Results
Currently, trimeric motifs such as Foldon from non-human protein sources expressed in fusion with antigenic proteins can spontaneously form stabilizing trimers from antigens. However, the potent immunogenicity of these motifs can weaken the immune response of the target proteins, limiting their use in vaccine antigen design. In this study, we explored the use of humanized type III collagen (specifically, a partial sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPn of Rh3, a type III collagen, forming a homotrimer) and model collagen sequences (such as POGn, where O represents hydroxyproline) as innovative trimeric motifs, synthesized through synthetic biology approaches.
The Vp4 antigenic trimer was engineered via synthetic biology techniques by fusing it with the rotavirus Vp4 protein. Since prokaryotes lack proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)), and hydroxylation is essential for collagen stability, we co-expressed P4H derived from mega viruses with the Vp4 proteins described above to achieve stability of Vp4 oligomers.
Furthermore, we utilized to reported artificially designed proline-free heterotrimeric collagen-like protein motifs ((PKG)n, (DKG)n, (EPG)n), three different serological Vp4 proteins (P4, P8, P15) were fused to one of the heterotrimeric collagen-like motifs. The fusion-expressed proteins were purified and mixed in vitro to form oligomers. In this way, a multivalent-oligomeric rotavirus vaccine in E. coli is possible without P4H modification.
Using PET-Dual and PET-24a as vector plasmids, we put PPG, P4H, and Rh3C in PET-Dual, and EPG50 with p8, PKG50 with p4, and DKG50 with p15 were inserted into PET-24a. Based on the homologous recombination technique, we added restriction sites to primers and applied the same restriction enzymes to the plasmids; then, the PCR fragment and linearized vector can be linked via homologous recombinase.
We first constructed the pET-Dual-HisRh3CP15VP4-P4H plasmid, amplified the Rh3C-P15-26-476 and P4H fragments by PCR, and obtained the linear plasmid backbone pET-Dual-N-His-TEV (Dual protein prokaryotic expression plasmid) by double digestion, and then, by homologous recombination, the Subsequently, by homologous recombination, the HisRh3CP15VP4 fragment was inserted between Xba1 and Sal1, and the P4H fragment was inserted between Nde1 and Xho1, and the recombinant plasmid pET-Dual-HisRh3CP15VP4-P4H was finally obtained.
Then, we continued to construct the pET-Dual-HisPPGP15VP4-P4H plasmid, amplified the PPG-P15-26-476 and P4H fragments by PCR, and obtained the linear plasmid backbone pET-Dual-N-His-TEV (Dual-Protein Prokaryotic Expression Plasmid) by double-enzymatic cleavage, and then, using homologous recombination, we inserted the HisRh3CP15VP4 fragment between Xba1 and Sal1, and the P4H fragment between Nde1 and Xho1. Subsequently, by homologous recombination, the HisPPGP15VP4 fragment was inserted between Xba1 and Sal1, and the P4H fragment was inserted between Nde1 and Xho1, and the recombinant plasmid pET-Dual-HisPPGP15VP4-P4H was finally obtained
Moreover, we continued to construct pET-24a-DKG50-P15VP4 plasmid, amplified DKG50-P15-26-476-FLAG fragments by PCR and obtained linear plasmid backbone pET-24a by double digestion, and then, using homologous recombination, we inserted the DKG50-P15VP4 fragments between Xba1 and Xho1 and finally obtained the recombinant plasmid pET-24a-DKG50-P15VP4. Xho1 to obtain the recombinant plasmid pET-24a-DKG50-P15VP4.
Next, we continued to construct pET-24a-PKG50-P4VP4 plasmid, amplified PKG50-P4-26-476-HA fragments by PCR and obtained linear plasmid backbone pET-24a by double digestion, and then, using homologous recombination, we inserted the PKG50-P4VP4 fragments between Xba1 and Xho1 and finally obtained the recombinant plasmid pET-24a-PKG50-P4VP4.
Finally, we constructed pET-24a-EPG50-P8VP4 plasmid, amplified EPG50-P8-26-476-c-Myc fragments by PCR, and obtained linear plasmid backbone pET-24a by double digestion. Then, using homologous recombination, we inserted the EPG50-P8VP4 fragments between Xba1 and Xho1 and finally obtained the recombinant plasmid pET-24a- EPG50-P8VP4.
Figure 1. Electrophoresis for the target genes PKG50, DKG50, PPG, EPG50, and P4H.
Figure 1 shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the target genes. After electrophoresis, the bands that contain the target DNA produced the required DNA fragment by gel extraction. The DNA extracted from the gel was inserted into the plasmid pET24a or pET-Dual via homologous recombination.
Subsequently, the concatenated plasmids A-E were introduced into E. coli DH5α through transformation. In Figure 2A, successful growth of isolated colonies is depicted, with selected colonies undergoing verification. Figure 2B shows that the length of Rh3CP15VP4 is about 2000 bp, and P4H is about 750 bp. After the extraction of the plasmid from the positive colony, the sequencing results in Figure 2C showed that Rh3CP15VP4 and P4H do not contain any gene mutation. It proves that the plasmid pET-Dual-HisRh3CP15VP4-P4H was successfully constructed.
Figure 2. The culture plate for E. coli DH5α(A), verification of colonies(B), and the sequencing results of pET-Dual-HisRh3CP15VP4-P4H(C).
The plasmid pET-Dual-HisPPGP15VP4-P4H was successfully constructed the same way as pET-Dual-HisRh3CP15VP4-P4H via homologous recombination, as shown in Figure 3.
Figure 3. The culture plate for E. coli DH5α (A), verification of colonies (B), and the sequencing results of pET-Dual-HisPPGP15VP4-P4H (C).
The plasmid pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, and pET-24a-EPG50-P8VP4 were successfully constructed using homologous recombination, as shown in Figure 4.
Figure 4. The culture plates for E. coli DH5α (A), verification of colonies (B), and the sequencing results of pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, pET-24a-EPG50-P8VP4 (C).
We first transformed the five plasmids into E.coli BL21 (DE3) to test the protein's expression. Figure 5A shows that the isolated colonies were successfully grown and selected for colony verification. After the E.coli BL21 had been cultured for 12-16h, single colones were selected and then examined via PCR. Figure 5B shows the electrophoresis results after PCR.
Figure 5. The culture plates for E. coli BL21 (A), verification of colonies (B) results of pET-Dual-HisRh3CP15VP4-P4H, pET-Dual-HisPPGP15VP4-P4H, pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, pET-24a-EPG50-P8VP4.
After the colonies (BL21) were verified, they were correctly inoculated into liquid LB medium and cultured until reaching an OD600 of 0.6. Inducer IPTG was then added to induce protein expression. Following overnight cultivation at 16°C, the cells were lysed, and the protein was purified for subsequent analysis via SDS-PAGE and Native-PAGE electrophoresis.
SDS-PAGE gels showing the purification results. Protein purification of the five proteins A-E was examined using nickel column affinity chromatography. As can be seen from the figure, there is a single band at the size of the target proteins. This further indicates that the proteins can be correctly folded and expressed after induction and are solubilized for expression.
Figure 6. The expression of His-Rh3CP15VP4 (A), His-PPGP15VP4 (B), DKG50-P15VP4 (C), PKG50-P4VP4 (D), EPG50-P8VP4 (E) using E. coli BL21. Tips: The sample order from left to right is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.
Collagen-VP4 A, B, C, D, and E were fermented separately in 400 mL of inductive culture medium. The proteins were then purified using a one-step nickel affinity chromatography method, as shown in Figure 6. The yield rates for each collagen were 3.379, 2.557, 2.256, 3.723, and 1.085 mg/L, respectively. Collagen-VP4 A, B, and the C-D-E-VP4 complex (in a 1:1:1 ratio) were diluted to 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure 7, the samples were divided into two distinct clusters: high-molecular-weight and low-molecular-weight states. Each cluster contained multiple bands, indicative of varying degrees of proline hydroxylation.
Figure 7. Native-PAGE electrophoresis showing the protein trimer formed by Rh3CP15VP4(A) and PPGP15VP4(B). The heterotrimeric collagen is composed of DKG50-P15VP4(C), PKG50-P4VP4(D), and EPG50-P8VP4(E).
Based on the construct design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis, as depicted in Figure 8. The peaks for collagen-VP4 A and B ranged from 0.5 to 0.8 CV, with the main peaks at 0.56 CV and 0.66 CV, respectively, suggesting the presence of trimer and monomer macromolecules. The peaks for the C-D-E-VP4 complex also ranged from 0.5 to 0.8 CV, with the main peaks at 0.57 CV, 0.68 CV, 0.72 CV, and 0.77 CV, indicating a trimer at 0.57 CV and the other peaks corresponding to the monomers of C, D, and E. Additionally, we calculated the peak area ratio of trimer to monomer for the main peaks to assess the assembly efficiency of the collagens. The ratios were 1.27 for collagen-VP4 A, 1.24 for collagen-VP4 B, and 0.60 for the C-D-E VP4 complex/monomers.
Figure 8. Size exclusion chromatographic (SEC) analysis of homotrimeric collgen-VP4, Rh3CP15VP4(A), and PPGP15VP4(B), heterotrimeric C-D-E-VP4 complex.Tips: DKG50-P15VP4(C), PKG50-P4VP4(D), and EPG50-P8VP4(E).
Further research is essential for the advancement of a recombinant human rotavirus vaccine. Evaluation of immunogenicity in larger animal models such as pigs and monkeys is imperative, as certain rotavirus antigens that elicit robust protective immunity in small animals may not confer the same effectiveness in larger species. Collaboration with biotechnology firms to conduct animal trials for dose determination is recommended. Additionally, the timeframe for observing vaccine efficacy and the likelihood of adverse reactions post-vaccination are critical considerations. These aspects warrant careful investigation to determine the duration of vaccine protection and the associated risk of vaccine-related illness in the vaccinated population.
Our vaccine now consists of three different serological Vp4 proteins (P4, P8, P15); we can try more different proteins in the future. In the future, we can work with various hospitals to get their data and further improve our vaccines.
We should continue optimizing the vaccine's recombinant protein technology route, exploring more forms of VP4 protein to enhance its protection and determine the vaccine's antigen. Adjuvant and formulation development involves designing adjuvants to enhance immune responses and appropriate vaccine formulations. Animal experiments: The vaccine's safety, immunogenicity, and preliminary efficacy were tested in animal models. This stage aids in pinpointing potential side effects and dose responses.