Characterization of co-purified acellular pertussis vaccines

Figures & data

Figure 1. Two-dimensional gel electrophoresis proteome map of co-purified APV bulks. Separation and identification of proteins in co-purified APV bulk samples using 2-DE analysis followed by MALDI-TOF/TOF MS. Samples were resolved by IEF (pH 3–11) and 12.5% SDS-PAGE. Protein spots were visualized by colloidal Coomassie staining. The main stained spots were excised individually for identification. Protein spots are labeled as indicated in the legend for Table 1. (A) co-purified APV (S2) bulk, from manufacture 1; (B) co-purified APV (S3) bulk from manufacture 2.

Table 1. Proteins in the co-purified APV bulks identified by mass spectrometry and database research

Order	Protein name	NCBI GI identifier	Mass (Da)	pI	Sequence coverage (%)	Function	Localization
1	Putative outer membrane ligand binding protein (BipA)	gi|33592249	137111	6.26	22–32	Uncharacterized	Outer membrane
2	Filamentous hemagglutinin (FHA)	gi|518079040	239347	8.59	18–36	Hemagglutinin	Outer membrane
3	Pertactin (PRN)	gi|72539781	68635	6.65	28–46	cell adhesion	Outer membrane
4	Autotransporter subtilisin-like protease (sphB1)	gi|408414445	99673	9.5	35–48	Hydrolase, serine-type endopeptidase activity	Outer membrane
5	Pertussis toxin S1 subunit	gi|33594638	30127	6.97	57–72	Virulence, NAD+ ADP-ribosyltransferase activity	Extracellular
6	Serotype 2 fimbrial subunit (FIM2)	gi|33592254	22448	8.39	58–64	cell adhesion	Extracellular
7	Serotype 3 fimbrial subunit (FIM3)	gi|33592658	21903	9.1	58–69	cell adhesion	Extracellular
8	ABC transporter substrate-binding protein	gi|33594007	33389	5.99	51	cell adhesion, metal ion transport	Cytoplasmic membrane
9	Sulfate-binding protein (Sbp)	gi|33592122	37891	7.82	68	Secondary active sulfate transmembrane transporter activity	Periplasmic

Figure 2. Levels of antigen-specific IgG in mice immunized with different pertussis vaccines. Sera from control mice and immunized mice were determined by ELISA. Antibody level (EU/mL) is calculated against mouse serum reference NIBSC 97/642. Results represent the geometric mean antibody titres for 5 mice per group. *indicates a statistically significant difference (P < 0.05) compared with WPV (S1) group; # indicates a statistically significant difference (P < 0.05) compared with co-purified APV (S2 and S3) groups.

Figure 3. Cytokine responses in mice immunized with different pertussis vaccines. Seven days after the second immunization, spleen cells were prepared from 5 mice from each group. The cytokines were determined by ELISA. Results are the mean responses for 5 mice per group. *indicates a statistically significant difference (P < 0.05) compared with co-purified (S2 and S3) and purified (S4) APV groups. # indicates a statistically significant difference (P < 0.05) compared with purified (S4) APV groups.

Figure 4. Protective effects of pertussis vaccines in the murine intracelebral challenge model. The results of each test sample were analyzed by the probit method, using the proportion of mice that survived the challenge for estimation of relative potency against Chinese National Standard, and expressed in IU/SHD. Number in bracket is 95% limit.

Table 2. Vaccine samples used in this study*

No.	Vaccine types	Indicated pertussis component content per single human dose
S1	DTwP	4.5 × 10^9 CFU of B. pertussis
S2	Co-purified DTaP	9 PNμg of pertussis antigens
S3	Co-purified DTaP	9 PNμg of pertussis antigens
S4	purified DTaP	PT 25 μg, FHA 25 μg, PRN 8 μg

*Note: DTwP, diphtheria, tetanus and whole cell pertussis vaccines; DTaP, diphtheria, tetanus toxoids and acellular pertussis vaccines.