3.2 Expression and purification of recombinant NTD of G1 and G2 PEDV S protein
The recombinant plasmids, pET-G1-NTD and pET-G2-NTD were transferred into E. coli BL21 (DE3) host. Recombinant G1-NTD and G2-NTD were expressed with a six histidine tag on the C-terminus, yielding fusion proteins of 383 and 386 aa, which gave bands corresponding to the expected molecular masses of approximately 42.60 kDa and 42.61 kDa, respectively. Both the recombinant G1-NTD and G2-NTD were expressed mostly as soluble forms in the cytosolic fraction of E. coli(Figure 2). The recombinant G1-NTD and G2-NTD proteins in the cleared cell lysate can be purified by using the HisPur Ni-NTA Spin Purification Kit (Thermo Fisher Scientific, MA, USA) according to the instructions of manufacture.
Evaluation of cross-reactivity of antibodies against G1 and G2 PEDV S proteins by Western blot
As shown in Figure 3, under same conditions, the G1-NTD antisera reacted to the G1 NTD protein very well, but hardly to the G2-NTD protein; however, the G2-NTD antisera reacted to both G1 and G2 NTD protein, with to G2 NTD protein better. The results indicated that the NTDs of G1 and G2 PEDV S displayed different antigenicity.
Evaluation of cross-reactivityof antibodies against G1 and G2 PEDV S proteins by ELISA
The ELISA results also indicated significant different cross-reactivity of antibodies against G1 and G2 PEDV S proteins. As shown in Figure 4A and 4B, the OD450 values of anti-G1-NTD antibody reacting with the G1-NTD was about six fold of those reacting with the G2-NTD. The OD450 values of anti-G2-NTD antibody reacting with the G2-NTD was about 2-fold of those reacting with the G1-NTD.
Evaluation of cross-reactivity of antibodies against G1 and G2 PEDV strain by IFA
The cross-reactivity of G1- and G2-NTD antisera against G1 and G2 PEDV strain was evaluated by use of IFA. The IFA results confirmed that the G1- and G2-NTD antisera cross-reacted differently with native S proteins of G1 strain CV777 and G2 PEDV strain CH/ZMDZY/11. The G1-NTD antisera reacted strongly with CV777-infected cells (Figure 5B), but weakly with CH/ZMDZY/11-infected cells (Figure 5F). Conversely, the G2-NTD antisera reacted very strongly with CH/ZMDZY/11-infected cells (Figure 5H), and moderately with CV777-infected cells (Figure 5D). Vero cells infected with G1 and G2 PEDV strains showed distinct morphology of cytopathic effects (CPE). G2 PEDV strain CH/ZMDZY/11-infected cells had higher cell fusion activity than G1 strain CV777. At the same postinoculation time, the CPE and syncytia induced by PEDV CH/ZMDZY/11 were much larger than those induced by CV777 vaccine strain. Positive signals of G2 PEDV strain CH/ZMDZY/11 S protein antigens were mainly detected as diffuse fluorescence in the center of the syncytia by G1-NTD antisera but as bright and concentrated fluorescence within the cytoplasm of syncytia by G2-NTD antisera.
Evaluation of cross-neutralization of antibodies against G1 and G2 PEDV strain by focus reduction neutralization (FRNT) assay
As the CPEs induced by G1 and G2 PEDV strain were different, the G1- and G2-NTD antisera were tested for their cross-neutralizing activity against the G1 strain CV777 and G2 strains CH/ZMDZY/11 by a FRNT assay rather than conventional plaque reduction neutralization assay. The PEDV-specific foci were identified by using immuno-peroxidase monolayer assay (IPMA). As shown in Figure 6, the single focus in CV777-infected cells and syncytia focus formed in CH/ZMDZY/11-infected cells could be identified clearly.
As shown in Figure 7A and 7B, infection of the G1 strain CV777 and G2 strain CH/ZMDZY/11 to Vero cells was effectively inhibited by the G1- and G2-NTD antisera respectively. The G1-NTD antisera was highly effective in inhibiting G1 strain CV777 infection in Vero cells with mean NA titers of 172. The mean NA titer of G2-NTD antisera against G2 strain CH/ZMDZY/11 reached 68. Both the mean NA titer of G1-NTD antisera against G2 strain CH/ZMDZY/11 and that of G2-NTD antisera against G1 strain CV777 were only 8 (Figure 8). The mean titer of G1-NTD antisera neutralizing G1 strain CV777 was significantly higher than that of cross-neutralizing G2 strain CH/ZMDZY/11 (p <0.01). The mean titer of G2-NTD antisera neutralizing G2 strain CH/ZMDZY/11 was also significantly higher than that of cross-neutralizing G1 strain CV777 (p <0.01)
DISCUSSION
Outbreaks of novel genotype 2 PEDV have resulted in significant economic losses to the swine industry in Asia and North America. All reported PEDV strains have been clarified into two genotypes, G1 and G2, based on the phylogenetic analysis of the S gene. The S protein plays important roles in infection and inducing virus neutralizing antibodies (Li et al., 2020). The G2 PEDV comprises the post-2010 global epidemic isolates including mutations mainly in the N terminal domain of S1 (S1-NTD) (Fan et al., 2017, Zhang et al., 2021. These mutations affect the conformational structure and N-linked glycosylation of S1-NTD, which may alter the pathogenicity and antigenicity of the G2 PEDV (Chen et al., 2019, Suzuki et al., 2018). Vaccination is an effective strategy for control and prevention of PED during epidemic or endemic outbreaks. Despite the administration of killed or attenuated vaccines based on G1 PEDV strain CV777, the G2 PEDV outbreaks in vaccinated herds in China still increased dramatically since 2010 (Li et al., 2012). A recent study evaluated the cross-protection between G1a- and G2a-genotype PEDVs in suckling piglets (Zhang et al., 2020). They demonstrated that all piglets from the sows immunized with CH/JX/01 (G2) could not only survive when challenged with the homologous PEDV, but also be fully protected when challenged with heterogenous G1 PEDV. However, the piglets from the sows immunized with CV777 (G1) could be protected when challenged with homologous PEDV and only partially protected when challenged with heterologous G2 strain of PEDV (CH/JX/01). This implied that immunization failure of G1 PEDV strain-based vaccines could be due to the mutations in the S gene contributing to the antigenicity difference between G1 and G2 PEDV strains. Identification of the role of the NTD of S protein in antigenicity difference between G1 and G2 PEDV strains will lead to the development of an effective PEDV vaccine with better protection against prevalent genotype of PEDV.
Most of previous S protein functional studies have focused on the core neutralizing epitope (COE), S1, full-length S and S2 proteins (Wang et al., 2016, Oh et al., 2014, Makadiya et al., 2016). To the best of our knowledge, our present study is the first work focused on the contribution of NTD (aa 1-380) of S protein to the antigenicity difference between G1 and G2 PEDV strains. In the present study, A G1 PEDV CV777 vaccine strain and G2 strain CH/ZMDZY/11 isolated from a CV777-immunized herd (Wang et al., 2013). Comparison of antigenic index profiles of the S proteins PEDV G1 strain CV777 and G2 strain CH/ZMDZY/11 indicated the most dissimilar regions were in NTD of the S protein, and there were two regions in NTD exhibited higher degree of antigenic change than other regions. The aa mutations generated two new N-linked glycosylation sites (NST and NAT) in G2 PEDV strain CH/ZMDZY/11. This is consistent with previous comparison of antigenic index profiles of the NTD of S protein between the prototype strain CV777 and a US strain (Huang et al., 2013). We assumed that vaccines based on the historical CV777 strain became antigenically less related to G2 PEDV variant strains emerged post-2010 in China due to the mutations in the NTD of S protein.
To confirmed this hypothesis, the NTDs of S genes of the G1 PEDV strain CV777 and a G2 PEDV strain CH/ZMDZY/11 were expressed in E. coli. , respectively. Cross-reactivity of G1- and G2-NTD antisera against G1 and G2 PEDV strains was evaluated using Western blot, ELISA, IFA, and SN. The Western blot and ELISA results confirmed the antigenicity of the G1- and G2-NTD proteins and the significant difference in cross-reactivity between the NTD proteins of two PEDV genotypes. The IFA results indicated different cross-reactivity of G1- and G2-NTD antisera against native S proteins of PEDV. Notably, difference in morphology of cytopathic effects (CPE) was observed in G1 strain CV777 and G2 strain CH/ZMDZY/11 infected cells in IFA. CH/ZMDZY/11-infected cells developed CPE characterized by cell fusion and syncytium. However, CV777-infected cells developed CPE characterized by individual round cell with enlarged nuclei. This implied that mutations in the NTD may alter the pathogenicity of PEDV.
Regarding the effects of amino acid mutations in the NTD on the neutralizing activity of anti-NTD polyclonal antibodies, G1- and G2-NTD antisera also displayed significant differences in SN titers against G1 and G2 PEDV strains. The SN assay indicated antigenic differences of 8- to 20-fold between the two PEDV genotypes. The NA titer of the G1-NTD antisera against G1 CV777 vaccine strain was more than 20-fold higher than that of against G2 CH/ZMDZY/11 strain. Interestingly, the NA titer of G2-NTD antisera against G2 strain CH/ZMDZY/11 was only 8-fold higher than that of against G1 strain CV777. This could be due to the two new N-linked glycosylation sites in the NTD of CH/ZMDZY/11 strain S protein. It is believed that glycosylation sites on viral surface glycoproteins play a role in glycan shielding which is a primary mechanism by which the virus evades neutralizing immune responses (Wei et al.,2012). Alteration of a glycosylation site can have dramatic consequences for a virus. It has been demonstrated that alteration of a glycosylation site can impact protein folding and conformation (Hebert et al., 1997) and affect distant parts of a protein through masking or conformational changes. This could result in tighter packing of glycoprotein regions involved in neutralization epitopes, reduce accessibility, and so also facilitate immune escape (Ye et al., 2000).
A previous study showed antigenic differences of twofold between the two PEDV genotypes, in which the immunogenicity and antigenic relationships among full-length S proteins of G1 strain CV777 and G2 strain LNCT2 was investigated (Wang et al.,2016). Neutralizing epitopes reside in other domains of S protein might lower the differences between the two PEDV genotypes. Our results suggested that there were neutralizing epitopes in the NTD (aa 1-380) of the S protein and new neutralizing epitopes had emerged in the post-2010 G2 PEDV variant strains. This result is consistent with a previous study in which potent neutralization was observed with antibodies targeting the NTD (1-220aa) (Li et al., 2017). Sialic acid (Sia) binding activity has been mapped to the amino-terminal 246 residues of the PEDV S protein (Deng et al., 2016, Li et al., 2016, Liu et al., 2015). This suggested that cell attachment domains of PEDV could reside in the NTD of S protein. Cell attachment domains of the S protein are key targets of neutralizing antibodies. Potent neutralization against G1 and G2 PEDV straachieved by antibodies targeting the NTD further underline the importance of NTD of S protein as major targets of potent neutralizing antibodies.
Taken together, our data indicated that the NTD of S protein contributes to the antigenicity difference between G1 and G2 PEDV strains, and the existence of neutralizing epitopes in the NTD of S protein. An ideal candidate strain used for PEDV vaccine is expected to have the ability to confer excellent protection against different field strains of PEDV. Results in the present study also highlights the necessity of choosing currently circulating G2 PEDV for the development of novel PEDV vaccine candidates with improved efficacy.