Enhancement of immunogenicity through conjugation of complement fragment C4d to porcine epidemic diarrhea virus surface protein antigen, S0

Abstract

돼지유행성설사병 바이러스의 외피단백질 일부분인 S0 아단위 백신의 면역원성을 높이기 위해서, 보체 절편인 C4d가 결합형 어쥬번트로 이용되었다. S0, S0-mC4d1, S0-mC4d2, mC4d를 E. coli 발현 벡터에 성공적으로 클로닝하였다. 각 벡터의 시퀀스는 Sanger 시퀀싱과 제한효소 소화 후 전기영동 확인방법을 통해 확인하였다. 그 이후, 단백질을 soluble하게 발현하는 것이 중요하므로 3 가지 요소를 통해 solubility를 증진시키고자 하였다. 첫 번째 방법은 샤페론 단백질의 도입이고, 두 번째 방법은 최적의 단백질 유도 optical density를 확인하는 것이며, 세 번째 방법은 단백질 유도 최적의 IPTG 농도를 찾는 것이다. 연구를 통해 샤페론의 발현이 soluble 단백질 발현에 필수적임을 알았고, 최적의 조건이 optical density 0.6, IPTG 0.1 mM임을 확인하였다. 이렇게 확인한 조건으로 S0, S0-mC4d1, S0-mC4d2, mC4d 단백질은 500 ml scale로 생산되어 정제, 투석, 농축 과정을 거쳐 동물실험에 사용되었다. 6 그룹의 마우스에 동일 몰의 단백질을 투여한 후 mC4d의 어쥬번트 효과를 보았다. 그룹은 PBS 음성 대조구, S0, S0-mC4d1, S0-mC4d2, S0와 mC4d 혼합물, S0와 mC4d 2배의 혼합물이었다. 면역접종 후 2,4,6 주 후에 혈청을 통해 S0 특이적 IgG를 분석했고, 6주에 비장을 분리하여 T 세포 분석을 하였다. 그 결과 mC4d를 결합 그룹은 S0 그룹에 비해 S0 특이적 IgG가 유의적으로 많았으며, mC4d 혼합물 그룹 역시 다량의 S0 특이적 IgG를 생산했다. IL-4 ELISpot 분석을 통해 비장 T 세포를 분석한 결과 mC4d 결합의 경우 mC4d 특이적 T 세포가 어쥬번트 역할의 원인이 된 것으로 파악된다. 결론적으로, mC4d 결합형 단백질은 높은 purity와 S0에 대한 높은 면역원성을 가지며, mC4d 혼합물 역시 높은 면역원성을 기록함으로써 어쥬번트 활용의 가치가 있음을 확인했다. 주요어: 돼지유행성설사병, 돼지유행성설사병 바이러스, 어쥬번트, 아단위 백신, 보체 절편, C4d, 결합형 어쥬번트, 외피단백질, 재조합 단백질, 최적화, solubility

The current landscape for vaccines is colored with concerns about the safety of vaccines in the wake of live attenuated or inactivated vaccines. From these concerns, subunit vaccines have risen into the next hot topic for vaccine production. The main advantage of a subunit vaccine is its inherent characteristic of consisting of the antigen-neutralizing subunit of the whole virus, being an inherently safer alternative to other types of vaccines. However, the biggest disadvantage a subunit vaccine must overcome is its low immunogenicity compared to live attenuated vaccines. In order to increase the immunogenicity of the subunit vaccine consisting of the S0 spike protein from PEDVs (Porcine Epidemic Diarrhea Virus) coronavirus, complement fragment C4d was used as a fusion adjuvant. Cloning by using isocaudomers was successful and was confirmed by fragment size distribution through agarose gel electrophoresis and Sanger sequencing. It is important to express recombinant proteins in a soluble way, so three factors were optimized to increase solubility. The first factor was a chaperone, the second was optical density, and the third was IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration. After optimization experiments, it was determined that trigger factor chaperone must be co-expressed with target proteins. In addition, the optimal optical density for inducing proteins is A600 = 0.6, which is during the mid-log phase of growth, using 0.1 mM IPTG. The new, optimized protocol was used for expressing the recombinant proteins S0, S0-mC4d1, S0-mC4d2, and mC4d in a large scale 500 mL flask. The proteins were purified using Ni-NTA (Nickel-nitrilotriacetic acid) resin and dialyzed in PBS (phosphate buffered saline) before being concentrated down. After finding the concentration of the proteins, 6 groups of mice were immunized using the same molar concentration of proteins. The groups were, PBS control, S0 control, the two fusion protein groups, and two mixture groups. After immunization, serum and spleens were taken from the mice and used for ELISA (enzyme-linked immunosorbent assay) and ELISpot (enzyme-linked immunospot), respectively. ELISA shows that end point titers of IgG increased significant in S0-mC4d2 and two mixture groups after 4 weeks and in all mC4d treatment groups after 6 weeks. ELISpot shows the presence of mC4d-specific Th2 cells in the two fusion protein groups but not in the S0 or the mixture groups. In conclusion, recombinant proteins were produced well with relatively high yield and purity and immunization results show that S0-mC4d1 and S0-mC4d2 increases humoral immunity through C4d-specific auto-reactive Th2 cells.