Immunogenicity And Protective Efficacy Of Tuberculosis Subunit Protein Vaccine Candidates

[Objective]Different strategies have been proposed for the development of protein subunit vaccine candidates of tuberculosis (TB), which shows better safety than other types of candidates and currently used Bacillus Calmette-Guerin (BCG) vaccine. Thus far, there are many strategied for the development of TB subunit protein vaccines, and five of thirteen novel TB vaccine candidates are all at different phases of clinical trials. In order to develop more effective protein subunits depending on the mechanism of cell mediated immunity against TB, a polyprotein CTT3H based on five immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8+epitopes of M. tb were constructed in this study. The immunogenicity and protective efficacy of TB subunit protein CTT3H in adjuvant of Dimethyldioctadecylammonium/monophosphoryl lipid A/Trehalose6,6’-dibehenate (DDA/MPL/TDB, DMT) liposome vaccinated C57BL/6mice was investigated.[Methods]1. Bacterial culture:M. bovis BCG China and M. tb H37Rv were grown in either Middlebrook7H9medium or enumerated for CFU on Middlebrook7H11agar, supplemented with10%ADC,0.5%glycerol and0.05%Tween80. Eschricha coli strains DH5a and BL21(DE3) were used for cloning and expression, respectively.2. Construction, expression and purification of subunit antigen CTT3H, which was demonstrated by SDS-PAGE and Western blotting.3. Adjuvant DMT (100μL/dose) was consisted of250μg DDA,25μg MPL and50μg TDB. DDA liposome was firstly prepared by the film method and then was mixed by adding MPL and TDB. Finally,200μL DMT liposome adjuvanted CTT3H vaccine was prepared as a mixture of100μL DMT and100μL CTT3H protein (20μg).4. Evaluation the protective effect of the CTT3H/DMT subunit vaccine against M. tuberculosis H37Rv infection (n=6). The C57BL/6mice were immunized by subcutaneous (s.c.) injection with CTT3H/DMT subunit vaccine to each mouse twice at3-week intervals. BCG China was used as a positive control and inoculated s.c. once with1.2×106CFU at the time of the first vaccination. PBS and DMT adjuvant were used as the negative and adjuvant controls. Three weeks after the last immunization, differentially vaccinated mice were challenged by aerosol exposure to60CFU of virulent M. tb H37Rv. Four weeks later, the protective effect of the CTT3H/DMT subunit vaccine was evaluated by the analysis of bacterial load and histopathology.5. The analysis of the cellar immune responses induced by the CTT3H/DMT subunit vaccine. After the last immunized the C57BL/6mice3weeks, the splenocytes were collected.1) The level of CTT3H and PPD specific IFN-y secreted from splenocytes was detected byELISA(n＝3).2) The numbers of CTT3H and PPD specific CD4+and CD8+T cells which secrete TNF-a, IFN-y and IL-2were counted by flow cytometry (FCM) and intracellular cytokines staining (ICS)(n=3).3) The cytotoxicity of TB10.4peptide-specific CD8+T cells was analyzed by the CFSE-labeled method in vivo (n=3).6. Evaluation the humoral immune response of the CTT3H/DMT subunit vaccine (n=3). The titers of CTT3H specific IgG, IgGl and IgG2c (replaced by IgG2a when analyzing the results) in mice sera were detected by ELISA.7. Statistical analysis was performed with SPSS18.0software, and one-way ANOVA analysis was used to compare the difference between different immunized groups, p□0.05was considered significant.[Results]1. The purified protein with the expected molecular weight of about61kDa was revealed a single major band by SDS-PAGE analysis. The identity of the expected bands was confirmed by Western blotting.2. The protective effect of the CTT3H/DMT subunit vaccine against M. tb infection. Among all groups, the highest bacterial load in both lung and spleen was separately obtained in PBS control as expected (p<0.05). Interestingly, DMT adjuvanted CTT3H vaccinated mice inhibited more significantly the growth of in both organs compared to PBS control and DMT adjuvant (p<0.05). The most severe histopathology based on the area of consolidation was detected in the lung of control mice as expected. The acid-fast positive bacteria were found throughout the lung section of control mice and a large number of AF positive bacteria focused on the region of consolidation. Less and more sporadic granuloma-like consolidation was recorded in CTT3H in adjuvant of DMT vaccinated mice than PBS control and DMT adjuvant. Notably, DMT adjuvanted CTT3H vaccinated mice even provided comparable pathological scores with BCG immunized mice and only a few AF positive bacteria were concentrated in the region of consolidation, which is consistent with the order of lung bacterial load in different groups.3. Antigen-specific cell-mediated immune responses induced by the CTT3H/DMT subunit vaccine.1) As expected, splenocytes from PBS group only produced very low levels of IFN-y, whatever stimulation with positive control PPD or specific antigen CTT3H (p<0.05). Of all groups, BCG vaccinated mice elicited the highest levels of IFN-y responses to PPD (p<0.05). Higher levels of IFN-y responses to either PPD or CTT3H were elicited in CTT3H/DMT vaccinated mice compared to PBS control and DMT adjuvant, respectively (p<0.05).2) When compared to PBS control and DMT adjuvant, BCG and CTT3H/DMT subunit vaccine induced much more numbers of PPD or CTT3H specific IFN-y+or IL-2+CD4+and CD8+T cells and TNF-a+CD4+T cells (p<0.05). In particular, CTT3H/DMT provided higher number of IFN-y+or TNF-a+CD4+and CD8+T-cell responses to PPD or CTT3H than BCG group, respectively (p<0.05). In addition, there was no statistical difference between DMT adjuvanted CTT3H subunit vaccinated group and BCG vaccinated mice in antigen-specific IL-2+CD4+and CD8+T cells, respectively.3) Lower level of killing rate was obtained in BCG and DMT groups than DMT adjuvanted subunit CTT3H vaccinated mice (p<0.05).4. The humoral immune response of the CTT3H/DMT subunit vaccine. The highest levels of CTT3H specific IgG, IgGl, or IgG2a antibodies were obtained in DMT adjuvanted CTT3H vaccinated mice as expected (p<0.05). Although the genes encoding five antigens except CFP10also presents in the genome of BCG, BCG group only elicited low levels of antibodies response to CTT3H. There was no CTT3H specific antibody response in DMT group. The increased ratio of IgG2a/IgGl was only detected in subunit CTT3H vaccinated mice, which indicates a switch of IgG subclass to Thl-based response (p<0.05).[Conclusions]DMT adjuvant is an effective adjuvant for the assist of TB candidate subunit protein vaccine, and DMT-liposome adjuvanted subunit CTT3H is a promising candidate for TB vaccine. [Objective]So far, BCG, as the unique clinical vaccine for the prevention of TB, has been used more than50years. However, TB remains one of the most serious diseases worldwide. Subunit vaccines can be used for individual, who are not suitable for the vaccination of BCG or other vaccines, and may replace the effect of BCG. Previously, Four subunit vaccines A1D3R1(Rv3407-PhoY2-Ag85A-Rv2626c-RpfB)/MTO, A1D4(Rv1813-Rv2660c-Rv2623-HspX)/MTO, CTT3H (CFP10-TB10.4-TB8.4-Rv3615c-HBHA)/DMT, and CMFO (CFP32-MTB70-Rv3044-Rv2073c)/DMT (or MTO) possessed acceptable immunogenicity. However, the protection of subunit vaccines was inferior to BCG. In consideration of the situation, four fusion proteins (A1D3R1, A1D4, CMFO and CTT3H) were combined according to the ratio of1:1:1:1and adjuvanted with DMT, and a subunit vaccine AACC/DMT containing19antigens was constructed. Finally, the immunogenicity and the protection of subunit vaccine AACC/DMT were evaluated.[Methods]1. Purification and demonstration of A1D3R1, A1D4, CMFO and CTT3H proteins.2. Preparation of AACC/DMT subunit vaccine.3. Evaluation the protective effect of the AACC/DMT subunit vaccine against M. tb H37Rv infection (n=6). The C57BL/6mice were immunized by subcutaneous (s.c.) injection with AACC/DMT subunit vaccine to each mouse twice at3-week intervals. BCG China was used as a positive control and inoculated s.c. once with106CFU at the time of the first vaccination. PBS was used as negative control.18weeks after the first immunization, differentially vaccinated mice were challenged by aerosol exposure to60CFU of virulent M. tb H37Rv. Four weeks later, the protective effect of the AACC/DMT subunit vaccine was evaluated by the analysis of bacterial load and histopathology.4. The analysis of the cellar immune responses induced by the AACC/DMT subunit vaccine. After the first immunized the C57BL/6mice9and18weeks, the splenocytes were collected.1) The level of A1D3R1, A1D4, CMFO, CTT3H and PPD specific IFN-γ secreted from splenocytes was detected by ELISA (n=6).2) The numbers of A1D3R1, A1D4, CMFO, CTT3H and PPD specific CD4+and CD8+T cells which secrete IFN-γand IL-2were counted by flow cytometry (FCM) and intracellular cytokines staining (ICS)(n=6).3) The cytotoxicity of TB10.4and Ag85B peptide-specific CD8+T cells was analyzed by the CFSE-labeled method in vivo (n-6).5. Evaluation the immune memory level of the AACC/DMT subunit vaccine (n=6). The number of A1D3R1, A1D4, CMFO, CTT3H and PPD specific TEM(CD62Ll0CD44hi) and TCM (CD62Lhi CD44hi) in CD4+or CD8+T cells were measured by FCM. Further, the amount of IL-2production TCM and IFN-y production TEM were analyzed by intracellular cytokines staining.6. Evaluation the humoral immune response of the AACC/DMT subunit vaccine (n=6). The titers of A1D3R1, A1D4, CMFO and CTT3H specific IgG, IgG1and IgG2c (replaced by IgG2a when analyzing the results) in mice sera were detected by ELISA.7. Statistical analysis was performed with SPSS18.0software, and one-way ANOVA analysis was used to compare the difference between different immunized groups, p□0.05was considered significant.[Results]1. The purified proteins A1D3R1, A1D4, CMFO and CTT3H with the expected molecular weights of about121kDa,105kDa,117kDa and61kDa were revealed a single major band by SDS-PAGE analysis. The identity of the expected bands was confirmed by Western blotting.2. The protective effect of the AACC/DMT subunit vaccine against M. tb infection. Compared with PBS control, DMT adjuvanted AACC vaccinated mice inhibited more significantly the growth of M. tb in both organs of spleen and lung (p<0.05), and had a comparable protection with BCG. In particular, compared with BCG, the lung pathological histopathology against M. tb infection of AACC/DMT subunit vaccine was better.3. Antigen-specific cell-mediated immune responses induced by the AACC/DMT subunit vaccine.1) Compared with PBS control, AACC/DMT subunit vaccine induced higher levels of A1D3R1, A1D4, CMFO and CTT3H specific IFN-y, and persistent increased (p<0.05).2) After vaccination with AACC/DMT subunit vaccine, A1D3R1, A1D4, CMFO and CTT3H specific IFN-y+CD4+T cells were higher than that of PBS control, and increased standing (p<0.05).3)9weeks after the first immunization, the TB10.4and Ag85B peptides specific CTL response of AACC/DMT subunit vaccine were higher than BCG (p<0.05), which were opposed to18weeks after the first immunization (p<0.05). In addition, the TB10.4and Ag85B peptides specific CTL response of BCG increased (p<0.05), and that of AACC/DMT subunit vaccine decreased as time passed (p<0.05).4. The immune memory level of AACC/DMT subunit vaccine. Compared with PBS control, A1D3R1, A1D4specific CD4-TEM, CD4+TCM and CD8+TCM cells as well as CMFO, CTT3H and PPD specific CD8+TCM cells induced by AACC/DMT subunit vaccine were higher, and persistent increased(p<0.05). Moreover, AACC/DMT subunit vaccine induced higher A1D3R1, A1D4, CMFO and CTT3H specific IL-2+CD4+TCM or IL-2+CD8+TCM cells, which were promoted as time passed (p<0.05).5. The humoral immune response of AACC/DMT subunit vaccine. A1D3R1, A1D4, CMFO and CTT3H specific Thl-type antibody response were induced by AACC/DMT subunit vaccine (p<0.05).[Conclusion]In conclusion, TB subunit vaccine AACC assisted with DMT adjuvant has the comparable protection to BCG, and better lung pathological histopathology against M. tb infection. Therefore, AACC/DMT subumit vaccine can be used as TB candidate subunit vaccine for pre-clinical animal experiments and clinical trials.