Fc-based delivery system enhances immunogenicity of a tuberculosis subunit vaccine candidate consisting of the ESAT-6:CFP-10 complex

Abstract

Tuberculosis (TB) remains a major global health threat despite chemotherapy and Bacilli Calmette-Guérin (BCG) vaccination. Therefore, a safer and more effective vaccine against TB is urgently needed. This study evaluated the immunogenicity of a recombinant fusion protein consisting of early secreted antigenic target protein 6 kDa (ESAT-6), culture filtrate protein 10 kDa (CFP-10) and the Fc-domain of mouse IgG2a as a novel subunit vaccine. The recombinant expression vectors (pPICZαA-ESAT-6:CFP-10:Fcγ2a and pPICZαA-ESAT-6:CFP-10:His) were transferred into Pichia pastoris. After SDS-PAGE and immunoblotting, the immunogenicity of the recombinant proteins was evaluated in mice. When both recombinant proteins (ESAT-6:CFP-10:Fcγ2a and ESAT-6:CFP-10:His) were used for vaccination, Th1-type cellular responses were induced producing high levels of IFN-γ and IL-12. However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a small increase in IL-4 as compared to the BCG and ESAT-6:CFP-10:His groups. Moreover, mice primed with BCG and then supplemented with ESAT-6:CFP-10:Fcγ2a produced the highest levels of IFN-γ and IL-12 in immunized groups. The findings indicate that when Fcγ2a is fused to the ESAT-6:CFP-10 complex, as a delivery vehicle, there could be an increase in the immunogenicity of this type of subunit vaccine. Therefore, additional investigations are necessary for the development of appropriate Fc-based tuberculosis vaccines.

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

“…The results obtained by Azadi, Mahboubi, Naghdi, Solaimanian, and Mortazavi (2017) showed that sorbitol at a concentration of 50 g/L (5%) could significantly increase the expression of recombinant protein. Moreover, according to the results of other studies, 2.5% methanol with 1% sorbitol (Mosavat et al, 2016), 2% methanol with 0.5% sorbitol (Farsiani et al, 2016), and 2% methanol with 1% sorbitol (Soleimanpour et al, 2015) were considered optimal for the highest recombinant protein production in shake flask culture. Therefore, the presence of other carbon sources such as glycerol and sorbitol can be beneficial to increase protein production.…”

The recombinant fusion protein CFP10–ESAT6–dIFN has protective effect against tuberculosis in guinea pigs

“…Immunization of mice with the recombinant fusion protein CFP10-ESAT6-dIFN was shown to increase the T-cell immune response, and the effect was even more pronounced when supplementing the fusion protein with Fc receptor [11]. The protective effect of a recombinant protein comprising two Mtb antigens, ESAT6 and Ag85B, was demonstrated in a guinea pig model [29].…”

“…The undesirable side effects of the proteins ESAT6 and CFP10 can be reduced by the introduction of additional components into the hybrid antigens containing these proteins. In particular, administration of the recombinant fusion protein CFP10-ESAT6-dIFN with the mouse IgG2a Fc fragment to laboratory mice increased the intensity of Th-1 T-cell response accompanied by an increased IFN-γ, IL-12, and IL-4 production [11]. A statistically significant increase in the immune response to administration of the recombinant fusion protein CFP10-ESAT6-dIFN supplemented with modified human IFN-γ (dIFN, deltaferon) as compared with these immunogens used alone has been demonstrated.…”

Respons Sitokin TNF-Α Dan Il-4 Pasca Stimulasi Antigen Fusi Resat-6-CFP-10

“…Sampai saat ini beberapa vaksin mulai diteliti untuk mencegah TB atau meningkatkan efektivitas BCG. 11,12 Identifikasi antigen yang sesuai dan pemilihan cara pemberian yang sesuai dibutuhkan untuk mendesain vaksin subunit terbaru. Antigen yang digunakan adalah antigen yang disekresikan oleh M. tuberculosis.…”

“…Penelitian pada efek imunogenisitas, melalui stimulasi IFN-γ yang dihasilkan oleh Th1 dan sel T sitotoksik menunjukkan bahwa vaksin yang mengandung ESAT6 dan CFP10 merupakan kandidat vaksin terbaru yang terbaik. 12 Penelitian Torabi et al tahun 2013 pada hewan coba menunjukkan ada peningkatan respons imun protektif pasca vaksin yang mengandung ESAT6 dan CFP10. 11 Sitokin yang dilepaskan pasca stimulasi antigen spesifik secara in vitro menggambarkan respons imun terhadap antigen tersebut.…”