Construction And Expression Of Recombinant Epitope Vaccine And Development Of Their Purification Strategy From Bench To Pilot Scale

Recombinant epitope vaccine comprises on one hand the use of recombinant DNA technology, and on the other hand the utilization of synthetic peptides which constitue epitopes that induce protection against infection. The main benefit of immunization with such vaccine is the ability to immunize with a minimal structure, consisting of a well-defined antigen which can be thoroughly characterized with respect to its antigenicity and immunogenicity. This novel approache can overcome the crucial drawbacks of traditional vaccines which based on the intact pathogen and provide a safer vaccine.According to the comprising epitopes and immune responses, there are two kinds of recombinant epitope vaccines. One contains both B-cell epitopes that are important for the induction of neutralizing antibodies and T-cell epitopes for presentation by MHC class II molecules. The other contains T-cell epitopes that are important for the induction of CTL response. Comprising the appropriate epitopes, these vaccines will stimulate effective specific immune responses against infections and cancers.When applying the strategy of peptide-based vaccines and developing such vaccines for commercial use, it is important to take into consideration the identification of epitopes specific to the antigen, construction and conformation of the vaccine, and the downstream process of the recombinant proteins. In this paper, we focus on the identification of B-cell epitopes in S2 subunit of SARS coronavirus spike protein, structure-function relationships of three epitope-based vaccines against foot and mouth disease (FMD), and the development of downstream purification process of heat shock protein 65 (HSP65) fusion protein. In these studies, we solved some problems in the development of epitope vaccines and obtained valuable experience in design, construction and downstream development of such vaccines.This study was composed of following parts:1. Expression of predicted B-cell epitope peptide in S2 subunit of SARS coronavirus spike protein in E.coli and identification of its mimic antigenicityThe spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells. For some coronavirus, the S protein, which forms morphologically characteristic projections on the virion surface, is cleaved into S1 and S2 subunits. The S2 subunit is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine.To study the expression of predicted B-cell epitope peptide in S2 subunit in E.coli and its mimic antigenitcity to S2 protein, B-cell epitopes in S2 subunit was predicted by multi-parameter method using DNAStar software. The cDNA sequence encoding the B-cell epitope peptide was constructed artificially by PCR and then cloned into the downstream of chaperone 10 gene in vector pET28a to construct pET28-chap10-S2epi plasmid. The fusion protein, chap10-S2epi, was expressed in E.coli BL21(DE3) and identified by SDS-PAGE and Western blot. The rabbit was immunized by purified Chap10-S2epi for the preparation of antiserum, which was used to identify the mimic antigenicity of Chap10-S2epi to S2 protein by ELISA. The result showed that chap10-S2epi fusion protein was successfully constructed and expressed in E.coli. The antiserum from the animal immunized by Chap10-S2epi recognized the full length of SARS coronavirus S2 spike protein.2. Expression of HSP-MUC1 in the E.coli periplasmMUC1 is a tumor-associated antigen expressed on breast cancer cells recognized by cytotoxic T-cells. The most important epitopes that can induce cytotoxic T lymphocyte (CTL) reside in the variable number tandem repeats (VNTR). In our lab, a recombinant protein (HSP-MUC1) was constructed by fusing BCG derived HSP65 with MUC1 VNTR peptide. It was proved that the growth of MUC1-expressing tumors was significantly inhibited in mice immunized with HSP-MUC1 both before and after tumor challenge. In the human system, HSP-MUC1 loaded human dendritic cells induced the generation of autologous MUC1 specific CTL in vitro. These results suggest that HSP-MUC1 may be a promising therapeutics for breast cancer.HSP-MUC1 constructed in our lab was expressed in cytoplasm of E.coli BL21(DE3) and was degraded during expression. To solve this problem, we subclone the gene encoded HSP-MUC1 into the plasmid pET26b and expressed HSP-MUC1 in the periplasm of BL21(DE3). However, analyzed by Western blot, HSP-MUC1 expressed in the periplasm was degraded too. So, the strategy of periplasm expression could not improve the stability of HSP-MUC1. 3. Structural and functional study on FMD vaccines composed of same B-cell and T-cell epitopes in different permutations.Foot and mouth disease virus (FMDV) is the causative agent of the most economically disease of clovenhoofed animals wordwide. Neutralizing antibodies are important determinants of protection against FMD. One of the major antigenic sites is located in the G-H loop of capsid protein VP1 (positions 140-160). This loop includes a highly conserved Arg-Gly-Asp (RGD) triplet involved in recognition of an integrin receptor. In addition to this linear B-cell epitope, there are also two T-cell epitopes (position 20-40 and 200-213) associated with the stimulation of neutralizing antibodies.In our lab, the B-cell epitope was combined with the T-cell epitopes to yield 3 different permutational constructs, such as VP1epi-I, VP1epi-II and VP1epi-III. Guinea pigs were immunized and titers of neutralizing antibodies were determined by suckling mice protection test. Antibody titer elicited by VP1epi-I was 1:18 and the suckling mices were partially protected. VP1epi-II could not elicit neutralizing antibodies. And the antibody titer elicited by VP1epi-III was 1:645 which was comparable to the traditional vaccines composed of whole pathogen.To unravel the difference of these three VP1epi recombinant proteins, we predicted their 3D structures by de novo method and compared the predicted structures with the crystal structure of natural VP1 protein. It was found that VP1epi-III was a global protein which has one B-cell epitope located on the surface of the protein and the RGD sequence in the epitope was exposed. The 3D structure of the exposed B-cell epitope was similar to the one of the natural VP1 protein. These features in structure make VP1epi-III more effective for production of neutralizing antibody than other VP1epi proteins. We concluded that the T1BT2BT1BT2 moity of the VP1epi-III was crucial for the structure and function of VP1epi-III.4. Downstream purification of HSP65 fusion protein and its scaling up from bench to pilot scale.BCG derived heat shock protein 65 (HSP65) was unstable and vulnerable to degradation in downstream process. Because of the similarity in properties, it was difficult to remove the degraded from the intact. Purification tag or HPLC was applied by some researchers to purify HSP65 with high purity for the study of function and structure.HSP-Her2 and HSP-MUC1 were constructed in our lab by combine HSP65 with tumor antigen epitopes of Her2 and MUC1 respecively. And HSP-MUC1 has been approved by Chinese FDA for clinical trials. In purification of these two proteins, degraded bands which identified by Western blot and migrated faster than the intact ones were reproducibly observed on the Coomassie blue-stained SDS-PAGE. Because of the degradation, purity of the bulk HSP65 fusion protein could not satisfy the requirement for therapeutics. However, the purification tag in the fusion protein may cause undesirable effects and therefore will not be allowed for human use. And for the limitation in scale and economy, HPLC could not be applied for scaling-up to pilot or industrial scale. So we focus on establishing an"old fashioned"scheme for purifying HSP65 fusion proteins from E.coli.Although the degradation of HSP65 fusion proteins occurred in fermentation, proteins with high purity could be obtained by controlling of the degradation in steps of cell disruption and chromatography. In this study, we found that the pH and salt concentration of the buffers were keys to control degradation. A purification scheme consisting of 4 steps including butyl-Sepharose, DEAE-Sepharose, 1% Trion X-144 phase separation and Sephadex G-25 was used to purify HSP-Her2. The purity of HSP-Her2 was more than 95%, the yield was 5.2% and the endotoxin level was less than 100 Eu/mg. And HSP-MUC1 was purified by two steps of phenyl-Sepharose and Q-Seppharose. In pilot scale, 472 mg HSP-MUC1 with purity of 99% was purified from 100 g wet cell mass. The endotoxin level was less than 10 Eu/mg. We also established the quality control methods of HSP-MUC1 and determined the purity, activity, antigenicity and residues of purfied bulk sample.