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.

Design

According to Dr. Kwang-Poo Chang’ s research , we found an interesting sequence in leishmania genome, which contains two coding regions, p36 and NAGT. These two coding regions are regulated by their own 5’UTR and 3’UTR, between the p36 and NAGT region is a 2300 bp intrinsic sequence, which is composed of the 3’UTR of p36 and the 5’UTR of NAGT. The 2300 bp intrinsic sequence has several stage-independent splicing sites, and allowed the genes to be constitutively expressed in any stage of leishmania. According to its features, we decided to make it a leishmania shuttle vector which could be used in biobrick form.

The first coding region, p36, is replaced with hygromycin resistant gene as a selection marker in leishmania, and combined with 5’UTR as a biobrick part, since the 5’UTR may contains promoter and ribosome binding site and other important functions in leishmania. As for the second coding region, NAGT, is allowed to be replaced with any protein we want leishmania to carry. In our project, we want to put hemagglutinin (HA) of H1N1 and ovalbumin (OVA) into the site to prove our concept. The 3’UTR of the sequence was designed to become a terminator part while the 2300 intrinsic sequence was also needed to regulate the expression of the protein in the shuttle vector.

The leishmania gene would be put into pSB1C3 to become a shuttle vector that could quickly proliferate in e.coli and express proteins in leishmania. There are 3 advantages of our shuttle vector, the first is that it contains two coding regions originally, so our shuttle vector will be a good choice to express two proteins at the same time. Second, the 2300 intrinsic sequence in our shuttle vector has many stage-independent splicing sites to cut the polycistronic RNA into two transcripts in any stage of leishmania. The third is that, through the biobrick design, we can build a standard shuttle vector for leishmania and also introduce a new animal model into iGEM competition.

Results

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.

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.

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.

Results

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.

Discussion

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.

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.

Design

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.

Results

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 analyze 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.


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 incubating 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.