African Swine Fever Virus Protein Research Articles

A new B cell epitope of pC129R protein of African swine fever virus identified by monoclonal antibodies

African swine fever virus (ASFV) is a most important pathogen which causes huge damage in swine production in the world. pC129R protein is one of the most abundant ASFV proteins in infected Vero cells and WSL-HP cells, which consequently could be a target for ASF detection and surveillance. In this study, 5–6-week-old female BALB/c mice were immunized with rpC129R protein expressed by a prokaryotic system. And three hybridomas, 1B1, 1B4 and 4H4, steadily secreted anti-pC129R monoclonal antibodies were screened by an indirect enzyme linked immunosorbent assay (ELISA). Among them, 1B4 and 4H4 had IgG2a isotype with Kappa light chain, while 1B1 had IgG1 isotype with Kappa light chain. Western blot and indirect immunofluorescence assay showed that three monoclonal antibodies (mAbs) specifically reacted with ASFV. Epitope mapping was performed with truncated polypeptides. And a new B cell epitope, 18KHYVLIPK25 was identified by the mAbs, which was highly conserved in most genotypes of ASFV. These findings not only provide a monoclonal antibody tool for further study of the function of C129R, but also lay the foundation for serological diagnosis and vaccine development.

African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. Invivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

ASFV proteins presented at the surface of T7 phages induce strong antibody responses in mice

African swine fever virus (ASFV) infection causes substantial economic losses to the swine industry worldwide, and there are still no safe and effective vaccines or therapeutics available. The granulated virus antigen improves the antigen present process and elicits high antibody reaction than the subunit antigen. In this study, the SpyTag peptide-p10 fusion protein was altered and displayed on the surface of the T7 phage to construct an engineered phage (T7-ST). At the same time, ASFV antigen-Spycatcher C-terminal-fused protein (antigen-SC) was expressed and purified by an E. coli prokaryotic expression system. Five virus-like particles (VLPs) displaying the main ASFV antigenic proteins P30, P54, P72, CD2v, and K145R were reconstructed by the isopeptide bond between SpyTag and antigen-SC proteins. The stability of five ASFV VLPs in high temperature and extreme pH conditions was evaluated by transmission electron microscopy (TEM) and plaque analysis. All ASFV VLPs induced a high titer antigen-specific antibody response in mice. Our results showed that the granulated antigen displaying ASFV protein on the surface of the T7 phage provides a robust potential vaccine and diagnostic tool to address the challenge of the ASFV pandemic.

ASFV transcription reporter screening system identifies ailanthone as a broad antiviral compound

African swine fever (ASF) is an acute, highly contagious and deadly viral disease in swine that jeopardizes the worldwide pig industry. Unfortunately, there are no authoritative vaccine and antiviral drug available for ASF control. African swine fever virus (ASFV) is the etiological agent of ASF. Among the ASFV proteins, p72 is the most abundant component in the virions and thus a potential target for anti-ASFV drug design. Here, we constructed a luciferase reporter system driven by the promoter of p72, which is transcribed by the co-transfected ASFV RNA polymerase complex. Using this system, we screened over 3200 natural product compounds and obtained three potent candidates against ASFV. We further evaluated the anti-ASFV effects and proved that among the three candidates, ailanthone (AIL) inhibits the replication of ASFV at the nanomolar concentration (IC50 ​= ​15 ​nmol/L). Our in vitro experiments indicated that the antiviral effect of AIL is associated with its inhibition of the HSP90-p23 cochaperone. Finally, we showed the antiviral activity of AIL on Zika virus and hepatitis B virus (HBV), which supports that AIL is a potential broad-spectrum antiviral agent.

Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies

African swine fever virus causes an acute, highly contagious swine disease with high mortality, leading to enormous losses in the pig industry. The K205R, a nonstructural protein of African swine fever virus, is abundantly expressed in the cytoplasm of infected cells at the early stage of infection and induces a strong immune response. However, to date, the antigenic epitopes of this immunodeterminant have not been characterized. In the present study, the K205R protein was expressed in a mammalian cell line and purified using Ni-affinity chromatography. Furthermore, three monoclonal antibodies (mAbs; 5D6, 7A8, and 7H10) against K205R were generated. Indirect immunofluorescence assay and western blot results showed that all three mAbs recognized native and denatured K205R in African swine fever virus (ASFV)-infected cells. To identify the epitopes of the mAbs, a series of overlapping short peptides were designed and expressed as fusion proteins with maltose-binding protein. Subsequently, the peptide fusion proteins were probed with monoclonal antibodies using western blot and enzyme-linked immunosorbent assay. The three target epitopes were fine-mapped; the core sequences of recognized by the mAbs 5D6, 7A8, and 7H10 were identified as 157FLTPEIQAILDE168, 154REKFLTP160, and 136PTNAMFFTRSEWA148, respectively. Probing with sera from ASFV-infected pigs in a dot blot assay demonstrated that epitope 7H10 was the immunodominant epitope of K205R. Sequence alignment showed that all epitopes were conserved across ASFV strains and genotypes. To our knowledge, this is the first study to characterize the epitopes of the antigenic K205R protein of ASFV. These findings may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

African Swine Fever Virus MGF505-7R Interacts with Interferon Regulatory Factor 9 to Evade the Type I Interferon Signaling Pathway and Promote Viral Replication.

African swine fever (ASF) is an acute and severe infectious disease caused by the ASF virus (ASFV). The mortality rate of ASF in pigs can reach 100%, causing huge economic losses to the pig industry. Here, we found that ASFV protein MGF505-7R inhibited the beta interferon (IFN-β)-mediated Janus-activated kinase-signal transducer and activation of transcription (JAK-STAT) signaling. Our results demonstrate that MGF505-7R inhibited interferon-stimulated gene factor 3 (ISGF3)-mediated IFN-stimulated response element (ISRE) promoter activity. Importantly, we observed that MGF505-7R inhibits ISGF3 heterotrimer formation by interacting with interferon regulatory factor 9 (IRF9) and inhibits the nuclear translocation of ISGF3. Moreover, to demonstrate the role of MGF505-7R in IFN-I signal transduction during ASFV infection, we constructed and evaluated ASFV-ΔMGF505-7R recombinant viruses. ASFV-ΔMGF505-7R restored STAT2 and STAT1 phosphorylation, alleviated the inhibition of ISGF3 nuclear translocation, and showed increased susceptibility to IFN-β, unlike the parental GZ201801 strain. In conclusion, our study shows that ASFV protein MGF505-7R plays a key role in evading IFN-I-mediated innate immunity, revealing a new mode of evasion for ASFV. IMPORTANCE ASF, caused by ASFV, is currently prevalent in Eurasia, with mortality rates reaching 100% in pigs. At present, there are no safe or effective vaccines against ASFV. In this study, we found that the ASFV protein MGF505-7R hinders IFN-β signaling by interacting with IRF9 and inhibiting the formation of ISGF3 heterotrimers. Of note, we demonstrated that MGF505-7R plays a role in the immune evasion of ASFV in infected hosts and that recombinant viruses alleviated the effect on type I IFN (IFN-I) signaling and exhibited increased susceptibility to IFN-β. This study provides a theoretical basis for developing vaccines against ASFV using strains with MGF505-7R gene deletions.

Structure and function of African swine fever virus proteins: Current understanding.

African swine fever virus (ASFV) is a highly infectious and lethal double-stranded DNA virus that is responsible for African swine fever (ASF). ASFV was first reported in Kenya in 1921. Subsequently, ASFV has spread to countries in Western Europe, Latin America, and Eastern Europe, as well as to China in 2018. ASFV epidemics have caused serious pig industry losses around the world. Since the 1960s, much effort has been devoted to the development of an effective ASF vaccine, including the production of inactivated vaccines, attenuated live vaccines, and subunit vaccines. Progress has been made, but unfortunately, no ASF vaccine has prevented epidemic spread of the virus in pig farms. The complex ASFV structure, comprising a variety of structural and non-structural proteins, has made the development of ASF vaccines difficult. Therefore, it is necessary to fully explore the structure and function of ASFV proteins in order to develop an effective ASF vaccine. In this review, we summarize what is known about the structure and function of ASFV proteins, including the most recently published findings.

Identification of p72 epitopes of African swine fever virus and preliminary application.

African swine fever virus (ASFV) causes a highly lethal hemorrhagic viral disease (ASF) of pigs that results in serious losses in China and elsewhere. The development of a vaccine and diagnosis technology for ASFV is essential to prevent and control the spread of ASF. The p72 protein of ASFV is highly immunogenic and reactive, and is a dominant antigen in ASF vaccine and diagnostic research. In this study, 17 p72 monoclonal antibodies (mAbs) were generated. Epitope mapping by a series of overlapping peptides expressed in Escherichia coli showed that these mAbs recognized a total of seven (1-7) linear B cell epitopes. These mAbs did not show significant neutralizing activity. Epitopes 1 (249HKPHQSKPIL258), 2 (69PVGFEYENKV77), 5 (195VNGNSLDEYSS205), and 7 (223GYKHLVGQEV233) are novel. Sequence alignment analysis revealed that the identified epitopes were highly conserved among 27 ASFV strains from nine genotypes. Preliminary screening using known positive and negative sera indicated the diagnostic potential of mAb-2B8D7. The results provide new insights into the antigenic regions of ASFV p72 and will inform the diagnosis of ASFV.

Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies.

African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The ASF Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones. The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools. ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest. The expression of recombinant p30 protein was assessed using direct immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively. In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays.

Complete Structural Predictions of the Proteome of African Swine Fever Virus Strain Georgia 2007.

Here, we announce the predicted structures of the 193 proteins encoded by African swine fever virus (ASFV) strain Georgia 2007 (ASFV-G). Previously, only the structures of 16 ASFV proteins were elucidated.

Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

African swine fever virus (ASFV), a DNA double-stranded virus with high infectivity and mortality, causing a devastating blow to the pig industry and the world economy. The CD2v protein is an essential immunoprotective protein of ASFV. In this study, we expressed the extracellular region of the CD2v protein in the 293F expression system to achieve proper glycosylation. Monoclonal antibodies (mAbs) were prepared by immunizing mice with the recombinant CD2v protein. Eventually, four mAbs that target the extracellular region of the ASFV CD2v protein were obtained. All four mAbs responded well to the ASFV HLJ/18 strain and recognized the same linear epitope, 154SILE157. The specific shortest amino acid sequence of this epitope has been accurately identified for the first time. Meaningfully, the 154SILE157 epitope was highly conformed in the ASFV Chinese epidemic strain and Georgia2008/1 strains according to the analysis of the conservation and have a fair protective effect. These findings contribute to further understanding of the protein function of CD2v and provide potential support for the development of diagnostic tools and vaccines for ASFV.

Identification and characterization of nanobodies specifically against African swine fever virus major capsid protein p72.

African swine fever virus (ASFV) is a large and very complex DNA virus. The major capsid protein p72 is the most predominant structural protein and constitutes the outmost icosahedral capsid of the virion. In the present study, the nanobodies against ASFV p72 protein were screened from a camelid immune VHH library by phage display technique. Nine distinct nanobodies were identified according to the amino acid sequences of the complementary determining regions (CDRs), and contain typical amino acid substitutions in the framework region 2 (FR2). Six nanobodies were successfully expressed in E. coli, and their specificity and affinity to p72 protein were further evaluated. The results showed that nanobodies Nb25 had the best affinity to both recombinant and native p72 protein of ASFV. The Nb25 possesses an extremely long CDR3 with 23 amino acids compared with other nanobodies, which may allow this nanobody to access the hidden epitopes of target antigen. Furthermore, the Nb25 can specifically recognize the virus particles captured by polyclonal antibody against ASFV in a sandwich immunoassay, and its application as a biosensor to target virus in PAM cells was verified by an immunofluorescence assay. Nanobodies have been proven to possess many favorable properties with small size, high affinity and specificity, easier to produce, low costs and deep tissue penetration that make them suitable for various biotechnological applications. These findings suggest that nanobody Nb25 identified herein could be a valuable alternative tool and has potential applications in diagnostic and basic research on ASFV.

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

Proteome Analysis of Swine Macrophages after Infection with Two Genotype II African Swine Fever Isolates of Different Pathogenicity

Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observed.

African swine fever virus MGF505-7R protein interacted with IRF7and TBK1 to inhibit type I interferon production

African swine fever virus (ASFV) employs diverse strategies to confront or evade host type I interferon (IFN-I)-induced antiviral responses. Moreover, the mechanisms of this process are largely unknown. Here, we assessed 27 ASFV proteins to determine whether any of them suppressed the cGAS-STING pathway to facilitate immune evasion. Using dual-luciferase assays, we found that ASFV MGF505-7R suppressed the activity of the IFN-β and ISRE promoters and the expression of IFN-I and ISGs. MGF505-7R interacted with IRF7 and TBK1, degrading IRF7 by autophagy, cysteine, and proteasome pathways and TBK1 by the proteasome pathway. Moreover, TBK1 and IRF3 were phosphorylated by cGAS-STING stimulation. Finally, small interfering RNA (siRNA)-based silencing of MGF505-7R enhanced IFN-I antiviral activity. Taken together, these results preliminarily clarified the immune escape mechanism of ASFV MGF505-7R, which provides a potential target for developing antiviral agents.

African Swine Fever Vaccinology: The Biological Challenges from Immunological Perspectives.

African swine fever virus (ASFV), a nucleocytoplasmic large DNA virus (NCLDV), causes African swine fever (ASF), an acute hemorrhagic disease with mortality rates up to 100% in domestic pigs. ASF is currently epidemic or endemic in many countries and threatening the global swine industry. Extensive ASF vaccine research has been conducted since the 1920s. Like inactivated viruses of other NCLDVs, such as vaccinia virus, inactivated ASFV vaccine candidates did not induce protective immunity. However, inactivated lumpy skin disease virus (poxvirus) vaccines are protective in cattle. Unlike some experimental poxvirus subunit vaccines that induced protection, ASF subunit vaccine candidates implemented with various platforms containing several ASFV structural genes or proteins failed to protect pigs effectively. Only some live attenuated viruses (LAVs) are able to protect pigs with high degrees of efficacy. There are currently several LAV ASF vaccine candidates. Only one commercial LAV vaccine is approved for use in Vietnam. LAVs, as ASF vaccines, have not yet been widely tested. Reports thus far show that the onset and duration of protection induced by the LAVs are late and short, respectively, compared to LAV vaccines for other diseases. In this review, the biological challenges in the development of ASF vaccines, especially subunit platforms, are discussed from immunological perspectives based on several unusual ASFV characteristics shared with HIV and poxviruses. These characteristics, including multiple distinct infectious virions, extremely high glycosylation and low antigen surface density of envelope proteins, immune evasion, and possible apoptotic mimicry, could pose enormous challenges to the development of ASF vaccines, especially subunit platforms designed to induce humoral immunity.

African swine fever virus pE301R negatively regulates cGAS-STING signaling pathway by inhibiting the nuclear translocation of IRF3

African swine fever (ASF) is a highly contagious and lethal infectious disease of domestic pigs and wild boars by the African swine fever virus (ASFV). ASFV infects domestic pigs with the mortality rate approaching 100 % at acute stage of infection. The cGAS-STING-mediated antiviral responses are wildly accepted that cGAS acts as DNA sensor for sensing of viral DNA during DNA virus infection. However, the molecular mechanisms underlying negatively regulation of cGAS-STING signaling and type I IFN (IFN-I) production by ASFV proteins are not fully understood. In this study, we demonstrated that ASFV pE301R antagonize the activities of IFN-β-, NF-κB-, ISRE-luciferase (Luc) reporters-induced by cGAS-STING in a dose dependent manner. Consistent with these results, the mRNA levels of Ifnb1, Isg15, Isg56 are attenuated by ASFV pE301R. Furthermore, ASFV pE301R executes its inhibitory function at the downstream of IFN-regulatory factor 3 (IRF3) phosphorylation. Mechanistically, pE301R interacts with IRF3 via its amino acid (aa) 1–200 region, resulting in inhibition of the nuclear translocation of IRF3 induced by cGAMP and poly(dA:dT). Overall, our findings reveal that pE301R acts as a negatively regulator to inhibit IFN-I production and to subvert host antiviral innate immunity during ASFV infection.

African Swine Fever Virus: A Review.

African swine fever (ASF) is a viral disease with a high fatality rate in both domestic pigs and wild boars. ASF has greatly challenged pig-raising countries and also negatively impacted regional and national trade of pork products. To date, ASF has spread throughout Africa, Europe, and Asia. The development of safe and effective ASF vaccines is urgently required for the control of ASF outbreaks. The ASF virus (ASFV), the causative agent of ASF, has a large genome and a complex structure. The functions of nearly half of its viral genes still remain to be explored. Knowledge on the structure and function of ASFV proteins, the mechanism underlying ASFV infection and immunity, and the identification of major immunogenicity genes will contribute to the development of an ASF vaccine. In this context, this paper reviews the available knowledge on the structure, replication, protein function, virulence genes, immune evasion, inactivation, vaccines, control, and diagnosis of ASFV.

Inhibition of BET Family Proteins Suppresses African Swine Fever Virus Infection.

ABSTRACTAfrican swine fever (ASF), an acute, severe, highly contagious disease caused by African swine fever virus (ASFV) infection in domestic pigs and boars, has a mortality rate of up to 100%. Because effective vaccines and treatments for ASF are lacking, effective control of the spread of ASF remains a great challenge for the pig industry. Host epigenetic regulation is essential for the viral gene transcription. Bromodomain and extraterminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, are epigenetic “readers” critical for gene transcription regulation. Among these proteins, BRD4 recognizes acetylated histones via its two bromodomains (BD1 and BD2) and recruits transcription factors, thereby playing a pivotal role in transcriptional regulation and chromatin remodeling during viral infection. However, how BET/BRD4 regulates ASFV replication and gene transcription is unknown. Here, we randomly selected 12 representative BET family inhibitors and compared their effects on ASFV infection in pig primary alveolar macrophages (PAMs). These were found to inhibit viral infection by interfering viral replication. The four most effective inhibitors (ARV-825, ZL0580, I-BET-762, and PLX51107) were selected for further antiviral activity analysis. These BET/BRD4 inhibitors dose dependently decreased the ASFV titer, viral RNA transcription, and protein production in PAMs. Collectively, we report novel function of BET/BRD4 inhibitors in inducing suppression of ASFV infection, providing insights into the role of BET/BRD4 in the epigenetic regulation of ASFV and potential new strategies for ASF prevention and control.IMPORTANCE Due to the continuing spread of the ASFV in the world and the lack of commercial vaccines, the development of improved control strategies, including antiviral drugs, is urgently needed. BRD4 is an important epigenetic factor and has been commonly used for drug development for tumor treatment. Furthermore, the latest research showed that BET/BRD4 inhibition could suppress replication of virus. In this study, we first showed the inhibitory effect of agents targeting BET/BRD4 on ASFV infection with no significant host cytotoxicity. Then, we found four BET/BRD4 inhibitors that can inhibit ASFV replication, RNA transcription, and protein synthesis. Our findings support the hypothesis that BET/BRD4 can be considered as attractive host targets in antiviral drug discovery against ASFV.

Two-Dimensional Protein Nanoarray as a Carrier of Sensing Elements for Gold-Based Immunosensing Systems.

Homogeneous and high-density immobilization of proteins on gold-based sensing surface without the loss of protein activity is of great significance for high-performance immunosensing but remains challenging. To realize more sensitive immunosensing, an improved method for protein immobilization on the gold surface is urgently required. Here, we propose a biological and mild approach by combining a genetically encoded SpyTag-SpyCatcher interaction system with a redesigned S-layer of bacteria. This method allows proteins of interest to be covalently linked with the S-layer in a biological manner and arranged orderly in a two-dimensional nanoarray on the gold surface. The activity of African swine fever virus proteins was significantly preserved after immobilization. In addition, our S-layer-based immobilization method exhibited an eightfold increase in detection sensitivity compared with the conventional chemical cross-linking for protein immobilization during serological tests. Together, our S-layer-based immobilization method provides an innovative approach for building a quality gold-based biosensing interface and should greatly contribute to the high-sensitivity sensing for a deeper understanding of pathogen infection and host immunity.