaccine components for human immune response. Combination with DTaP-IPV showed various e#ects on immunogenicity of Hib-conjugated vaccines in the mouse model. A decrease in anti-HibCP antibody levels was observed when Act-Hib, in which TT was as active as non-conjugated TT, was combined with DTaP-IPV vaccine. Mixture of Amvax Hib-TT with DTaP-IPV in a same syringe showed higher levels of anti-HibCP antibodies than Amvax Hib-TT alone. Likewise, combination
of Hib-TITER with DTaP-IPV produced higher serum antibodies to HibCP than Hib-TITER alone. Moreover, the ine#iciency of the TT component of Amvax Hib-TT, and of the CRM197 component of Hib-TITER in induction of anti-TT and anti-DT antibodies, respectively, has already been demonstrated.15 In summary, our results in the mouse model and those of others in chicken and human18,19 demonstrate that potentiation of the antibody responses to carrier, either due to large dose of carrier (existence of active B cell epitopes on the carrier and combination with non-conjugated carrier) or existence of anti-carrier antibodies prior to administration of Hib-conjugated vaccines will result in suppression of serum antibody responses to HibCP. These observations are in agreement with carrier specific B- and T-cell clonal dominance hypothesis20–22 which suggest that inactivation of carrier B-cell epitopes and consequently prevention of clonal dominance of carrier-specific B cells leads to prevention of epitopic suppression.22 It is reasonable that during secondary immune response, memory B cells interact with the carrier epitopes more e#iciently than other non-immune antigen presenting cells.20 Our results in the mouse system seem in accordance with this and confirm that removing the carrier B-cell-epitopes elevates the likelihood for HibCP epitopes of Hib-conjugated vaccines to interact with HibCP-specific B cells, and thereby, presentation of carrier T cell epitopes by HibCP-specific B cells will result in anamnestic anti-HibCP antibody responses. Also, the same specific B cell precursors are activated either by a polysaccharide in a thymus-independent way or by conjugated-polysaccharide in a thymus-dependent way,23 and only carrier-specific T cells modify the result of immune responses. Thus, conjugation to a protein does not increase the chance of polysaccharides for primary interactions with B cells. The latter is a possible explanation for non-dose dependency for anti-HibCP antibody responses in our experiments and those of others.2 Vogel et al. have reported a modulatory e#ect of whole-cell Bordetella pertussis on carrier-induced epitopic suppression.24 So, the discrepancy between our results and those of Redhead et al.,3,4 who reported the increase in anti-HibCP antibodies in simultaneous injection of Act-Hib and DTP vaccines in a mouse model might be attributable to whole cell pertussis component of DTP vaccine. Lyng and Heron12 found that potency results depend on di#erent factors including animal
DTaP-IPV & Hib vaccines in animal models
species. In the current study, we found that concurrent immunization with Act-Hib and DTaP-IPV in the mouse model resulted in reduction of antiHibCP antibody responses, while co-administration of Act-Hib with DTaP-IPV did not have a remarkable e#ect on anti-HibCP responses in the guinea pig model. Selected dose in the guinea pig model and in the mouse model, on a weight to weight basis was almost the same, and the levels of anti-carrier antibodies (anti-TT) in the International Unit basis in the guinea pig model was much higher than in the mouse model. This is tempting to speculate that the repertoire of the murine immune system in response to the Hib-conjugated vaccines is relatively restricted, and therefore, induction of suppression with the Hib-conjugated vaccines in the mouse model is easier than in the guinea pig model. In the current study, anti-HibCP responses of the guinea pig model in two di#erent protocols (‘‘short interval’’ and ‘‘long interval’’ protocols) were compared. Immunization of guinea pigs with either Act-Hib alone or combination of Act-Hib with DTaP-IPV did not show any striking di#erence in anti-HibCP responses between the two protocols. In the Amvax Hib-TT groups (Amvax Hib-TT alone and combination of Amvax Hib-TT with DTaP-IPV), animals which had been immunized in the ‘‘short interval’’ protocol responded even better than the groups which had been immunized in ‘‘long interval’’ protocol. These demonstrate that the ‘‘long interval’’ protocol does not have any advantage over the ‘‘short interval’’ protocol for induction of anti-HibCP antibodies in the guinea pig model. Additional studies are required to clarify the factors that determine di#erential immune responses as a consequence of mutual interactions between components of combined vaccines in humans and laboratory animals. Acknowledgements
Authors wish to thank Henrick Aggerbeck, Gert Albert Hansen, Max Kristiansen, and Gitte Stawski for worthwhile arguments; Adam Gottschau for statistical analysis and interpretation of results; and Halina L. Pedersen and Vita Skov for their excellent technical assistance.
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polyribosylribitol phosphate (PRP) conjugated vaccines in the mouse model. APMIS 1998; 106: 526–534. Anderson P, Pichichero ME, Insel RA. Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197. J Clin Invest 1985; 26: 52–59. Clemens JD, Ferreccio C, Levine MM et al. Impact of Haemophilus influenzae type b polysaccharidetetanus protein conjugate vaccine on responses to concurrently administered diphtheria-tetanuspertussis vaccine. JAMA 1992; 267: 673–678. Barington T, Gyhrs A, Kristensen K et al. Opposite e#ect of actively and passively acquired immunity to the carrier on responses of human infants to a Haemophilus influenzae type b conjugate vaccine. Infect Immun 1994; 62: 9–14. Sarvas H, Ma¨ kela¨ O, Toivanen P et al. E#ect of carrier preimmunization on the anti-hapten response in the chicken. Scand J Immunol 1974; 3: 455–460.
20. Schutze MP, Deriaud E, Przewlock G et al. Carrierinduced epitopic suppression is initiated through clonal dominance. J Immunol 1989; 142: 2635–2640. 21. Schutze MP, Leclerc C, Lolivert M et al. Carrierinduced epitopic suppression, a major issue for future synthetic vaccines. J Immunol 1985; 135: 2319–2322. 22. Schutze M, Leclerc C, Vogel FR et al. Epitopic suppression in synthetic vaccine models: Analysis of the e#ector mechanisms. Cell Immunol 1987; 104: 79–90. 23. Willers J, Weiler E, Kolb C. Stimulation of the same B-cell population by thymus-independent dextran and by thymus-dependent oligosaccharide-carrier. Scand J Immunol 1995; 42: 345–352. 24. Vogel FR, Leclerc C, Schutze MP et al. ‘‘Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muranyl peptide’’. Cell Immunol 1987; 107: 40–51.
Received for publication 22 December 1998; accepted 3 June 1999