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.