Composition for Oral or Nasal Delivery of Tetanus, Diphtheria, and Pertussis Vaccine alone or in combination using Neurotoxin Associated Proteins

A process for the preparation of second generation tetanus toxoid vaccine, i.e., tetanus heavy chain comprising steps of;

• a) Induction of E. Coli culture OD600=0.5 by adding 0.2 mM IPTG
• b) Growing culture for 14-16° C. at 14 to 20 hours
• c) the cell paste suspends in 25 mM phosphate buffer containing 200 mM sodium chloride
• d) add 1% of triton −X-100 to the buffer
• e) Sonicate for a period of 3 minutes (5 sec on/off pulse) at 4° C. on cold beads.
• f) Centrifuge the culture for 60 to 90 minutes.
• g) Supernatant is collected
• h) Supernatant purified using Ni-NTA affinity column using an eluant;
• and
• i) Combine the pool which has less contaminated bands and concentrate the pool by using Centriprep-30.

A process for preparation of third generation detoxified recombinant tetanus toxoid vaccine comprising steps of;

• a. pBN3 vector was used for cloning TeTx Light chain in a BL21(DE3) strain for expression of the plasmid.
• b. Two active sites mutation, E234A and E271A, was done which corresponds to E224 and E262 of BoNT/A active sites.
• c. HC portion of TeNT was fused to construct full length tetanus neurotoxin plasmid with two active sites mutation, E234A and E271A.
• d. Purified the large band at 150 kDa protein position using Ni-NTA affinity column.

A process for the preparation of vaccine and NAPs composition comprises

• a. mixing equimolar ratio of vaccine and NAPs at pH 5.5 to 6.2
• b. Reacting at an ambient temperature for 2-3 hours
• c. 25 mM phosphate buffer of pH 5.8 containing 200 mM sodium chloride solution charged to step 2
• d. reacting above obtained reaction mixture with 10 mM imidazole solution
• e. The reaction mixture further loaded to Ni-NTA affinity column;
• and
• f. eluted the column to get the pure NAPs-vaccine composition.