Pneumococcal fusion protein vaccines
A technology of fusion protein and amino acid, applied in the field of pneumococcal fusion protein vaccine, can solve the problem of no consistent observed effect on pneumonia prevention
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Embodiment 1
[0256] Example 1: Induction and Induction of Th17 Responses in Mice Mediated by SP0785 and SP1500 Components of Fusion Protein CP1 Defense against pneumococcal nasal colonization
[0257] Purpose:
[0258] This example compares the ability of individual S. pneumoniae proteins SP0785 and SP1500 to stimulate Th17 responses and protect mice from S. pneumoniae nasal colonization with the exemplary fusion protein CP1 following nasal immunization with adjuvanted cholera toxin (CT). ability. An exemplary fusion protein CP1 is a fusion protein comprising truncated Rhizobium avidin (amino acids [45-179], designated Rhavi), a SP0785 polypeptide, and a SP1500 polypeptide. In some embodiments, the fusion protein CP1 is or comprises Rhavi-linker(GGGGSSS)-SP1500-linker(AAA)-SP0785.
[0259] Overview:
[0260] 1. Streptococcus pneumoniae proteins SP0785 and SP1500 and fusion protein CP1 can each produce robust Th17 in mice as demonstrated by interleukin 17A (IL-17A) secretion followin...
Embodiment 2
[0283] Example 2: Killing activity of antiserum on fusion protein CP1
[0284] Materials and methods:
[0285] bacterial growth
[0286] S. pneumoniae strains 6B, 15A, 16F and 35B were inoculated into a 10 mL Todd Hewitt Broth (THB) culture with yeast extract. Place the culture in 5% CO 2 Incubate at 37°C for 4-7 hours until OD 600 Achieving 0.5-0.8 (mid-log). Bacteria were harvested by spinning at 3,000 g for 7 min at 4 °C, and the bacterial pellet was resuspended in 10 mL of THB containing 10% glycerol and stored at -80 °C. Frozen stock solutions on casein soy agar were trypsinized by serial dilution with 5% sheep blood (Becton, Dickenson, and Company) and incubated at 37°C and 5% CO 2 Colony forming unit (CFU) estimates were determined by incubation for 18 to 24 hours.
[0287] Centralized Opsonophagocytosis Assay (COPA)
[0288] Thaw the frozen stock of Streptococcus pneumoniae and make 2 x 10 5 CFU / ml resuspended in assay buffer (Hank's buffered saline with ...
Embodiment 3
[0294] Example 3: Mixture with SP0785, SP1500 and Rhavid truncated avidin (unconjugated) Or in mice immunized with fusion protein CP1 or SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT Comparison of induction of Th17 responses
[0295] Materials and methods:
[0296] Recombinant protein production
[0297] Histidine-tagged recombinant proteins were expressed in Escherichia coli and purified using Ni-nitrilotriacetic acid affinity chromatography. A second purification was performed using size exclusion chromatography with a Superdex 200 column. Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (Bio-Rad).
[0298] formulate
[0299] Cholera toxin (CT) is used as an adjuvant for intranasal immunization to facilitate the induction of T cell responses to proteins alone. Prior to administration, proteins (amounts indicated in Table 6) were mixed with 1 μg CT in saline solution in a final volume of 20 μl per dose.
[0300] Intra...
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