Outbreaks of CSFV cause significant economic losses in the swine industry. E2 based subunit vaccines have been shown to be a promising strategy for the control and eradication of this disease, as live attenuated vaccines fail to elicit differentiable immunity between infected and vaccinated animals [27, 28]. In contrast to prokaryotic systems, baculovirus infected insect cells produce recombinant proteins with correct 3D structure and glycosylation as the original eukaryotic system, which leads to the proper biological function and immunogenicity in protein products, making them functional vaccine candidates [29]. As the main immunogenic protein inducing neutralizing antibodies against CSFV, E2 protein is widely used for vaccine production [30]. Secretion of E2 protein is preferred for the large-scale protein production and purification. Studies performed here demonstrated an efficient E2 secretion system in baculovirus relying on the newly identified signal peptide SPZJ. Together with the selected E2 antigen, SPZJ-E2ZJ presents improved immunogenicity and protective immunity against CSFV infection.
Currently E2 subunit vaccines produced in insect cells have not been widely available yet due to the limited yield [30]. Many strategies have been applied to improve E2 expression, including codon optimization of E2 gene in yeast [32] and promoter selection, such as a polyhedrin, p10, and a minimal Drosophila melanogaster Hsp70 promoter, for the modulated expression [33]. Besides, efforts have been made to signal peptide engineering. Previously, with the deletion of the transmembrane region [34] and the fusion with a certain signal peptide, such as honeybee melittin signal peptide [25] or immunoglobulin kappa (Igκ) signal peptide [35], E2 was adapted to secretion. In this study, compared with a series of truncated signal peptides, results showed that SP23 was important for E2 efficient secretion (Fig. 1B). SPZJ was identified as the most efficient one for E2 secretion in baculovirus system of candidates tested. Besides E2ZJ, SPZJ increase the expression of other E2 types as compared to their native signal peptides (Fig. 2B). E2 yield with SPZJ has been significantly enhanced in comparison to conventional methods. In protein alignment of multiple signal peptides, results indicated that SPZJ shared 86.9% homology with SPC and 95.6% homology with SPHZ (Fig. 2A). 3 amino acids were involved in sequence variations of signal peptides. It was believed that these amino acids be key points in the guidance of protein secretion.
Antigen immunogenicity is mainly determined by epitopes. In antigenic regions of E2, E2ZJ of group 2.1 presents several unique mutations which are not found in other strains within the same group. These substitutions in identified or predicted epitopes should contribute to the enhancement in the immunogenicity of E2ZJ. E2ZJ displays antigenic features of both genotype I and II CSFV regarding to E2, which may help to improve the cross immunogenicity against different E2 strains. Besides, E2ZJ carries epitopes glycosylation in envelope proteins contributes to the regulation of the immunogenicity as well. E2 glycoprotein protein contains one putative O-linked glycosylation site (O1) and six N-linked glycosylation sites (N1, N2, N3, N4, N5 and N6) [36]. Among them, O1, N1, N2, N3 and N4 are involved in structural unit assembling, which affects the induction of neutralizing antibody [37]. In this study, E2ZJ protein induced the higher level of CSFV specific antibody at the earlier stage than other E2 types tested, confirming the excellent immunogenicity of E2ZJ. The level of glycosylation of E2ZJ detected is between E2C and E2HZ, suggesting the moderate level of glycosylation are favorable for the good immunogenicity of E2 proteins. Although all the identified neutralizing epitopes are conserved among variable strains, including E2ZJ [38], the improved immunogenicity of E2ZJ may result from other neighboring variation related to epitope folding for eases in immune recognition. These findings confirmed that with the good immunogenicity, E2ZJ protein serves as a potential candidate vaccine against CSFV infection.
Rational vaccine dosage is critical to determine the vaccine efficacy. Currently, multiple doses are required for most veterinary subunit vaccines and a large amount of antigen in each dose was used to achieve the sufficient efficacy [39]. Due to the incorrect protein folding or modification as the original viral antigens, subunit vaccines produced in prokaryotic systems usually exploit significantly high effective dose. For example, two doses of E.coli expressed CAP of 200 μg were required for piglets against PCV2 challenge [40]. 300 μg of E2 from yeast was used to induce neutralizing antibodies after booster immunization against CSFV infection [20]. For other E2 types, booster dose at 40 μg of baculovirus expressed genotype 1.1 and 2.1 E2 was recommended to elicit immune responses against CSFV challenge [17]. However, excessive antigen immunization may cause the tolerance against the target antigen in hosts, leading to the inefficient antibody induction. Besides, the risks of side effects will be much lifted with overdose from other unpredicted ingredients in the vaccine formulation, such as toxin from E.coli. Meanwhile, multiple doses at large amounts of antigen will raise the vaccine cost, causing extra economic burden to pig farms. Encouragingly, as shown in the vaccine trials here, a single dose of 5 μg of E2ZJ in piglets is sufficient to elicit complete protection against CSFV lethal challenge. The group of 5 μg-E2ZJ developed sufficient CSFV specific antibody at 21 dpi and neutralizing antibody at 7 dpi. Previous studies indicated that CSFV specific antibody response from single dose of 32 μg of E2 last for 6 to 13 month after immunization in pigs [19, 41]. In this study, the immunogenic durability of single dose of 5 μg of E2ZJ in pigs was evaluated until 28 dpi, and the protective durability was monitored until 44 dpi (16 dpc, days post challenge). No CSFV related clinical symptoms were observed in any E2ZJ vaccinated piglets upon CSFV challenge, indicating the sufficient durability of the efficacy of the single dose E2ZJ. Besides, neutralization antibody against both homologous and heterologous strains were detected in all E2ZJ vaccinated animals, confirming the cross protective immunogenicity of E2ZJ. Meantime, for another E2 vaccine candidate (non-ZJE2), which was expressed in non-baculovirus system, one dose of 60 μg failed to induce comparable antibody response as E2ZJ, confirming the advantage of E2ZJ in immunogenicity and vaccine efficacy. Therefore, the single dose of 5 μg of E2ZJ in pigs will be recommended as CSFV vaccine for the complete protection against the disease, which will significantly reduce the cost in vaccination and enhance the vaccine quality.
In summary, the study presented a novel efficient E2 signal peptide for E2 secretion and the lowest effective dose of E2 reported against CSFV lethal challenge in pigs. A single dose of 5 μg of E2ZJ developed a complete protective immune response in pigs and conferred broad protection against the homologous and heterologous CSFV strains. This replies on robust SPZJ-E2ZJ secretion and efficient immunogenicity against CSFV. Hence, serving as a promising vaccine platform, baculovirus expressed SPZJ-E2ZJ is an economical and effective vaccine candidate. It will also be a useful tool for CSF eradication in China, together with other strategies, including differential diagnosis, regional or national-wide eradication campaigns, serological surveillance, and biosecurity procedures.