Streptococcus suis polysaccharide-protein conjugate composition
A technology of protein conjugates and Streptococcus suis, applied in the direction of multivalent vaccines, bacterial antigen components, antibody medical components, etc., can solve the problem of no effective vaccines
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Embodiment 1
[0119] Preparation of conjugate vaccine:
[0120] Depolymerization of Type 1.2 CPS:
[0121] In 2010, Van Calsteren et al. reported the precise structure of the repeat unit of serotype 2 CPS (Byrd and Kadis. Infect Immun. 1992; 60:3042-51). The CPS repeat unit consists of a unique arrangement of 1 rhamnose:1 glucose:3 galactose:1 N-acetylglucosamine:1 sialic acid (also known as N-acetylneuraminic acid [Neu5Ac]). Sialic acid was found to be terminal on the branch with an α2,6-linkage to galactose. Precise knowledge of the structure of the CPS of S. suis serotype 2 provides the chemical basis for the construction of glycoconjugates.
[0122] Using highly purified CPS from S. suis type 2 containing less than 1% w / w protein or nucleic acid as previously described by Calzas et al. (Calzas et al. Infect Immun. 2013; 81:3106-18), the affix Whether conjugation to a carrier protein such as TT avoids the T-cell-independent (TI) antigenicity of CPS and instead induces a T-cell-depende...
Embodiment 2
[0141] Conjugate detection and purification
[0142] Gel shift, Western blot and HPLC analysis confirmed the successful conjugation of CPS to TT: Gel shift and Western blot experiments ( Figures 1A-1D ) and analyzed by HPLC ( figure 2 ) confirms the presence of the conjugate in different preparations. For gel shift experiments, Coomassie blue ( Figure 1A ) and silver staining ( Figure 1B ) both show considerable migration from the purified TT monomer at 150 kDa (lane 2) to a thick band in the conjugate exceeding 250 kDa (lanes 3-4). This migration is caused by the covalent addition of a random number of 115 kDa CPS chains to the protein. Coomassie blue ( Figure 1A ), silver staining ( Figure 1B ) or Western blot using anti-CPS mAb ( Figure 1C ) did not show any bands of depolymerized CPS included as a control in all gels (lane 5), indicating its poor ability to migrate through the gel under these assay conditions. Control staining with anti-TT mAb ( Figure 1D ...
Embodiment 3
[0145] Mouse immunization:
[0146] C57BL / 6 female mice (Charles River, Wilmington, MA, USA) aged 5-6 weeks were immunized subcutaneously with different doses of S. suis conjugate preparations in 0.1 ml PBS on day 0 and on day 21. strengthen. In the first set of experiments aimed at comparing different adjuvants, 3 groups (n=10) received 20 μg of CpG oligodeoxyribonucleic acid (ODN) 1826 (InvivoGen, San Diego, CA, USA) according to the manufacturer's recommendation, (Prionics, La Vista, NE, USA) or 25 μg of the 2:1 conjugate vaccine formulation in PBS adjuvanted by Gold (CytRx Corporation, Norcross, GA, USA). Three placebo groups (n=5) received only PBS adjuvanted as described above. In the second set of experiments, using Groups of mice (n=8) immunized with 1, 2.5, 5 or 25 μg of the 2:1 conjugate vaccine emulsified in Gold 1:1 (v / v) were subjected to a dose-response study. Included for comparison purposes with similar doses of Gold emulsified free (unconjugated) dis...
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