African swine fever virus (ASFV) is a large double-stranded DNA virus belonging to the
The disease caused by this virus, African swine fever, is associated with many symptoms, including high fever, lesions, and haemorrhagic skin discolouration in the host organism, or rarely is not associated with any signs (29). This peracute infection usually occurs in animals that die even before the immune system can react to the infection (25). For this reason, several vaccines have been developed, but none has been effective enough to protect pigs against a virulent strain of the virus.
Our present study focuses on the ASFV p22 protein, which may become a subunit vaccine candidate. Typically, subunit vaccines contain proteins originating from the virus infecting the host organism. Many structural antigens from ASFV have been tested as subunit vaccines, including p22, p30, p54, p72, CD2v and other proteins (20, 21, 24). Of these proteins, p22, p30 and CD2v were capable of inducing at least a minimal immune response (4, 12, 22). Although none of the antigens successfully protected the host against a virulent virus challenge, they could still be considered potential antigens for future vaccine development.
The p22 protein was initially believed to be part of the outer membrane (5). However, recently it has been found in the inner membrane (1). An initial experiment using a non-ionic detergent showed that the p22 protein is part of the outer envelope; however, as was learned later, the detergent could disrupt not only the outer but also the capsid envelope (2, 5). The structure of the p22 antigen consists of three subparts: a 20-amino-acid-long (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) N-terminal domain, a 21-amino-acid (21…41) transmembrane α-helix, and an outer 148-amino-acid (42…189) C-terminal part. Recently, it has been found that the p22 protein interacts with host proteins related to several biological processes, including virus binding, signal transduction, and cell adhesion (30). Some of these proteins participate in ribosome, spliceosome, and actin filament organisation and movement, possibly also in DNA replication, or affect phagocytosis and endocytosis (30). Even though the p22 protein can interact with many host proteins, it was shown recently not to be essential for ASFV replication or virulence (27).
The gene encoding the p22 protein was initially discovered on the left end of the BA71 genome and was named then based on the Kʹ177 open reading frame (ORF) (5, 11), but it was later renamed KP177R and termed the early membrane protein. The KP177R encoding gene was also found on the right end of the Malawi LIL120/1 genome (28). Vydelingum
As the p22 protein takes part in virus binding (30), it deserves further attention for its possible recognition by the immune system. We therefore decided to prepare a recombinant protein based on the outer C-terminal part of p22 and examine its immunogenicity in mice.
A large-scale expression was performed to produce higher protein quantities for purification and further experiments. An aliquot of 20 mL of the culture was inoculated into 1 L of the LB medium and allowed to grow at 37°C with shaking until the optical density (OD600) reached 0.7–0.8. Recombinant protein production was induced by adding IPTG at a final concentration of 0.2 mM. The culture was grown at 28°C for 16 h and then collected by centrifugation (8,000 ×
Nickel-nitrilotriacetic acid (Ni-NTA) Agarose (Qiagen, Hilden, Germany) was pre-equilibrated with 25 mM Tris-HCl pH 9.0, containing 300 mM NaCl, 2% glycerol, 0.1% Triton X-100, and 15 mM imidazole. The soluble fraction was loaded twice onto the matrix to increase the binding of the desired proteins. Unbound proteins were washed out with the same buffer, and further elution was performed with buffers containing 30, 40, 50 mM, and finally 300 mM imidazole. The eluted protein samples were concentrated and buffer-exchanged for the buffer without imidazole using Amicon 10,000 Mw centrifugal filters (Thermo Fisher Scientific). Subsequently, the samples were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The purified proteins were stored at −80°C. Protein concentrations were determined by Bradford reagent (Bio-Rad, Hercules, CA, USA) and band density on SDS-PAGE gels using ImageLab software (Bio-Rad) with bovine serum albumin (BSA) as the standard.
For Western blot analysis, the electrophoresed proteins were transferred to a polyvinylidene difluoride membrane using a wet transfer apparatus (Bio-Rad) for 16 h at a constant voltage of 20 V. The membrane was rinsed twice with 25 mM Tris-HCl buffer at pH 8.0 containing 0.9% NaCl (Tris-buffered saline – TBS), blocked with 5% non-fat milk in TBS containing 0.05% Tween 20 for 90 min, and then incubated with a 1:500 diluted His-tag primary antibody (MA1-21315; Thermo Fisher Scientific) for 90 min. Immunodetection was performed for 90 min with a 1 : 4000 diluted murine immunoglobulin G kappa -anti-mouse secondary antibody conjugated to horseradish peroxidase (sc-516102; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an enhanced chemiluminescent substrate (ECL kit; Bio-Rad). As a molecular mass marker, the Novex Sharp Pre-Stained Protein Standard (Thermo Fisher Scientific) was used.
The p22 protein of ASFV contains a short N-terminal domain, a single membrane-spanning helix and an outer large C-terminal domain. Using the Phyre2 protein fold recognition server (15), we predicted by comparing the amino acid sequence of the C-terminal domain of p22 with other known proteins from the database that the separate domain should fold into a stable tertiary structure (Fig. 1).
Two DNA constructs were prepared,
First, a small-scale expression in 5 mL cultures was performed. Gel electrophoresis showed that the expression level of the LTB-p22Ct fusion protein was much lower than that of the p22Ct protein (Fig. 3), while still being detectable.
Secondly, a large-scale expression was performed under the same conditions in 1 L cultures (Fig. 3). The larger-scale expression yields of p22Ct and LTB-p22Ct in 1 L cultures, estimated with BSA as a standard, were 4.9 and 3.8 mg per L of culture.
In the subsequent immunoblot testing of purified p22Ct and LTB-p22Ct recombinant proteins for their ability to interact with serum antibodies (IgG) from ASFV infected pigs, both proteins showed positive reactions with the four sera used as primary antibodies (Fig. 5).
When the serological activity of the two groups’ samples against the recombinant proteins was assayed, the antibody titre was high for the mice group immunised with p22Ct, while the samples from the LTB-p22Ct group did not react at all in ELISA (Fig. 6). The mice allocated to the p22Ct protein group were fully immunised with three doses. However, in the case of the LTB-p22Ct group, abscesses occurred after the first dose. Due to this development, only one dose was administered.
Due to the difference in immune response induction in the mice, the oligomerisation of both recombinant proteins was examined by SDS-PAGE under non-reducing conditions (Fig. 7). As calculated by comparing the mobility of recombinant proteins with those of protein molecular mass standards, the 33 kDa LTB-p22Ct fusion most probably forms hexameric complexes of 198 kDa. In contrast, the p22Ct protein prevalently retains the monomeric form of 23 kDa.
The global swine pandemic caused by the African swine fever virus has a negative impact on the economy of many countries throughout the world; therefore, the virus needs to be eliminated. Many researchers worldwide have tried to design a new effective vaccine for combating the disease caused by this virus; however, they have not been successful in protecting the pigs against virulent viral strains. A previous study showed that administration of ASFV proteins from the inner and capsid membranes could somewhat delay the onset of clinical symptoms from the time of viral challenge, but could not stop the further progress of the disease; the pigs administered the proteins eventually died. The four proteins investigated (p22, p30, p54 and p72) were expressed in a baculovirus system. The individual proteins were not purified to homogeneity but used as a mixture to immunise the pigs (20). The p22 protein chosen for our study was previously considered a potential antigen for new vaccine development (4, 22). A study using DNA prime and recombinant vaccinia virus boost (12) showed that p22 (the KP177R early membrane protein) is a potential antigen for inducing a protective immune response or can serve as an infection serological marker. However, the role of p22 in the infection process is still unknown. Recently the focus on developing an effective vaccine turned to live attenuated strains developed using a genetically modified virulent parental virus (3).
In contrast to previous studies, we selected only the large C-terminal globular part of the protein, p22Ct (amino acids 42 to 189). We also prepared its fusion with the heat-labile enterotoxin B-subunit, LTB-p22Ct, which was expected to stimulate the immune response in the host (9, 13).
After cloning the corresponding constructs into the
Our results show that p22Ct, the C-terminal globular part of the p22 protein, but not the fusion protein LTB-p22Ct, can induce an immune response in mice. The exact cause of the observed difference is unclear. The most probable reason is that mice immunised with LTB-p22Ct were injected with only one dose rather than the three doses of p22Ct administered. A significant problem appeared in the form of abscess formation in the mice vaccinated with the fusion protein; therefore, these mice received only one dose of antigen. This is probably also the reason why the mice immunised with LTB-p22Ct could not produce specific antibodies against the protein. Another reason may be that LTB could become less active when produced by recombinant technology in
Our results also show that p22Ct can produce a high antibody titre in mice, and is thereby indicated to be a highly potent and immunogenic region. It can be considered a candidate primarily in serological diagnostics for the development of specific antigen-based detection techniques such as fluorescent antibody tests, ELISA and immunoblotting (23). However, further experiments should be conducted to investigate its possible link to other immunostimulatory domains.
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