Effects of fusion adjuvants to enhance immunogenicity of porcine epidemic diarrhea virus subunit vaccine

Abstract

Porcine epidemic diarrhea (PED) is one of the most catastrophic swine diseases characterized by severe diarrhea, vomiting, dehydration and even death. The mortality rate is as high as 80-100 % especially in neonatal piglets, which has potential to cause in tremendous economic burden to swine farms. Since, 1992, the PED outbreaks have been occurring every year in the South Korea. This outbreak became an endemic, which resulted substantial economic losses to Korean domestic swine industry. Since, early 2000, PED prevention program using inactivated and modified attenuated vaccines has been adopted, which derived a decline of PED virus (PEDV) associated diarrheal disease outbreak. However, the highest mortality of pigs from PEDV infection was recorded in the year 2018, demanding a development of an effective PED vaccine. The most recently used PED vaccines are live attenuated vaccine (LAV) or inactivated form, which carries issues related to its safety and cost-effectiveness. Especially, the effectiveness of some vaccines has become controversial after an outbreak of PED in the United States, which consequently stressed the demand for the development of a novel PED vaccine. Subunit vaccines have many advantages over traditional LAVs, including safety, simplicity and mass production through Escherichia. coli (E. coli) system. In particular, the cost-effectiveness of subunit vaccines are essential interests in association with the economic objective of the livestock industry. However, antigen candidates of PED subunit vaccine, such as S0, have been expressed as insoluble aggregates in E. coli, which are not suitable to be used as a vaccine. In order to overcome the low solubility and immunogenicity of the PED subunit vaccine, introduction of a molecular fusion adjuvant was necessary. In the study I, the bacterial flagellin, Vibrio vulnificus FlaB, was introduced to S0, a truncated region of PEDV spike protein, and its conjugation effect on the enhancement of solubility and immunogenicity was examined. From previous researches, the conjugation effect of flagellin has been known to be varied depending on the target antigen or the direction of its conjugation, therefore flagellin was conjugated to the C- or N-terminal of S0 in order to generate S0-F and F-S0, respectively. The flagellin conjugation to C-terminus of S0 displayed improved the solubility around 59 % in E. coli expression system compared to generic condition. In particular, flagellin conjugation improved the solubility of recombinant proteins up to 99 %, regardless of its fusion direction in the presence of trigger factor chaperone tig. Particularly, flagellin conjugated to the N-terminus of S0 (F-S0) induced significantly higher S0-specific antibodies in serum compared to other recombinant antigens including the flagellin conjugated to the C-terminus of S0 (S0-F) in mice. Therefore, it is evident that the superior ability of S0-F to activate the NLRC4/NAIP5 pathway may interfere with flagellins adjuvancy and reduce serum S0-specific IgG despite of its advantage in a conjugated form. In the study II, the conjugation effect of C4d as well as C3d on the solubility and immunogenicity was investigated. The complement fragment C4d was selected as a novel fusion adjuvant candidate, since it has high frequency of T cell epitope in its intra-structure evaluated through in silico study. C3d is a split product derived from the complement C3 protein. It has been proved that the human C3d has unusually high frequency of T cell epitopes. C3ds are responsible for many heterogeneous MHC class II molecules in human, and there are circulating memory type of autoreactive helper T cells that recognize these peptide:MHC class II complexes. These characteristics leads C3d to be utilized as a potent fusion adjuvant by donating T cell epitopes to antigen up-taking cells when it is conjugated with target antigen. To explore another novel adjuvant, which act as C3d, various serum protein candidates were evaluated through in silico study to identify T cell epitope frequencies. Among serum protein candidates, C4d, a split fragment derived from C4, was selected as a novel fusion adjuvant. Following the selection, C3d or C4d was conjugated in tandem repeats with S0, to generate S0-mC3d1, S0-mC3d2 (two tandem repeats), S0-mC3d3 (three tandem repeats), S0-mC4d1, and S0-mC4d2. The conjugation of C4d to S0 improved the solubility of the recombinant proteins regardless of the existence of chaperone tig, while the conjugation of C3d had no effect on the solubility enhancement. Thus, both C3d and C4d conjugated to S0 (S0-mC3d1, S0-mC3d2, S0-mC3d3, S0-mC4d1, and S0-mC4d2) induced significantly higher S0-specific antibodies in serum compared to S0 alone in mice. In addition, the involvement of mC4d-specific autoreactive T cell in the context the adjuvant effect of mC4d has been also confirmed. In contrast, C3d or C4d alone also elicited an adjuvant effect. In study III, the recombinant protein candidates from the study I and II were compared at the same time to select the best suitable PED subunit vaccine protein in terms of immunogenicity. F-S0, S0-mC3d2 and S0-mC4d2 were selected as fusion adjuvant candidates considering their high immunogenicity. mC3d and mC4d was used as non-conjugated mixture control and also selected as adjuvant candidates as they have greater yield compare to other fusion proteins. The adjuvanticity after intramuscular administration, all of the recombinant protein candidates induced high level of S0-specific antibody titers in serum compare to control group. From the production yield data, mC3d and mC4d were finally selected as the best immunogenic adjuvant for systemic PED subunit vaccine. Meanwhile, S0-mC4d2 displayed the highest S0-specific IgA titers at various mucosal areas compare to the other recombinant proteins after intranasal administration. Therefore, from the mucosal IgA data results, S0-mC4d2 was finally selected as the best candidate for the mucosal PED subunit vaccine. Meanwhile, following intranasal administration, mC3d or mC4d had no effect on humoral immune response contrast to intramuscular administration. Therefore, T cell epitope donation due to the conjugation of mC4d with target antigen S0 could be responsible for the enhancement of IgA in mucosal fluids, which demonstrates that mC4d-specific T cell was inferred to be involved in the adjuvant effect of mC4d conjugation. In this study, the best recombinant proteins for PED subunit vaccine has been selected. In particular, the novel complement fragment adjuvant C4d could be applied to not only to PEDV antigen, but also to various antigens for other vaccines. These findings provide critical insights for the development of a novel PED vaccine and the immunotherapeutics based on flagellin or complement fragments.