Team:CGU Taiwan/Results
Leijuvant
Results
Shuttle Vector
(I) Introduction
In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.
A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others’ future application.
A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others’ future application.
(II) Design
(III) Result
Leijuvant
– the immune response of photo-inactivated leishmania as adjuvant in mice
(I) Introduction
To prove our concept, we tested the efficiency of the antibody immune response and T cell immune response of the photo-inactivated Leishmania as a vaccine adjuvant. Ovalbumin (OVA) has been commonly used as the antigen for testing the efficiency of antibody response and T cell activation in previous immunology experiments. Also, OVA is the only foreign antigen carried by Leishmania that has been shown to load the major histocompatibility complex class I molecules (MHC I) after phagocytosis by APCs, since the transgenic mutants of Leishmania is a new way to deliver antigens into antigen-presenting cells (APC). Thus, we want to use OVA in our in vivo test to validate our hypothesis of photo-inactivated Leishmania as an adjuvant. We co-injected OVA recombinant protein and photo-inactivated Leishmania that is genetically modified to present OVA protein into mouse.
Serum is collected every 5 days after the second injection to test the antibody immune response with Anti-OVA ELISA and further tested the T cell response with dissected splenic cell. The outcome will be compare to the of Alum adjuvant.
(II) Design
We subcutaneously co-injected leish-OVA (Leishmania expressing OVA) and OVA protein into mice and compare the outcome to the of Alum adjuvant with OVA protein as a positive control. We immunized the mice twice, the second boost will be injected on the 15th day after the first shot and after the second shot we will collect serum from the mice on the 5th 10th 13th day after. The serum will be tested for anti-OVA IgG1 and IgG2a as the antibody response and the cell-mediated immune response, respectively. We will dissect the spleen on the 10th day after the second boost and culture the splenic cells for 6 days. Culture supernatant will be tested for cytokines specific for T cell response.
(III) Result
The result of a particular IgG subclass secreted from B cells is determined by which type of T cell is induced. Proliferating helper T cells will develop into effector T cells and differentiate into two major subtypes, T helper 1(Th1) and T helper2(Th2). Th1 cells mainly promote cellular immune pathway which maximizes the killing efficacy of macrophages, the proliferation of cytotoxic cluster of differentiation 8 (CD8) T cells and stimulate the production of Immunoglobulin G 2a (IgG2a)[1]. Th2 cells dominantly facilitate humoral immune pathway, stimulating B-cells into proliferation and production of neutralizing antibodies such as Immunoglobulin G (IgG), Immunoglobulin M (IgM) and Immunoglobulin A (IgA) and Immunoglobulin E (IgE) antibodies.
Th1 can secrete cytokines, e.g. Interferon-γ (IFN-γ), to help stimulate other immune cells which give rise to the positive feedback. Therefore, promotes the Th1 profile. Th2 can secrete Interleukin 10 (IL-10) which down regulates the expression of Th1 cytokines but enhances B cell survival, proliferation, and antibody IgG1 production [2]. Therefore, higher IgG1/IgG2a ratio indicates increasing activity of the Th2-pathway.
Antibody titer of serum samples collected from immunized mice were measured with anti-OVA IgG1 and IgG2a ELISA. The antibody titer of Alum+OVA group on the 25th day is set as 100%.
The IgG1 antibody titer increased drastically after the second boost on the 15th day. And the antibody titer reached to peak on the 25th day (Figure 2A.). Inactivated Leishmania can achieve over 60% of the antibody titer. As for OVA-expressing inactivated Leishmania, the antibody titer can reach about 80% on the 25th day. The OVA expressed in the Leishmania will be spliced once it is engulfed by microphage and directly activate the CD8 and CD4 T cell. The IgG2a antibody titer of inactivated Leishmania increased extremely on the 20th day and is significantly higher compare to Alum on the 25th day. The productions of IgG2a from inactivated Leishmania groups are much higher than Alum. Alum is able to induce a good Th2 response (IgG1), but it has little capacity to stimulate Th1 immune responses[1] and thus has a lower production of IgG2a. Inactivated Leishmania as the adjuvant has a higher tendency to stimulate Th1 immune responses than Alum.
The concentration of IL-10 and IFN γ released from splenocytes were measured by IL-10 and IFN γ ELISA. The splenocytes were collected from spleens dissected form mice on the 25th day of immunization (Figure 2B.). The IL-10 secretion of inactivated Leishmania stimulated with OVA is significantly higher than the positive control (ConA). IL-10 secreted form Th2 may help the production of IgG1. As for the IFN γ, the secretion concentration was low and don’t have a significant difference.
Due to the limited sample size, there wasn’t any significant difference between inactivated Leishmania only and inactivated OVA-expressing Leishmania. The OVA-expressing Leishmania wasn’t a stable transfectant cell line yet when we performed the in vivo test. Owing to the time limits, there was still 1/3 of the complete drug selection process remaining to select the stable tranfectants with high expression of the target antigen. Therefore, the result of injecting stable tranfectants of inactivated OVA-expressing Leishmania may have a much higher antibody production. It has shown the co-injection of normal saline with OVA has only a slight increase of antibody, but the sample size is too small to show the significant difference of it with other groups.
We conclude that Leijuvant is a potential bi-pathway adjuvant that can stimulate both Th1 and Th2 immune responses.
Th1 can secrete cytokines, e.g. Interferon-γ (IFN-γ), to help stimulate other immune cells which give rise to the positive feedback. Therefore, promotes the Th1 profile. Th2 can secrete Interleukin 10 (IL-10) which down regulates the expression of Th1 cytokines but enhances B cell survival, proliferation, and antibody IgG1 production [2]. Therefore, higher IgG1/IgG2a ratio indicates increasing activity of the Th2-pathway.
Antibody titer of serum samples collected from immunized mice were measured with anti-OVA IgG1 and IgG2a ELISA. The antibody titer of Alum+OVA group on the 25th day is set as 100%.
The IgG1 antibody titer increased drastically after the second boost on the 15th day. And the antibody titer reached to peak on the 25th day (Figure 2A.). Inactivated Leishmania can achieve over 60% of the antibody titer. As for OVA-expressing inactivated Leishmania, the antibody titer can reach about 80% on the 25th day. The OVA expressed in the Leishmania will be spliced once it is engulfed by microphage and directly activate the CD8 and CD4 T cell. The IgG2a antibody titer of inactivated Leishmania increased extremely on the 20th day and is significantly higher compare to Alum on the 25th day. The productions of IgG2a from inactivated Leishmania groups are much higher than Alum. Alum is able to induce a good Th2 response (IgG1), but it has little capacity to stimulate Th1 immune responses[1] and thus has a lower production of IgG2a. Inactivated Leishmania as the adjuvant has a higher tendency to stimulate Th1 immune responses than Alum.
The concentration of IL-10 and IFN γ released from splenocytes were measured by IL-10 and IFN γ ELISA. The splenocytes were collected from spleens dissected form mice on the 25th day of immunization (Figure 2B.). The IL-10 secretion of inactivated Leishmania stimulated with OVA is significantly higher than the positive control (ConA). IL-10 secreted form Th2 may help the production of IgG1. As for the IFN γ, the secretion concentration was low and don’t have a significant difference.
Due to the limited sample size, there wasn’t any significant difference between inactivated Leishmania only and inactivated OVA-expressing Leishmania. The OVA-expressing Leishmania wasn’t a stable transfectant cell line yet when we performed the in vivo test. Owing to the time limits, there was still 1/3 of the complete drug selection process remaining to select the stable tranfectants with high expression of the target antigen. Therefore, the result of injecting stable tranfectants of inactivated OVA-expressing Leishmania may have a much higher antibody production. It has shown the co-injection of normal saline with OVA has only a slight increase of antibody, but the sample size is too small to show the significant difference of it with other groups.
We conclude that Leijuvant is a potential bi-pathway adjuvant that can stimulate both Th1 and Th2 immune responses.
MHC Presentation
(I) Introduction
The biobrick construct of E.coli-Leishmania shuttle vector is meant to express
the targeting antigen protein in Leishmania through amplification in E.coli and
transfection into Leishmania. Therefore, total size of the shuttle vector can
significantly affect the efficiency of transformation and transfection during the
procedure. To enhance the efficiency, we’ve tried to focus on shortening the
targeting antigen sequence which will then be sub-cloned into the shuttle vector. In
order to identify antigen sequence with the highest MHC binding affinity, researchers
have to utilize several bioinformatics tools to figure out or predict the protein
properties. In our project, we have generated an integrated protein information
website, McHug, to help users in searching for peptide sequences that can optimally
activate immune response. Other than providing users with all basic protein
information, McHug features the visualized interface which can transform loads of
numerical data into legible charts. The ultimate goal of McHug website is to mark all
the protein annotations on the given protein sequence and display the relative
immune properties such as MHC binding affinity in every position of the protein.
Selection of the most suitable sequence for MHC presentation can be easily
accomplished with McHug. The apllicability of McHug website was further tested in
vitro using OVA-loaded dendritic cells. MHC molecules were immunoprecipitated and
the associated peptide sequences were then analyzed by mass spectrometry. You can
learn faster and more about your targeting antigen while experiencing McHug.
(II) Design
Our in vitro experiment is to focus on immunoprecipitating MHCII and MHC I molecules to prepare LC/MS samples. The peptides on the MHC molecules will be isolated and sequenced to analyse the viability of our software, McHug. In figure 1, the schematic diagram of our co-culture and lysis system is described. Inactivated Leishmania was co-cultured with dendritic cells for several time points and be lysed by CHAPS buffer to collect the supernatant for further experiment. The ultimate goal of our in vitro experiment is to isolate the peptides on MHC molecules and sequenced by MS spectrometry. Therefore, immunoprecipitation should be proven to be workable.
(III) Result
First, we wanted to test what kind of protein G bead is able to conjugate our MHCII antibody. As the result shown in figure 2, MHCII antibody was detectable when incubate antibody with protein G Sepharose bead. Whereas the protein G magnetic bead can not bind to MHCII antibody.
Next, we planned to measure the MHCII protein level after co-culturing Leishmania with dendritic cells. The outcome will provide us an appropriate time point to incubate Leishmania and dendritic cells. Judging from figure 3, MHCII protein level was increased in 6 hours and 48 hours. We then took 6 hours as our incubation time in the following few experiments.
After making sure of the co-culturing time and bead binding ability, we started our MHCII and MHCI immunoprecipitation. In figure 4, MHCII protein was detectable in immunoprecipitated supernatant while the beads plus antibody group had no MHCII signal. This render the success of immunoprecipitating MHCII molecules. On the other hand, MHCI was unable to be pull down by immunoprecipitation since the antibody can’t bind to MHCI efficiently. Only the MHCII sample hence be further proceed to LC/MS sample.
After MHCII immunoprecipitation, the MHCII complexes were eluted by acetic acid. Figure 5 shows that the eluted fractions contain MHCII molecule which means MHCII complexes are successfully eluted. The eluted fractions were then desalted by HPLC cartridge set (RP-18 ADS) to prepare the final LC/MS samples. However, the first LC/MS outcome interprets no peptide signal. That might because of the low eluted MHCII concentration and the experiment will be further modified.
Next, we planned to measure the MHCII protein level after co-culturing Leishmania with dendritic cells. The outcome will provide us an appropriate time point to incubate Leishmania and dendritic cells. Judging from figure 3, MHCII protein level was increased in 6 hours and 48 hours. We then took 6 hours as our incubation time in the following few experiments.
After making sure of the co-culturing time and bead binding ability, we started our MHCII and MHCI immunoprecipitation. In figure 4, MHCII protein was detectable in immunoprecipitated supernatant while the beads plus antibody group had no MHCII signal. This render the success of immunoprecipitating MHCII molecules. On the other hand, MHCI was unable to be pull down by immunoprecipitation since the antibody can’t bind to MHCI efficiently. Only the MHCII sample hence be further proceed to LC/MS sample.
After MHCII immunoprecipitation, the MHCII complexes were eluted by acetic acid. Figure 5 shows that the eluted fractions contain MHCII molecule which means MHCII complexes are successfully eluted. The eluted fractions were then desalted by HPLC cartridge set (RP-18 ADS) to prepare the final LC/MS samples. However, the first LC/MS outcome interprets no peptide signal. That might because of the low eluted MHCII concentration and the experiment will be further modified.
