Shanghai_Metropolis & Shanghai_HS_ID
Inspiration and Common Goal
Lactobacillus casei is commensal bacteria that have long been utilized as probiotics and are generally regarded as safe for use in humans, making them prime candidates for vector design. Recent research has, however, focused on the probiotic properties rather than their specific interactions with the mucosal immune system.
In order to rationally develop L. casei as vaccine vectors, EV71 terminator and Pro-CASei decided to partner with each other in developing the HFMD Oral Vaccines using L. casei as a substitute for E. coli. EV71 terminator will construct plasmids containing DNA expressing the protein coat of the virus, and convert the plasmids into the experimental strain of L. casei cultivated by Pro-CASei. The wild type of L. casei exhibits low expression rates of plasmids, since it is protected by the Restriction Modification system, which destroys foreign DNA and prevents its expression in the L. casei bacteria. Pro-CASei designs to remove a specific segment of L. casei's DNA which codes for a pivotal enzyme participating in the Restriction Modification system. Hopefully, this will create a strain of new L. casei with higher expression rates along with its immense health benefits.
Experiment Design
Figure 1. Visual Representation of Experimental Design from EV71 Terminator
We designed to introduce the constructed plasmid into E.coli to verify the viability of the concept of producing oral vaccines. The plasmids were then transformed into L.casei to detect for the correct protein expression.
For plasmid constructions, we first conducted PCR to obtain the VP1, VP1-linker and LTB fragments, then OE PCR to obtain VP1-LTB fragments. The VP1 fragments, VP1-LTB fragments and pGEX-6P-1 will undergo restriction enzyme double digestion and ligation to finally construct the expected plasmids, pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, which would be shortly transformed into E. coli BL21 as well as L. casei ATCC334 for the antibody expression. To evaluate the performance of the protein expression, we will conduct the SDS-PAGE procedure and the Western Blot experiments, combined with Coomassie Brilliant Blue Staining as function tests.
Figure 2: Visual Representation of Experimental Design from Pro-CASei
For Pro-CASei, they plan to utilize CRISPR-Cas9 to reform wild Lactobacillus. casei by knocking out the gene LSEI-2094 in order to improve the conversion and expression efficiency of exogenous genes.
Pro-CASei obtained sgRNA with pLCNICK as the template by PCR. In the same way, the upstream and downstream homologous arms could be obtained with L. casei as the template. After the ligation experiment, sgRNA, the upstream and downstream homologous arms were inserted into pLCNICK to be the expected plasmid. Initially, they transformed the plasmid into E. coli DH5α for culturing and then it will be sent for sequencing after the plasmid extraction. Once they obtain the correct plasmid, they would introduce it into the L. casei ATCC334 by electrotransformation in order to knock out gene LSEI-2094.
In the final stage where they obtain the LSEI-2094 gene defect L. casei ATCC334, they will evaluate its performance by transferring a foreign plasmid - pIB165 with the wild L. casei ATCC334 as a comparison.
By cooperating, the two teams will design an experiment to transform the plasmid constructed by EV71 Terminator before implanting it into the transformed colony of L. casei bacteria which was modified by Pro-CASei.
Progress and Collaboration
EV71 Terminator
Figure 3. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions in E. coli
Figure 4. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining in L. casei ATCC334
Firstly we succeeded in the first stage that we obtained the expected plasmids, pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, which were proved by colony PCR and sequencing. Besides, these plasmids were successfully transformed into E.coli BL21 and L.casei ATCC334 which were also verified by colony PCR, respectively.
After that, we transformed them into E.coli BL21 and L. casei ATCC334 to verify the protein expression by SDS PAGE with Coomassie Brilliant Blue Staining and Western Blot, respectively, to stain the bands. By selecting bacteria solutions of different concentrations and inducting them with IPTG solutions of different concentrations, we determined the optimum conditions under which the proteins GST-VP1 and GST-VP1-LTB were expressed better. In addition, it indicated that LTB could promote the expression of VP1 during the late growth period of E.coli.
However, there was no obvious band in certain KD ranges of the L. casei PAGE gel (figure 4), which meant there was no obvious expression of GST-VP1 and GST-VP1-LTB in L. casei, which is probably because of the restriction-modification system in wild L. casei. Therefore, it is necessary to modify the L. casei and the task of the Pro-CASei team is significant.
Pro-CASei
Figure 5. results from Pro-CASei
Toughing through several failures, Pro-CASei team successfully constructed the plasmid pNCas9-LSEI-2094, the core task which could be used for the later Gene Knock-out experiment. During the time, they also prepared L. casei competent cells and MRS media which were also provided to us to continue our experiments about L. casei.
After electrotransforming into L.casei ATCC334, it took several days for culture and those strains were verified as positive by sequencing. In the next stage, they decided to test the performance of the modified L. casei by being transformed with a foreign plamid pIB165 meantime comparing to the wild L. casei. As showing above, we can see that the modified L. casei has higher transformation efficiency than the wild.
Collaboration
EV71 Terminator
Our team explored and practiced the transformation incubation procedures of L.casei, an elementary step of the functional test designed by Pro-CASei. These protocols helped Pro-CASei perfect the experiment procedures, and laid down the foundation for further steps. Besides, our attempt to use the wild L. casei could serve as a comparison to their modified L. casei in the future.
Pro-CASei
Pro-CASei supplied EV71 Terminator with necessary materials, such as the L. casei competent cells and MRS media to cultivate both the wild strain and experimental strain of L. casei. This simplified the complex experiment procedures and accelerated the project process.
Other Collaboration
We two teams published articles on our official accounts introducing the topics, and future plans.
Challenge and Future Plan
Even though the EV71 Terminator's carrier pGEX-6P-1 is generally said to be able to express in Lactic Acid Bacteria, no evidence specifies its viability in L.casei. Possible reasons behind the scarce research on L. casei can be the relatively low electroporation efficiency, slow growth rate, and the Restriction Modification system in L.casei. These are all challenges that the two teams together will need to tackle in the future to transform the plasmid containing VP1-LTB into the new experimental strain L. casei which would be pre-modified and cultivated by Pro-CASei.
Currently, EV71 terminator has verified the feasibility of the oral vaccine and determined the optimum condition for the protein expression in E.coli. Further steps would be to substitute E. coli with L.casei to improve the vaccine and protect patients from the nasty effects of bacteria imbalance.
Pro-CASei had obtained the modified L.casei and is now conducting more functional tests to evaluate the performance.
Because of the delicate conditions required for optimized protein expression, the two teams will either need to improve the incubation and electroporation techniques or reconstruct the plasmids to maximize the expression of GST-VP1-LTB in L. casei.
No doubt that both teams strive to improve the transformations rate of the foreign plasmids to L. casei than that seen in the wild-type bacteria, allowing us two teams to step closer to our common goal - HFMD Oral L. casei-Based Vaccines.
Assuming we two teams are able to detect a higher expression rate in the L. casei, the next step would be to gavage feed the transformed L. casei to groups of mice and measure the level of anti-VP1-LTB IgG in their serum and the level of IgA in their feces. With statically significant differences between the levels of the experimental group mice and control group, this experiment might have profound evidence to prove whether immunogenicity in the mice that consumed the transformed L. casei. In other words, this will prove the feasibility of the whole project.
Reference
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Adachi K , Kawana K , Yokoyama T , et al. Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7[J]. Vaccine, 2010, 28(16):2810-2817.
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Stoeker L L . Immunogenicity and Safety of Lactobacilli as Oral Mucosal Vaccine Vectors.[D]. North Carolina State University. 2011.