Immunogenic compositions

Abstract

The invention provides an immunogenic composition comprising one or more GBS conjugates and one or more antigens selected from: a) cellular or acellular pertussis antigen, b) a tetanus toxoid, c) a diphtheria toxoid and d) an inactivated polio virus antigen, wherein each GBS conjugate is a group B streptococcus capsular saccharide conjugated to a carrier protein. The invention also provides a method for raising an immune response in a patient, comprising the step of administering to the patient a composition of the invention.

Claims

1. An immunogenic composition comprising: (A) a bacterial saccharide conjugate component comprising: (i) a capsular saccharide from Group B streptococcus (GBS) serotype Ia conjugated to a carrier protein; (ii) a capsular saccharide from GBS serotype Ib conjugated to a carrier protein; (iii) a capsular saccharide from GBS serotype III conjugated to a carrier protein; and (B) a protein antigen component consisting of: (i) unconjugated tetanus toxoid (TT), (ii) unconjugated diphtheria toxoid (DT), and (iii) acellular pertussis antigens.

2. The immunogenic composition according to claim 1, wherein each GBS capsular saccharide is present at an amount from 0.1 to 30 g per dose.

3. The immunogenic composition according to claim 1, wherein the conjugate that is a capsular saccharide from GBS serotype Ia conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 1:1 to 1:2; the conjugate that is a capsular saccharide from GBS serotype Ib conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 1:1 to 1:2; and the conjugate that is a capsular saccharide from GBS serotype III conjugated to a carrier protein has a saccharide:protein ratio (w/w) between about 3:1 to 1:1.

4. The immunogenic composition according to claim 1, wherein the carrier protein is a diphtheria toxoid, a tetanus toxoid or CRM197.

5. The immunogenic composition according to claim 4, further comprising a conjugate selected from: a conjugate that is a capsular saccharide from GBS serotype II conjugated to a carrier protein; and a conjugate that is a capsular saccharide from GBS serotype V conjugated to a carrier protein.

6. The immunogenic composition according to claim 1, wherein the acellular pertussis antigens consist of detoxified pertussis toxin, filamentous hemagglutinin and pertactin.

7. The immunogenic composition according to claim 1, wherein the unconjugated diphtheria toxoid is present at a concentration of between 4 Lf/ml and 8 Lf/ml per 0.5 ml dose.

8. The immunogenic composition according to claim 1, wherein the unconjugated tetanus toxoid is present at a concentration of about 5 Lf per 0.5 ml dose.

9. The immunogenic composition according to claim 4, wherein the immunogenic composition contains an aluminium salt adjuvant.

10. The immunogenic composition according to claim 4, wherein the composition is a vaccine.

11. The immunogenic composition according to claim 4, wherein the carrier protein for each GBS capsular saccharide is the same.

12. A method for raising an immune response in a patient, comprising the step of administering to the patient a composition according to claim 1.

13. A process for preparing the immunogenic composition according to claim 1, comprising mixing said bacterial capsular saccharide antigen component with said protein antigen component.

14. The process according to claim 13, wherein the GBS conjugates in the bacterial capsular saccharide antigen component are lyophilised.

15. A kit for preparing the immunogenic composition according to claim 1, comprising said bacterial capsular saccharide antigen component and said protein antigen component; wherein the two components are in separate containers.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows IgG titers against (A) GBS Ia, (B) GBS Ib and (C) GBS III in the mouse groups described in Table 1. Sera were pooled for all mice in groups 5 and 6. GMT titers are indicated by the central bar. Upper and lower bars indicate 95% confidence intervals.

(2) FIG. 2 shows IgG titers against (A) Diptheria Toxoid, (B) Tetanus Toxoid, (C) Pertussis Toxoid, (D) Pertussis FHA and (E) Pertussis 69K in mouse groups described in Table 1. Statistical significance is indicated by * (p<0.05). GMT titers are indicated by the central bar. Upper and lower bars indicate 95% confidence intervals.

(3) FIG. 3 shows OPK titers against (A) GBS Ia, (B) GBS Ib and (C) GBS III in the mouse groups described in Table 1. Sera were pooled from all mice in each group (except for groups 5 and 6) for each experiment (experiment repeated twice). Sera from both experiments were pooled for each of groups 5 and 6. GMT titers are indicated by the central bar.

(4) FIG. 4 shows IgG titers against (A) GBS Ia, (B) GBS Ib and (C) GBS III in mouse groups described in Table 3. Statistical significance is indicated by * (p<0.05). GMT titers are indicated by the central bar. Upper and lower bars indicate 95% confidence intervals.

(5) FIG. 5 shows IgG titers against (A) Diptheria Toxoid, (B) Tetanus Toxoid, (C) Pertussis Toxoid, (D) Pertussis FHA and (E) Pertussis 69K in mouse groups described in Table 3. Statistical significance is indicated by ** (p<0.01) and * (p<0.05). GMT titers are indicated by the central bar. Upper and lower bars indicate 95% confidence intervals.

(6) FIG. 6 shows OPK titers against (A) GBS Ia, (B) GBS Ib and (C) GBS III in mouse groups described in Table 3. Sera were pooled from all mice in each group for each experiment. GMT titers are indicated by the central bar.

(7) FIG. 7 shows IgG titers against (A) GBS Ia, (B) GBS Ib and (C) GBS III in mouse groups described in Table 5. Sera were pooled for groups 5, 6 and 7. Statistical significance is indicated by ** (p<0.01). GMT titers are indicated by the central bar. Upper and lower bars indicate 95% confidence intervals.

(8) FIG. 8 shows IgG titers against (A) Tetanus Toxoid and (B) Diptheria Toxoid in mouse groups described in Table 5. Statistical significance is indicated by * (p<0.05). GMT titers are indicated by the central bar. Upper and lower bars indicate 95% confidence intervals.

(9) FIG. 9 shows OPK titers against (A) GBS Ia, (B) GBS Ib and (C) GBS III in mouse groups described in Table 5. Sera were pooled from all mice in each group for each experiment. GMT titers are indicated by the central bar.

MODES FOR CARRYING OUT THE INVENTION

(10) Materials and Methods

(11) Vaccines

(12) The GBS trivalent vaccine used in the following experiments is composed of capsular polysaccharides derived from three major serotypes: Ia, Ib and III, each conjugated to CRM197. The TdaP(H4) vaccine is adjuvanted with aluminium hydroxide and contains Tetanus toxoid, Diphtheria toxoid and Acellular Pertussis antigens (PT, FHA and 69K).

(13) The TdaP(H4)-IPV vaccine is adjuvanted with aluminium hydroxide and contains Tetanus toxoid, Diphteria toxoid, Acellular Pertussis antigens (PT, FHA and 69K) and Polio antigens (IPV1, IPV2 and IPV3).

(14) Commercially available Tetanus and Tetanus/Diphteria liquid vaccines were used.

(15) ELISA

(16) IgG titers against GBS polysaccharides Ia, Ib and III in the sera from immunized mice were measured by ELISA as explained in reference 159. Generally, there is good correlation between ELISA IgG Abs and OPK titers.

(17) Opsonophagocytic Killing (OPK) Assay

(18) Measuring the opsonizing activity of serum antibody is a useful indicator of vaccine activity since protection is likely afforded by opsonophagocytic killing. The opsonophagocytosis killing assay measures the ability of serum antibody to opsonize GBS for killing by effector cells in the presence of complement. OPK assays to measure the functional activity of antibodies elicited in immunized mice against GBS polysaccharides Ia, Ib and III were performed using HL-60, a pro-myelocytic leukemia cell line obtained from the American Type Culture Collection (ATCC, CCL-240). The strains GBS 515, GBS H36b and GBS COH1 were used to measure killing of serotype Ia, Ib and III isolates, respectively. Negative control reactions were performed either in the presence of heat inactivated complement or in the absence of antibody or effector cells, or using pre-immune or placebo sera. Reactions were plated in trypticase soy agar plate and bacterial counts were determined. The percentage of killing was calculated as (mean CFU at T0mean CFU at T60)/(mean CFU at T0). OPK titers were expressed as the reciprocal serum dilution leading to 50% killing of bacteria. The OPK assay was performed according to the killing-based opsonophagocytosis assay (kOPA) protocol described in reference 160.

(19) LUMINEX Based Multiplex Assay

(20) A LUMINEX-based multiplex assay for IgG quantification of tetanus, diphtheria and pertussis antigens was used. Diphteria Toxoid (DT), Tetanus Toxoid (TT), Pertussis Toxoid (PT), Pertussis FHA and Pertussis 69K antigens were each covalently conjugated to microspheres (Luminex Corporation, Austin, Tex.). Mouse serum samples were analyzed to assess antibody titers specific to each antigen. Experiments were performed in duplicate and values from duplicates were averaged. A reference serum was prepared by pooling sera from CD1 mice immunized with TdaP antigens+Alum.

(21) Study 1: GBS Reconstituted in TdaP and TdAP-IPV

(22) This study investigated the immunogenicity of lyophilized GBS trivalent vaccines (Ia, Ib, III polysaccharides conjugated to CRM197) reconstituted with: (i) Alum adjuvanted liquid vaccine containing Tetanus, Diphteria and Acellular Pertussis antigens (TdaP) and (ii) Alum adjuvanted liquid vaccine containing Tetanus, Diphteria, Acellular Pertussis and Polio antigens (TdaP-IPV).

(23) The immunization protocol is reported in Table 1 and was repeated two times. In each experiment, six groups of 8 CD1 female mice were immunized intraperitoneally on days 0 and 21, with 2 doses of the vaccines shown in Table 1, and bled on days 0 and 35.

(24) TABLE-US-00001 TABLE 1 Immunization protocol for study 1. Vaccine Volume Adjuvant Group N.sup.o Vaccine Type composition route Antigens Dose Dose N.sup.o mice 1 GBS + PSIa-CRM 5 g/ml i.p. 200 l PSIa-CRM 1 g Alum 8 TdaP(H4) PSIb-CRM 5 g/ml PSIb-CRM 1 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 1 g T 10 Lf/ml T 2 Lf D 8 Lf/ml D 1.6 Lf PT 8 g/ml PT 1.6 g FHA 8 g/ml FHA 1.6 g 69K 16 g/ml 69K 3.2 g Alum 2 g/ml 2 GBS + PSIa-CRM 5 g/ml i.p. 200 l PSIa-CRM 1 g Alum 8 TdaP(H4)-IPV PSIb-CRM 5 g/ml PSIb-CRM 1 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 1 g T 10 Lf/ml T 2 Lf D 8 Lf/ml D 1.6 Lf PT 8 g/ml PT 1.6 g FHA 8 g/ml FHA 1.6 g 69K 16 g/ml 69K 3.2 g IPV1 80 dU/ml IPV1 16 dU IPV2 16 dU/ml IPV2 3.2 dU IPV3 64 dU/ml IPV3 12.8 dU Alum 2 mg/ml 3 GBS only PSIa-CRM 5 g/ml i.p. 200 l PSIa-CRM 1 g Alum 8 PSIb-CRM 5 g/ml PSIb-CRM 1 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 1 g Alum 2 mg/ml 4 GBS only PSIa-CRM 5 g/ml i.p. 200 l PSIa-CRM 1 g Alum 8 PSIb-CRM 5 g/ml PSIb-CRM 1 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 1 g Alum 2 mg/ml 5 No antigen Alum 2 mg/ml i.p. 200 l Alum 8 400 g 6 TdaP(H4)-IPV T 10 Lf/ml i.p. 200 l T 2 Lf Alum 8 D 8 Lf/ml D 1.6 Lf 400 g PT 8 g/ml PT 1.6 g FHA 8 g/ml FHA 1.6 g 69K 16 g/ml 69K 3.2 g IPV1 80 dU/ml IPV1 16 dU IPV2 16 dU/ml IPV2 3.2 dU IPV3 64 dU/ml IPV3 12.8 dU Alum 2 mg/ml

(25) Sera from immunized mice were analyzed for the presence of IgG antibodies against each GBS serotype specific antigen (types Ia, Ib, and III) by ELISA. Antibodies against tetanus toxoid (TT), diphtheria toxoid (DT) and acellular pertussis antigens (PT, FHA and 69K) were quantified by LUMINEX assay. Functional activity of antibodies against GBS antigens was evaluated by opsonophagocytic killing (OPK) assay.

(26) FIGS. 1 and 2 show the IgG titers against the various antigens of the six groups of mice tested in study 1 (merged results from 8+8 mice from the two experiments). The GMT titers are indicated in Table 2 below.

(27) TABLE-US-00002 TABLE 2 GMT serum IgG titers after 2 immunizations in mice tested in study 1. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 GBS Ia 112 97 87 82 13 13 GBS Ib 561 97 229 185 13 13 GBS III 683 537 625 762 13 13 DT 187.55 48.94 N/A N/A <LLOQ 89.95 TT 560.67 268.97 N/A N/A <LLOQ 626.72 PT 280.43 121.10 N/A N/A 2.10 232.60 FHA 789.00 450.81 N/A N/A <LLOQ 749.95 69K 789.0 450.8 N/A N/A <LLOQ 88.6 <LLOQ = below lower limit of quantification

(28) Overall, all vaccine formulation provided immunogenicity that was comparable with the control, and no significant immunological interference was observed. For IgG titers post 2nd immunization against GBS Ia, Ib and III, no significant differences in Ig responses were detected by Mann-Whitney U test between the vaccine groups for any of the serotypes, indicating absence of interference. For IgG titers post 2nd immunization against DT, TT, PT, FHA and 69K, GBS+TDaP elicited about 2 fold higher GMT titers than GBS+TDaP+IPV (P<0.05 by Mann-Whitney U test), suggesting minor negative effects of IPV on all TDaP antigens. Additionally, for TT, the vaccine TDaP+IPV yielded 2 fold higher titers than TDaP+IPV+GBS (U test, P<0.026), suggesting minor negative effects of GBS on TT in the presence of IPV.

(29) FIG. 3 shows the OPK titers against GBS Ia, Ib and III of the six groups of animals tested in study 1. As shown, no major differences in OPK titers against GBS Ia, Ib or III (in the range of assay and biological variability) were detected for any of the vaccine formulations.

(30) Study 2: GBS Reconstituted in TdaP

(31) This study investigated the immunogenicity of different amounts of lyophilized GBS trivalent vaccines (Ia, Ib, III polysaccharides conjugated to CRM197) reconstituted with Alum adjuvanted liquid vaccine containing Tetanus, Diphteria and Acellular Pertussis antigens (TdaP).

(32) Eight groups of 16 CD1 female mice were immunized subcutaneously on days 0, 21 and 35, with 3 doses of vaccines, and bled on days 0, 35 (post-2) and 49 (post-3). The immunization protocol is reported in Table 3.

(33) TABLE-US-00003 TABLE 3 Immunization protocol for study 2. Group Vaccine Volume Adjuvant N.sup.o Vaccine Type composition route Antigens Dose Dose N.sup.o mice 1 GBS + PSIa-CRM 5 g/ml s.c. PSIa-CRM 1 g Alum 8 TdaP(H2) PSIb-CRM 5 g/ml 200 l PSIb-CRM 1 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 1 g T 10 Lf/ml T 2 Lf D 4 Lf/ml D 0.8 Lf PT 8 g/ml PT 1.6 g FHA 8 g/ml FHA 1.6 g 69K 16 g/ml 69K 3.2 g Alum 2 mg/ml 2 TdaP(H2) T 10 Lf/ml s.c. T 2 Lf Alum 8 D 4 Lf/ml 200 l D 0.8 Lf 400 g PT 8 g/ml PT 1.6 g FHA 8 g/ml FHA 1.6 g 69K 16 g/ml 69K 3.2 g Alum 2 mg/ml 3 GBS + PSIb-CRM 0.25 g s.c. PSIII-CRM 0.25 g Alum 8 TdaP(L2) PSIII-CRM 5 g/ml 200 l T 2 Lf 400 g T 10 Lf/ml D 0.8 Lf D 4 Lf/ml PT 0.4 g PT 2 g/ml FHA 0.4 g FHA 2 g/ml 69K 0.8 g 69K 4 g/ml Alum 2 mg/ml 4 TdaP(L2) T 10 Lf/ml s.c. T 2 Lf Alum 8 D 4 Lf/ml 200 l D 0.8 Lf 400 g PT 2 g/ml PT 0.4 g FHA 2 g/ml FHA 0.4 g 69K 4 g/ml 69K 0.8 g Alum 2 mg/ml 5 GBS (H) PSIa-CRM 5 g/ml s.c. PSIa-CRM 1 g Alum 8 PSIb-CRM 5 g/ml 200 l PSIb-CRM 1 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 1 g Alum 2 mg/ml 6 GBS (L) PSIa-CRM 5 g/ml s.c. PSIa-CRM 0.25 g Alum 8 PSIb-CRM 5 g/ml 200 l PSIb-CRM 0.25 g 400 g PSIII-CRM 5 g/ml PSIII-CRM 0.25 g 7 No antigen Alum 2 mg/ml s.c. Alum 8 (1) 200 l 400 g 8 No antigen Alum 2 mg/ml s.c. Alum 8 (2) 200 l 400 g

(34) FIGS. 4 and 5 show the IgG titers against the various antigens for all groups after 3 immunizations for the groups of mice tested in study 2. The GMT titers are indicated in Table 4 below.

(35) TABLE-US-00004 TABLE 4 GMT serum IgG titers after 3 immunizations in mice tested in study 2. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 GBS Ia 155.5 12.5 237.0 N/A 598.6 418.3 20.4 12.5 GBS Ib 107.5 15.1 90.6 N/A 250.4 271.7 15.7 12.5 GBS III 588.2 12.5 677.7 N/A 958.9 946.4 23.1 12.5 DT 141.3 164.3 164.5 228.2 N/A N/A N/A 0.1 TT 333.6 382.1 457.2 509.6 N/A N/A N/A 0.1 PT 191.4 187.1 149.9 128.1 N/A N/A N/A 0.2 FHA 423.8 544.6 476.1 314.9 N/A N/A N/A 0.1 69K 374.7 435.4 328.2 337.9 N/A N/A N/A 0.1

(36) For IgG titers post 3rd immunization against GBS Ia, Ib and III, the first observation is that two mice from group 7 probably received vaccine by mistake (high IgG titers to all three antigens, never observed previously in similar placebo groups). Secondly, a trend of lower IgG titers to all three GBS antigens in the groups reconstituted with TdaP (2-4 fold GMT, P<0.05 in some of the cases) compared to GBS vaccines without TdaP was observed. The same trend was observed for post-2 IgG titers to all serotypes.

(37) For IgG titers post 3rd immunization against DT, TT, PT, FHA and 69K, respectively, no significant differences in the IgG responses between any of the groups were detected by Mann-Whitney test, indicating the complete absence of interference. No dose response was observed for DT and TT, while slightly higher titers were observed for higher doses compared to lower doses of pertussis PT, FHA and 69K antigens.

(38) When the IgG titers against GBS Ia, Ib and III in groups 5 and 6, where responses to GBS 1 g (high, L) and 0.25 g (low, L) vaccine doses were compared, the variability in individual responses to GBS Ia and Ib was higher than that to GBS III. No significant differences between low and high doses were observed for any of the serotypes, while a lower number of non-responders and significantly higher responses at post-3 compared to post-2 were detected for all serotypes (Mann-Whitney U test).

(39) FIG. 6 shows the OPK titers against GBS Ia, Ib and III of the seven groups of mice tested in study 2. There is a trend of slightly lower titers in GBS vaccines formulated with TdaP compared to GBS alone.

(40) Study 3: GBS Reconstituted in Tetanus and Tetanus/Diphteria Liquid Vaccines

(41) This study investigated the immunogenicity of lyophilized GBS trivalent vaccines (Ia, Ib, III polysaccharides conjugated to CRM197) reconstituted with Alum adjuvanted liquid vaccines containing (i) Tetanus antigen or (ii) Tetanus and Diphtheria antigens.

(42) The immunization protocol is reported in Table 5 and was repeated two times.

(43) In each protocol, seven groups of 8 CD1 female mice were immunized subcutaneously on days 0 and 21 with 2 doses of the vaccines shown in Table 5, and bled on days 0 and 35.

(44) TABLE-US-00005 TABLE 5 Immunization protocol for study 3. Vaccine Vaccine Volume Adjuvant Group N.sup.o Type composition route Antigens Dose Dose N.sup.o mice 1 GBS + T PSIa-CRM 5 g/ml s.c. PSIa-CRM 1 g Alum 8 PSIb-CRM 5 g/ml 200 l PSIb-CRM 1 g 600 g PSIII-CRM 5 g/ml PSIII-CRM 1 g T 40 Ul/ml T 8 Ul Alum 3 mg/ml 2 GBS + TD PSIa-CRM 5 g/ml s.c. PSIa-CRM 1 g Alum 8 PSIb-CRM 5 g/ml 200 l PSIb-CRM 1 g 600 g PSIII-CRM 5 g/ml PSIII-CRM 1 g T 40 Ul/ml T 8 Ul D 4 g/ml D 0.8 Ul Alum 3 mg/ml 3 GBS only PSIa-CRM 5 g/ml s.c. PSIa-CRM 1 g Alum 8 (1) PSIb-CRM 5 g/ml 200 l PSIb-CRM 1 g 600 g PSIII-CRM 5 g/ml PSIII-CRM 1 g Alum 3 mg/ml 4 GBS only PSIa-CRM 5 g/ml s.c. PSIa-CRM 1 g Alum 8 (2) PSIb-CRM 5 g/ml 200 l PSIb-CRM 1 g 600 g PSIII-CRM 5 g/ml PSIII-CRM 1 g Alum 3 mg/ml 5 No Alum 3 mg/ml s.c. Alum 8 antigen 200 l 600 g 6 T only T 40 Ul/ml s.c. T 8 Ul Alum 8 Alum 3 mg/ml 200 l 600 g 7 TD only T 40 Ul/ml s.c. T 8 Ul Alum 8 D 4 Ul/ml 200 l D 0.8 Ul 600 g Alum 3 mg/ml

(45) FIGS. 7 and 8 show the IgG titers against the various antigens for all groups of mice tested in study 3 (merged results from 8+8 mice from the two experiments). The GMT titers are indicated in Table 6 below.

(46) TABLE-US-00006 TABLE 6 GMT serum IgG titers after 2 immunizations in mice tested in study 3. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 GBS 65 162 458 235 13 13 13 Ia GBS 217 402 550 417 13 13 13 Ib GBS 854 1018 1186 924 13 13 13 III TT 742.00 882.70 N/A N/A 6.60 1295.00 1038.00 DT 7.49 111.6 N/A N/A <LLOQ <LLOQ 28.52 <LLOQ = below lower limit of quantification

(47) For the IgG titers against GBS Ia, Ib and III measured by ELISA, the Mann-Whitney test did not reveal any significant difference between vaccine groups, except for serotype Ia, where the vaccine constituted by GBS alone (group 3) yielded significantly higher titers than the one containing GBS plus Tetanus Toxoid. In the OPK assay, no major differences in OPK titers against GBS Ia, Ib or III (in the range of assay and biological variability) were detected for any of the vaccine formulations.

(48) IgG titers against TT were significantly lower (P=0.03) in the group containing the GBS vaccine and TT compared to TT alone, although the difference in GMT titers was less than 2 fold. Concerning IgG responses to DT, they were higher in groups containing the GBS vaccine, as expected by the effect of CRM197 (detoxified DT).

(49) FIG. 9 shows the OPK titers against GBS Ia, Ib and III in sera from all groups of mice tested in study 3. As shown, no major differences in OPK titers against GBS Ia, Ib or III (in the range of assay and biological variability) were detected for any of the vaccine formulations.

CONCLUSIONS

(50) The following observations were made regarding the immunogenicity of the investigated vaccine formulations: GBS trivalent, Diphtheria, Tetanus and Pertussis vaccines elicited specific antibody titers to the corresponding antigens, in mice immunized with all of the investigated formulations. Immunological interference between GBS and Tetanus/Diphteria/Pertussis/Polio vaccine combinations was investigated in studies 1 (intraperitoneal immunization with 2 vaccine doses) and 2 (subcutaneous immunization with 2 and 3 vaccine doses). No interference was observed in study 1, where IgG and functional antibody responses to GBS, Diphtheria, Tetanus and Pertussis antigens where comparable irrespective of their use, alone or in combination. In study 2, we observed a trend of lower IgG titers to all three GBS antigens reconstituted in TdaP compared to GBS alone, even though the differences in GMT titers were never higher than 4 fold. Immunological interference between GBS and Tetanus or Tetanus/Diphteria vaccines was investigated in study 3. The Mann-Whitney test did not reveal significant differences in GBS responses between any of the vaccine groups except for serotype Ia, where the vaccine constituted by GBS alone yielded significantly higher titers than the one containing GBS plus Tetanus Toxoid. The same study revealed a slight interference of the GBS vaccine towards TT (2 fold GMT difference, P=0.03 for the difference in the IgG titers against TT, in the group containing the GBS vaccine compared to TT alone). Concerning IgG responses to DT, they were higher in groups containing the GBS vaccine, as expected by the effect of CRM197 (detoxified DT).

(51) In conclusion, there was no evidence of strong interference between any of the investigated vaccines.

(52) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

(53) [1] Vaccines. (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0. [2] Paoletti et al. (1990) J Biol Chem 265:18278-83. [3] Wessels et al. (1990) J Clin Invest 86:1428-33. [4] Paoletti et al. (1992) Infect Immun 60:4009-14. [5] Paoletti et al. (1992) J Clin Invest 89:203-9. [6] Wessels et al. (1987) Proc Natl Acad Sci USA 84:9170-4. [7] Wang et al. (2003) Vaccine 21:1112-7. [8] Wessels et al. (1993) Infect Immun 61:4760-6 [9] Wessels et al. (1995) J Infect Dis 171:879-84. [10] Baker et al. (2004) J Infect Dis 189:1103-12. [11] Paoletti & Kasper (2003) Expert Opin Biol Ther 3:975-84. [12] WO2012/035519 [13] WO2006/050341 [14] Guttormsen et al. (2008) Proc Natl Acad Sci USA. 105 (15):5903-8. Epub 2008 Mar. 31. [15] WO96/40795 [16] Michon et al. (2006) Clin Vaccine Immunol. 2006 August; 13 (8):936-43. [17] U.S. Pat. Nos. 6,027,733 & 6,274,144. [18] www.polymer.de [19] Lewis et al. (2004) PNAS USA 101:11123-8. [20] Wessels et al. (1989) Infect Immun 57:1089-94. [21] WO2006/082527. [22] U.S. patent application 61/008,941, entitled FERMENTATION PROCESSES FOR CULTIVATING STREPTOCOCCI AND PURIFICATION PROCESSES FOR OBTAINING CPS THEREFROM filed on 20 Dec. 2007 and international patent application WO 2009/081276. [23] Ramsay et al. (2001) Lancet 357 (9251):195-196. [24] Lindberg (1999) Vaccine 17 Suppl 2:S28-36. [25] Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-68. [26] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-33, vii. [27] Goldblatt (1998) J Med. Microbiol. 47:563-7. [28] European patent 0477508. [29] U.S. Pat. No. 5,306,492. [30] WO98/42721. [31] Dick et al. in Conjugate Vaccines (eds. Cruse et al.) Karger, Basel, 1989, 10:48-114. [32] Hermanson Bioconjugate Techniques, Academic Press, San Diego (1996) ISBN: 0123423368. [33] U.S. Pat. No. 4,356,170. [34] WO2006/082530. [35] WO2005/000346 [36] Anonymous (January 2002) Research Disclosure, 453077. [37] Anderson (1983) Infect Immun 39 (1):233-238. [38] Anderson et al. (1985) J Clin Invest 76 (1):52-59. [39] EP-A-0372501. [40] EP-A-0378881. [41] EP-A-0427347. [42] WO93/17712 [43] WO94/03208. [44] WO98/58668. [45] EP-A-0471177. [46] WO91/01146 [47] Falugi et al. (2001) Eur J Immunol 31:3816-24. [48] Baraldo et al. (2004) Infect Immun 72:4884-87. [49] EP-A-0594610. [50] WO00/56360. [51] WO02/091998. [52] Kuo et al. (1995) Infect Immun 63:2706-13. [53] WO01/72337 [54] WO00/61761. [55] WO00/33882 [56] WO2004/041157. [57] WO99/42130. [58] WO2004/011027. [59] WO96/40242. [60] Lei et al. (2000) Dev Biol (Basel) 103:259-264. [61] WO00/38711; U.S. Pat. No. 6,146,902. [62] International patent application PCT/IB2008/02690, CONJUGATE PURIFICATION, claiming priority from GB-0713880.3 (NOVARTIS AG), published as WO 2009/010877. [63] Vaccines. (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0. [64] National Institute for Biological Standards and Control; Potters Bar, UK. www.nibsc.ac.uk [65] Sesardic et al. (2001) Biologicals 29:107-22. [66] NIBSC code: 98/560. [67] Module 1 of WHO's The immunological basis for immunization series (Galazka). [68] NIBSC code: 69/017. [69] NIBSC code: DIFT. [70] National Institute for Biological Standards and Control; Potters Bar, UK. www.nibsc.ac.uk [71] Sesardic et al. (2002) Biologicals 30:49-68. [72] NIBSC code: 98/552. [73] Module 1 of WHO's The immunological basis for immunization series (Galazka). [74] NIBSC code: TEFT. [75] NIBSC code: 66/303. [76] Rappuoli et al. (1991) TIBTECH 9:232-238. [77] Module 6 of WHO's The immunological basis for immunization series (Robertson) [78] WO2008/028956 [79] WO2008/028957 [80] Liao et al. (2012) J Infect Dis. 205:237-43 [81] Paoletti et al. (2001) Vaccine 19:2118-2126. [82] WO00/56365. [83] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [84] WO01/41800. [85] Paoletti (2001) Vaccine 19 (15-16):2118-26. [86] WO03/009869. [87] Almeida & Alpar (1996) J Drug Targeting 3:455-467. [88] Agarwal & Mishra (1999) Indian J Exp Biol 37:6-16. [89] WO00/53221. [90] Jakobsen et al. (2002) Infect Immun 70:1443-1452. [91] Bergquist et al. (1998) APMIS 106:800-806. [92] Baudner et al. (2002) Infect Immun 70:4785-4790. [93] Ugozzoli et al. (2002) J Infect Dis 186:1358-1361. [94] Nony et al. (2001) Vaccine 27:3645-51. [95] U.S. Pat. No. 6,355,271. [96] WO00/23105. [97] Vaccine Design . . . (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum. [98] U.S. Pat. No. 5,057,540. [99] WO96/33739. [100] EP-A-0109942. [101] WO96/11711. [102] WO00/07621. [103] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271. [104] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32:321-338. [105] Niikura et al. (2002) Virology 293:273-280. [106] Lenz et al. (2001) J Immunol 166:5346-5355. [107] Pinto et al. (2003) J Infect Dis 188:327-338. [108] Gerber et al. (2001) Virol 75:4752-4760. [109] WO03/024480 [110] WO03/024481 [111] Gluck et al. (2002) Vaccine 20:B10-B16. [112] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400. [113] WO02/26757. [114] WO99/62923. [115] Krieg (2003) Nature Medicine 9:831-835. [116] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185. [117] WO98/40100. [118] U.S. Pat. No. 6,207,646. [119] U.S. Pat. No. 6,239,116. [120] U.S. Pat. No. 6,429,199. [121] Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658. [122] Blackwell et al. (2003) J Immunol 170:4061-4068. [123] Krieg (2002) Trends Immunol 23:64-65. [124] WO01/95935. [125] Kandimalla et al. (2003) BBRC 306:948-953. [126] Bhagat et al. (2003) BBRC 300:853-861. [127] WO03/035836. [128] WO95/17211. [129] WO98/42375. [130] Beignon et al. (2002) Infect Immun 70:3012-3019. [131] Pizza et al. (2001) Vaccine 19:2534-2541. [132] Pizza et al. (2000) Int J Med Microbiol 290:455-461. [133] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313. [134] Ryan et al. (1999) Infect Immun 67:6270-6280. [135] Partidos et al. (1999) Immunol Lett 67:209-216. [136] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293. [137] Pine et al. (2002) J Control Release 85:263-270. [138] Domenighini et al. (1995) Mol Microbiol 15:1165-1167. [139] WO99/40936. [140] WO99/44636. [141] Singh et al] (2001) J Cont Release 70:267-276. [142] WO99/27960. [143] U.S. Pat. No. 6,090,406 [144] U.S. Pat. No. 5,916,588 [145] EP-A-0626169. [146] WO99/52549. [147] WO01/21207. [148] WO01/21152. [149] Andrianov et al. (1998) Biomaterials 19:109-115. [150] Payne et al. (1998) Adv Drug Delivery Review 31:185-196. [151] Stanley (2002) Clin Exp Dermatol 27:571-577. [152] Jones (2003) Curr Opin Investig Drugs 4:214-218. [153] WO04/60308 [154] WO04/64759. [155] Hennings et al. (2001) J Infect Dis. 183 (7):1138-42. Epub 2001 Mar. 1. [156] Glezen & Alpers (1999) Clin. Infect. Dis. 28:219-224 [157] Madoff et al. (1994) J Clin Invest 94:286-92. [158] Paoletti et al. (1994) Infect Immun 62:3236-43. [159] GB patent application no. 1121301.4 [160] Fabbrini et al. (2012) J Immun Methods 378:11-19