NON-CROSS-LINKED ACELLULAR PERTUSSIS ANTIGENS FOR USE IN COMBINATION VACCINES

Abstract

The present invention relates to stable compositions comprising acellular pertussis antigens that have not been cross-linked with a cross-linking agent such as formaldehyde or glutaraldehyde and their use as acellular pertussis components in combination vaccines. Processes for preparing these antigens and compositions are also disclosed.

Claims

1. A process for preparing an aP component, comprising: a. growing a culture of a B. pertussis strain expressing a genetically detoxified pertussis toxin; b. purifying two or more B. pertussis antigens from the culture to obtain two or more batches each containing a different purified B. pertussis antigen; and c. mixing the two or more batches to prepare the aP component, wherein the process is characterised in that the purified B. pertussis antigens are not treated with a cross-linking agent, and wherein the aP component comprises at least two non-cross-linked B. pertussis antigens selected from PT, FHA and pertactin with the proviso that any PT is genetically detoxified.

2. The process of claim 1, wherein the genetically detoxified pertussis toxin is PT-9K/129G.

Description

DESCRIPTION OF THE DRAWINGS

[0277] FIGS. 1 to 3 show IgG titres in mice immunised with a Tdap formulation containing either non-formylated or formylated aP antigens, as described in Example 6 below. IgG tires were determined by Luminex assay. Results are expressed in relative light units (RLU)/ml. The lower limit of quantification (LLOQ) is shown as a dotted line. Titres are shown for pertactin (FIG. 1), FHA (FIG. 2), and PT (FIG. 3). The numbers 1/5 or 1/50 indicate the antigen dose, relative to a human dose. The numbers 14 and 42 indicate the day after first immunisation on which the serum were assessed. The symbol + indicates a formylated antigen, whereas a indicates a non-formylated antigen. A * indicates p=0.02.

MODES FOR CARRYING OUT THE INVENTION

Example 1: Stability of Formylated and Non-Formylated FHA Batches

[0278] Two FHA batches were prepared by purifying FHA from the supernatant of a B. pertussis culture. The supernatant was concentrated and diafiltrated. The filtered concentrate was added onto a hydroxyapatite column. The FHA-containing eluate was further purified by a series of chromatographic steps including a butyl 650M Sepharose column and a Q Sepharose FF column. The resulting purified FHA batches were concentrated and subjected to diafiltration. One FHA batch was additionally incubated in the presence of formaldehyde and lysine, the other batch was left untreated.

[0279] Samples of each batch were incubated at room temperature (RT) for up to one month or at 2-8 C. for up to three months. The initial protein concentration of each sample was determined by measuring the adsorption at 280 nm. The measurements were repeated after incubation for two weeks and one month (samples incubated at RT) or two weeks, one month and three months (samples incubated at 2-8 C.). Structural stability was confirmed by 3-8% gradient SDS-PAGE. Stability at RT and 2-8 C. was comparable for both the formaldehyde-treated batch and the untreated batch. No statistically significant reduction of the protein concentration was observed for both batches after one month at RT and one or three months at 2-8 C.

[0280] Integrity of the samples was also confirmed by size exclusion high pressure liquid chromatography (SEC-HPLC) using a Superdex 200 HPLC column. Samples were filtered before running them over the column. Again no difference was apparent between the formaldehyde-treated and the untreated batch after incubating samples of each batch for one or three months at 2-8 C.

Example 2: Aggregate Formation in Formylated and Non-Formylated FHA Batches

[0281] Formation of FHA aggregates/precipitates during incubation was checked by dynamic light scattering (DLS). Samples were centrifuged before testing. Two peaks were identified: Peak 1 corresponded to the monomeric form and peak 2 corresponded to the aggregate form. A 60 C. angle was used to evaluate aggregate/precipitate formation. DLS analysis revealed that precipitation occurred during storage at 2-8 C. for 3 months whether the FHA was treated with formaldehyde or not. Precipitation could also be reduced by the addition of 0.05% Tween-80 to the formaldehyde-treated sample but not the untreated FHA sample. Aggregation only occurred subsequent to formaldehyde treatment, and further studies showed that aggregation could be reduced by lowering the concentration of formaldehyde used to treat FHA.

Example 3: Stability of Vaccine Composition Containing Formylated or Non-Formylated FHA

[0282] Two TdaP vaccine compositions were formulated comprising the following antigens: Tetanus Toxoid (T), Diphtheria toxoid (D), and three purified antigens from acellular Pertussis (aP) (PT, FHA and pertactin). Vaccines were formulated in histidine buffer (100 mM, pH 6.5) supplemented with NaCl (9 mg/ml) and adjuvanted with aluminium hydroxide (2 mg/ml). The FHA in one of the vaccine compositions was derived from a formaldehyde-treated batch, while the FHA in the other vaccine composition was FHA from a batch that had been left untreated.

[0283] Short-term stability studies were performed by incubating samples from each of the two vaccine compositions containing formylated and non-formylated FHA, respectively, for 2 or 4 weeks at 2-8 C., and at 36-38 C.).

[0284] Degree of adsorption of the aP antigens was quantitatively determined using sandwich ELISA. The results are summarised in Tables 1 and 2. No difference in the degree of adsorption was observed between TdaP vaccine compositions formulated with non-formylated or formylated FHA bulks at time 0 and after up to one month incubation at either 2-8 C. or 36-38 C.

[0285] Integrity/identity of the samples after incubation was confirmed by Western blotting using PT-, FHA-, and pertactin-specific antibodies. No differences were observed between the samples at time 0 and after two weeks or one month incubation at 2-8 C. and 36-38 C., respectively, whether the vaccine composition contained formylated or non-formylated FHA.

TABLE-US-00001 TABLE 1 Stability results for Tdap vaccine containing non-formylated FHA: Acceptable 2 weeks at 4 weeks at 2 weeks 4 weeks Analytical methods range Time 0 36-38 C. 36-38 C. at 2-8 C. at 2-8 C. pH 6.0-7.0 6.4 6.5 6.5 6.5 6.4 Degree of N/A >90% >90% >90% >90% >90% adsorption for DT Degree of N/A >90% >90% >90% >90% >90% adsorption for TT Degree of N/A >99.84% >99% >99% >99% >99% adsorption for PT (ELISA Test) Degree of N/A >99.84% >99% >99% >99% >99% adsorption for FHA (ELISA Test) Degree of N/A >99.84% >99% >99% >99% >99% adsorption for 69K (ELISA Test)

TABLE-US-00002 TABLE 2 Stability results for Tdap vaccine containing formylated FHA: Acceptable 2 weeks at 4 weeks at 2 weeks 4 weeks Analytical methods range Time 0 36-38 C. 36-38 C. at 2-8 C. at 2-8 C. pH 6.0-7.0 6.4 6.5 6.5 6.5 6.4 Degree of N/A >90% >90% >90% >90% >90% adsorption for DT Degree of N/A >90% >90% >90% >90% >90% adsorption for TT Degree of N/A >99% >99% >99% >99% >99% adsorption for PT (ELISA Test) Degree of N/A >99% >99% >99% >99% >99% adsorption for FHA (ELISA Test) Degree of N/A >99% >99% >99% >99% >99% adsorption for 69K (ELISA Test)

Example 4: In Vivo Immunogenicity Properties (ELISA) of aP Antigens

[0286] Groups of eight CD1 mice were immunized subcutaneously with TdaP vaccine compositions comprising either non-formylated or formylated FHA (dosage 1). In addition, the tested TdaP vaccines were diluted 4-fold (dosage 2) and 16-fold (dosage 3) in saline, and the dilutions were used immediately after preparation to inject the additional mice subcutaneously. All of the tested vaccines were prepared using the same batch of formylated PT and formylated pertactin.

[0287] The mice were bled five weeks following the injection. Sera from the bleeds were used to perform ELISA assays. In order to assess the efficacy of the two TdaP vaccines containing formylated or non-formylated FHA, respectively, the immunogenicity of each of the aP antigens was determined by measuring the mean geometric antibody titre against PT, FHA and pertactin. The results of the ELISA assays are summarised in Tables 3 to 5.

TABLE-US-00003 TABLE 3 Immunogenicity results (GMT (UI/ml) for PT antigen: FHA treatment Dosage 1 Dosage 2 Dosage 3 Non-formylated 220 218 51 Formylated 194 120 36

TABLE-US-00004 TABLE 4 Immunogenicity results (GMT (UI/ml) for FHA antigen: FHA treatment Dosage 1 Dosage 2 Dosage 3 Non-formylated 124 53 5 Formylated 167 71 5

TABLE-US-00005 TABLE 5 Immunogenicity results (GMT (UI/ml) for pertactin antigen: FHA treatment Dosage 1 Dosage 2 Dosage 3 Non-formylated 52 91 5 Formylated 97 52 3

[0288] Immunogenicity results of Tdap vaccine compositions containing non-formylated and formylated FHA were very similar, suggesting that omission of the formaldehyde-treatment step has no effect on the immunogenicity of the FHA component itself or on the PT and pertactin components.

Example 5: Relative Potency of aP Antigens

[0289] Single mouse titres (IU/ml) were used to determine the relative potency of each vaccine formulation compared to a reference Tdap vaccine using the methodology described in the European Pharmacopoeia. Results are summarised in Table 6.

TABLE-US-00006 TABLE 6 Relative potency results (Potency (95% CI)): FHA treatment PT FHA Pertactin Non-formylated 0.98 (0.51-1.88) 0.40 (0.21-0.69) 0.78 (0.38-1.55) Formylated 0.55 (0.31-0.92) 0.49 (0.30-0.76) 0.74 (0.45-1.21)

[0290] The omission of the formaldehyde-treatment step had no effect on the relative potency of the FHA and pertactin components in the tested Tdap vaccine. The unexpected higher PT potency in vaccine comprising non-formylated FHA will require further investigation.

Example 6: In Vivo Immunogenicity Properties of Non-Formulated and Formylated aP Antigens

[0291] To confirm that the above results obtained for non-formylated FHA were similarly applicable to other antigens commonly included in the aP antigen component of Tdap vaccines, two batches of an experimental Tdap vaccine were prepared, in which the three aP antigens were either formylated or non-formylated.

[0292] Five groups of 12 mice each (female, Balb/C mice, 6 weeks old) were immunised as follows: Groups 1 and 2 received a Tdap vaccine in the three aP antigens were non-formylated, whereas groups 3 and 4 received a Tdap vaccine in which all of the aP antigens were formylated. Mice in groups 1 and 3 received 1/5 of the human dose of the Tdap vaccine, but mice in groups 2 and 4 received 1/50 of the human dose. Mice were injected i.m. twice (days 0 and 28) with 100 L (250 l each time). Mice in group 5 were not immunised and served as a nave control group.

[0293] Pre-immunisation serum samples were taken on day 0. Post-immunisation serum samples were taken on days 14 and 42, and serum IgG titres were determined for the tested aP antigen (see FIGS. 1-3). Titres were statistically assessed by test t, Mann-Whitney test.

[0294] With the exception of PT at day 14, no statistically significant differences in IgG titres were observed between batches containing only formylated aP antigens and batches that contained one non-formylated aP antigen. For PT, the post-immunisation IgG titres at 1/5 of the human dose at day 14 were statistically significantly higher for the non-formylated antigen than the IgG titres obtained with the formylated antigen (see FIG. 3).

[0295] As expected, for each of the investigated antigens post-immunisation IgG titres at day 42 were statistically significantly higher than post-immunisation IgG titres at day 14. Similarly, titres at day 42 were in all cases significantly higher than in the nave mice, even at 1/50 dose (p0.003 in all cases), and titres at 1/5 dose were significantly higher than with a 1/50 dose (p0.01 in all cases).

[0296] 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.

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