African Swine Fever Virus Antigens Research Articles

Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody.

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.

Development of a quantum dots based immunochromatographic strip for rapid and on-site detection of African swine fever virus

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.

Saccharomyces cerevisiae oral immunization in mice using multi-antigen of the African swine fever virus elicits a robust immune response.

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens-KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L- induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV.

Development and evaluation of two rapid lateral flow assays for on-site detection of African swine fever virus.

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. In the absence of a widely available and definitively proven vaccine, rapid and early detection is critical for ASF control. The quick and user-friendly lateral flow assay (LFA) can easily be performed by following simple instructions and is ideal for on-site use. This study describes the development and validation of two LFAs for the rapid detection of ASF virus (ASFV) in pig serum. The highly immunogenic antigens (p30 and p72) of ASFV Georgia 2007/1 (genotype II) were expressed in plants (Nicotiana benthamiana) and were used to immunize BALB/c mice to generate specific monoclonal antibodies (mAbs) against the p30 and p72 proteins. mAbs with the strongest binding ability to each protein were used to develop p30_LFA and p72_LFA for detecting the respective ASFV antigens. The assays were first evaluated using a spike-in test by adding the purified p30 or p72 protein to a serum sample from a healthy donor pig. Further validation of the tests was carried out using serum samples derived from experimentally infected domestic pigs, field domestic pigs, and feral pigs, and the results were compared with those of ASFV real-time PCR. p30_LFA and p72_LFA showed no cross-reaction with common swine viruses and delivered visual results in 15 min. When testing with serially diluted proteins in swine serum samples, analytical sensitivity reached 10 ng/test for p30_LFA and 20 ng/test for p72_LFA. Using real-time PCR as a reference, both assays demonstrated high sensitivity (84.21% for p30_LFA and 100% for p72_LFA) with experimentally ASFV-infected pig sera. Specificity was 100% for both LFAs using a panel of PBS-inoculated domestic pig sera. Excellent specificity was also shown for field domestic pig sera (100% for p30_LFA and 93% for p72_LFA) and feral pig sera (100% for both LFAs). The results obtained in this study suggest that p30_LFA and p72_LFA hold promise as rapid, sensitive, user-friendly, and field-deployable tools for ASF control, particularly in settings with limited laboratory resources.

ASFV antigens selected from genotype I immunised pigs are immunogenic, but do not protect against genotype II challenge.

African swine fever virus (ASFV) has recent caused outbreaks of viral haemorrhagic fever in domestic pigs and wild boar in Africa, Asia, Europe, Oceania and North America. Control measures for African swine fever are limited in most countries to biosecurity at the farm gate followed by movement restrictions and selective or complete culling of pigs on affected premises. Modified live vaccines are being trialled in several countries, however development of safe and effective African swine fever subunit vaccines has been restricted by a poor understanding of the key antigens required for protection, particularly for the panzootic genotype II viruses. The cellular immune response is thought to be critical for protection against African swine fever and therefore to develop an effective subunit vaccine that stimulates an anti-ASFV T-cell response we screened lymphocytes from pigs which survived challenge with Georgia 2007/1. Using an overlapping peptide library corresponding to 168 annotated open reading frames and 24 potential minor open reading frames we identified seventeen proteins with strongly stimulated secretion of interferon gamma in an ELISpot assay. The phenotype of the T cells which were stimulated by these pools of peptides were then investigated by flow cytometry. Proteins stimulating predominantly CD8+ T cells were incorporated into bivalent replication deficient adenovirus vectors and tested as potential vaccine candidates in immunisation and challenge experiments in pigs.

A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 μg/mL and quadruple ASFV antigen combination of 1 μg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Comparative Analysis of Swine Antibody Responses following Vaccination with Live-Attenuated and Killed African Swine Fever Virus Vaccines.

African swine fever virus (ASFV) is circulating in many swine-producing countries, causing significant economic losses. It is observed that pigs experimentally vaccinated with a live-attenuated virus (LAV) but not a killed virus (KV) vaccine develop solid homologous protective immunity. The objective of this study was to comparatively analyze antibody profiles between pigs vaccinated with an LAV vaccine and those vaccinated with a KV vaccine to identify potential markers of vaccine-induced protection. Thirty ASFV seronegative pigs were divided into three groups: Group 1 received a single dose of an experimental LAV, Group 2 received two doses of an experimental KV vaccine, and Group 3 was kept as a non-vaccinated (NV) control. At 42 days post-vaccination, all pigs were challenged with the parental virulent ASFV strain and monitored for 21 days. All pigs vaccinated with the LAV vaccine survived the challenge. In contrast, eight pigs from the KV group and seven pigs from the NV group died within 14 days post-challenge. Serum samples collected on 41 days post-vaccination were analyzed for their reactivity against a panel of 29 viral structural proteins. The sera of pigs from the LAV group exhibited a strong antibody reactivity against various viral structural proteins, while the sera of pigs in the KV group only displayed weak antibody reactivity against the inner envelope (p32, p54, p12). There was a negative correlation between the intensity of antibody reactivity against five ASFV antigens, namely p12, p14, p15, p32, and pD205R, and the viral DNA titers in the blood of animals after the challenge infection. Thus, antibody reactivities against these five antigens warrant further evaluation as potential indicators of vaccine-induced protection.

Generation and characterization of a monoclonal antibody against an African swine fever virus protein encoded by the A137R gene

The ongoing African swine fever (ASF) pandemic continues to have a major impact on global pork production and trade. Since ASF cannot be distinguished from other swine hemorrhagic fevers clinically, ASF-specific laboratory diagnosis is critical. Thus ASF virus (ASFV)-specific monoclonal antibodies (mAbs) are critical for the development of laboratory diagnostics. In this study, we report one ASFV-specific mAb, F88ASF-55, that was generated and characterized. This mAb recognizes the ASFV A137R-encoded protein (pA137R). Epitope mapping results revealed a highly conserved linear epitope recognized by this mAb, corresponding to amino acids 111–125 of pA137R. We explored the potential use of this mAb in diagnostic applications. Using F88ASF-55 as the detection antibody, six ASFV strains were detected in an enzyme-linked immunosorbent assay (ELISA) with low background. In immunohistochemistry (IHC) assays, this mAb specifically recognized ASFV antigens in the submandibular lymph nodes of animals experimentally infected with different ASFV strains. Although not all ASFV genotypes were tested in this study, based on the conserved ASFV epitope targeted by F88ASF-55, it has the potential to detect multiple ASFV genotypes. In conclusion, this newly generated ASFV pA137R-specific mAb has potential value in ASF diagnostic tool development. It can be used in ELISA, IHC, and possibly-immunochromatographic strip assays for ASFV detection. It also suggests that pA137R may be a good target for diagnostic assays to detect ASFV infection.

Expression of ASFV p17 in CHO cells and identification of one novel epitope using a monoclonal antibody

As a highly pathogenic large DNA virus, African swine fever virus (ASFV) has huge particles and numerous encoded proteins. At present, few of the existing studies on ASFV proteins have investigated the function of p17. Specific antibodies against p17 to promote the development of prevention techniques against African swine fever (ASF) are urgently needed. Herein, we successfully expressed ASFV p17 in CHO cells using a suspension culture system and generated a monoclonal antibody (mAb) against p17. The mAb recognized a novel linear epitope (8LLSHNLSTREGIK20) and exhibited specific reactivity, which was conducive to the identification of ectopically expressed p17, the recombinant porcine reproductive and respiratory syndrome virus expressing p17, and the ASFV-SY18. The epitope was conservative among genotype I and genotype II ASFV strains. Overall, the mAb against p17 revealed efficient detection and promising application prospects, making it a useful tool for future vaccine research on ASF. Determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of ASFV antigen protein.

Immunization of pigs with replication-incompetent adenovirus-vectored African swine fever virus multi-antigens induced humoral immune responses but no protection following contact challenge.

African swine fever virus (ASFV) is a pathogen of great economic importance given that continues to threaten the pork industry worldwide, but there is no safe vaccine or treatment available. Development of a vaccine is feasible as immunization of pigs with some live attenuated ASFV vaccine candidates can confer protection, but safety concerns and virus scalability are challenges that must to be addressed. Identification of protective ASFV antigens is needed to inform the development of efficacious subunit vaccines. In this study, replication-incompetent adenovirus-vectored multicistronic ASFV antigen expression constructs that covered nearly 100% of the ASFV proteome were generated and validated using ASFV convalescent serum. Swine were immunized with a cocktail of the expression constructs, designated Ad5-ASFV, alone or formulated with either Montanide ISA-201™ (ASFV-ISA-201) or BioMize® adjuvant (ASFV-BioMize). These constructs primed strong B cell responses as judged by anti-pp62-specific IgG responses. Notably, the Ad5-ASFV and the Ad5-ASFV ISA-201, but not the Ad5-ASFV BioMize®, immunogens primed significantly (p < 0.0001) higher anti-pp62-specific IgG responses compared with Ad5-Luciferase formulated with Montanide ISA-201™ adjuvant (Luc-ISA-201). The anti-pp62-specific IgG responses underwent significant (p < 0.0001) recall in all the vaccinees after boosting and the induced antibodies strongly recognized ASFV (Georgia 2007/1)-infected primary swine cells. However, following challenge by contact spreaders, only one pig nearly immunized with the Ad5-ASFV cocktail survived. The survivor had no typical clinical symptoms, but had viral loads and lesions consistent with chronic ASF. Besides the limited sample size used, the outcome suggests that in vivo antigen expression, but not the antigen content, might be the limitation of this immunization approach as the replication-incompetent adenovirus does not amplify in vivo to effectively prime and expand protective immunity or directly mimic the gene transcription mechanisms of attenuated ASFV. Addressing the in vivo antigen delivery limitations may yield promising outcomes.

Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs

BackgroundAfrican swine fever (ASF) is a highly fatal disease in domestic pigs caused by ASF virus (ASFV), for which there is currently no commercial vaccine available. The genome of ASFV encodes more than 150 proteins, some of which have been included in subunit vaccines but only induce limited protection against ASFV challenge.MethodsTo enhance immune responses induced by ASFV proteins, we expressed and purified three fusion proteins with each consisting of bacterial lipoprotein OprI, 2 different ASFV proteins/epitopes and a universal CD4+ T cell epitope, namely OprI-p30-modified p54-TT, OprI-p72 epitopes-truncated pE248R-TT, and OprI-truncated CD2v-truncated pEP153R-TT. The immunostimulatory activity of these recombinant proteins was first assessed on dendritic cells. Then, humoral and cellular immunity induced by these three OprI-fused proteins cocktail formulated with ISA206 adjuvant (O-Ags-T formulation) were assessed in pigs.ResultsThe OprI-fused proteins activated dendritic cells with elevated secretion of proinflammatory cytokines. Furthermore, the O-Ags-T formulation elicited a high level of antigen-specific IgG responses and interferon-γ-secreting CD4+ and CD8+ T cells after stimulation in vitro. Importantly, the sera and peripheral blood mononuclear cells from pigs vaccinated with the O-Ags-T formulation respectively reduced ASFV infection in vitro by 82.8% and 92.6%.ConclusionsOur results suggest that the OprI-fused proteins cocktail formulated with ISA206 adjuvant induces robust ASFV-specific humoral and cellular immune responses in pigs. Our study provides valuable information for the further development of subunit vaccines against ASF.

Molecular Signatures in Swine Innate and Adaptive Immune Responses to African Swine Fever Virus Antigens p30/p54/CD2v Expressed Using a Highly Efficient Semliki Forest Virus Replicon System.

African swine fever virus (ASFV) causes a devastating viral hemorrhagic disease in domestic pigs and Eurasian wild boars, posing a foremost threat to the swine industry and pig farming. The development of an effective vaccine is urgently needed, but has been hampered by the lack of an in-depth, mechanistic understanding of the host immune response to ASFV infection and the induction of protective immunity. In this study, we report that immunization of pigs with Semliki Forest Virus (SFV) replicon-based vaccine candidates expressing ASFV p30, p54, and CD2v, as well as their ubiquitin-fused derivatives, elicits T cell differentiation and expansion, promoting specific T cell and humoral immunity. Due to significant variations in the individual non-inbred pigs in response to the vaccination, a personalized analysis was conducted. Using integrated analysis of differentially expressed genes (DEGs), Venn, KEGG and WGCNA, Toll-like receptor, C-type lectin receptor, IL17 receptor, NOD-like receptor and nucleic acid sensor-mediated signaling pathways were demonstrated to be positively correlated to the antigen-stimulated antibody production and inversely correlated to the IFN-γ secreting cell counts in peripheral blood mononuclear cells (PBMCs). An up-regulation of CIQA, CIQB, CIQC, C4BPA, SOSC3, S100A8 and S100A9, and down-regulation of CTLA4, CXCL2, CXCL8, FOS, RGS1, EGR1 and SNAI1 are general in the innate immune response post-the second boost. This study reveals that pattern recognition receptors TLR4, DHX58/DDX58 and ZBP1, and chemokines CXCL2, CXCL8 and CXCL10 may play important roles in regulating this vaccination-stimulated adaptive immune response.

Immune response following safer administration of recombinant Salmonella Typhimurium harboring ASFV antigens in pigs

African swine fever virus (ASFV) is a contagious epizootic pathogen adversely affecting porcine industry in Asian and European countries. Till date, 8 serotypes and 24 genotypes of the virus have been reported. Few live attenuated virus vaccine studies have reported to provide complete protection against ASFV infection but biohazard concern still remain. Recombinant subunit antigens are capable of providing cellular and humoral immunity in porcine, but not a single vaccine has hit the market yet. In the present study, we attempted to use recombinant Salmonella Typhimurium JOL912 strain harboring ASFV antigens (rSal-ASFV) to investigate its immunostimulant effect in porcine. Post intramuscular administration, we observed significant increment in the levels of helper T cells, cytotoxic T cells, natural killer (NK) cells, and immunoglobulin (i.e. IgG, IgA, and IgM) levels in rSal-ASFV treated groups. Further RT-PCR analysis indicated the increased expression of MHC-I, MHC-II, CD80/86, NK cell receptors (NKp30, NKp44, and NKp46) and cytokines while ELIspot analysis revealed significant production of IFN-γ in rSal-ASFV treated groups. Taken together, we are able to demonstrate that rSal-ASFV could elicit a non-specific cellular as well as humoral immune response. However, additional antigen specific immunity data is needed to evaluate its efficacy. Intramuscular administration of rSal-ASFV was found to be safe and immunostimulant in nature without any side-effects and may serve as an excellent option for in-vivo antigen delivery in pigs.

Dynamics of Serological and Mucosal Antibody Responses against African Swine Fever Viruses in Experimentally Infected Pigs

African swine fever virus (ASFV) is a lethal swine pathogen, and there is no effective vaccine or treatment available for ASFV infection. Recently, the occurrence of ASFV genotype I and genotype II natural mutants that manifest as subacute, longer-incubation, or persistent infections poses threats to preventing ASFV infection. The dynamics of antibody responses to ASFV are still completely unrevealed, especially the secretion of mucosal antibodies in oral fluid. Here, a systematic analysis was performed of serological and mucosal antibody secretion against 6 ASFV antigens after direct or indirect infection with four different ASFV strains or genotypes, namely, the field virulent genotype II isolate ASFV HLJ/18, the artificially attenuated genotype II strain HLJ/18-7GD, the naturally attenuated genotype II isolate HLJ/HRB1/20, and genotype I isolate SD/DY-I/21. Severe clinical signs of HLJ/18 infection were observed in pigs from 4 days postinoculation. However, no clinical signs were observed in HLJ/18-7GD-infected pigs. The contact pigs cohoused with the pigs intramuscularly infected with the isolate SD/DY-I/21 or HLJ/HRB1/20 only showed chronic clinical signs. Interestingly, the oral fluid sIgA responses to all the selected antigens were significantly stronger and earlier than the serum IgG responses in both HLJ/18- and HLJ/18-7GD-challenged pigs. Although significant fluctuations and individual differences appeared in oral swab sIgA responses in the contact transmission group, they were earlier than the corresponding serological IgG responses. Moreover, according to the comparative analysis of the three infection groups, P54 was proposed to be a dominant target for serological IgG diagnosis, while P30, CD2v, P54, P22, and P10 were more advantageous as mucosal sIgA diagnosis targets. These results highlight the important role of mucosal antibodies in the early diagnosis of ASFV infection and can provide references to screen appropriate targets for ASFV detection.

Orally administered recombinant Lactobacillus expressing African swine fever virus antigens that induced immunity responses.

African swine fever (ASF) is a highly contagious, acute, febrile disease caused by the African swine fever virus (ASFV), with morbidity and mortality rates approaching 100% in domestic and wild swine, resulting in massive economic losses to the pig industry worldwide. This study aimed to express the p30, p54, and p72 proteins encoded by ASFV in vitro using the Lactobacillus lactis (L. lactis) expression system. Here, six new functional recombinant L. lactis were constructed, and the expression of the p30 protein, p54 protein, p72 protein, p30-LTB (heat-labile enterotoxin B, LTB) fusion protein, p54-LTB fusion protein, and the p72-LTB fusion protein was successfully detected by Western blot analysis. Following oral immunization of rabbits with recombinant L. lactis, serum IgG, intestinal mucosal sIgA, cytokines (IL-4 and INF-γ), and splenocyte viability were higher than in the control group via ELISA. Notably, without the LTB adjuvant group, humoral and Th1 cellular immunity were promoted, whereas, with the LTB adjuvant group, local mucosal immunity, humoral immunity, and Th2 cellular immunity were promoted, providing new insights into the design and development of an ASFV subunit vaccine.

A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R.

African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150-200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope (2VEPREQFFQDLLSAV16) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

The Indirect ELISA and Monoclonal Antibody against African Swine Fever Virus p17 Revealed Efficient Detection and Application Prospects.

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope (3TETSPLLSH11) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

Comparison of mucosal immune responses to African swine fever virus antigens intranasally delivered with two different viral vectors

Transmission of African swine fever virus (ASFV) in domestic swine occurs mainly via contact with mucosal surfaces. In this study, we constructed a pseudotyped surface-displaying BacMam-F1 vector expressing ASFV CD2v-p30-p54 fusion antigen, and compared its mucosal responses in pigs with that of rAd-F1 vector expressing the same antigen. From day 21 after intranasal immunization, the antigen-specific IgG and intranasal secretory IgA (S-IgA) antibody responses induced by BacMam-F1 were significantly stronger than that by rAd-F1. The significantly different S-IgA antibody responses were also detected in their tracheal washes and lung lavages. After stimulation with ASFV antigens, 4/6 S-IgA-promoting cytokine responses in porcine alveolar macrophages (PAMs) from BacMam-F1-immunized pigs were significantly stronger than that from rAd-F1-immunized pigs. The similar expression patterns of S-IgA-promoting cytokines were also detected in their lung lavages. After pretreating ASFV with different samples from immunized pigs, significant inhibitory effects were detected in tracheal washes, lung lavages and PAM cultures, but not serum samples with slight inter-group difference. These data suggest that the pseudotyped surface-displaying BacMam vector is more suitable for swine mucosal immunization.

Development of a chromatographic lateral flow immunoassay for detection of African swine fever virus antigen in blood

African swine fever (ASF) is a highly lethal disease of domestic and wild swine caused by African swine fever virus (ASFV). The disease currently circulates in Africa, Europe, Asia and on the island of Hispaniola. The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses. No effective vaccine is available for ASF, making rapid and accurate detection of ASFV essential for disease mitigation strategies. Currently available diagnostics for ASFV possess significant limitations related to assay performance, deployability, and/or turn-around time; therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity. A chromatographic lateral flow immunoassay (LFIA) was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein. The assay requires only water to perform and provides results in 25 min, making it well-suited for field use. The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection, though it was unable to detect a genotype X ASFV strain. Diagnostic sensitivity correlated with clinical disease severity, body temperature, and viral DNA levels, and was over 90% in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus. The LFIA also showed a robust diagnostic specificity of over 98%, which is essential to field testing for a high consequence to foregin animal disease. The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates, making it a promising candidate for use as a field-deployable antigen detection assay. Additional evaluation using field samples and different virus strains is required to further assess the utility of this rapid diagnostic test.

Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus.

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family that damages the immune system of pigs, resulting in the death or slaughter of millions of animals worldwide. Recent modern techniques in ASFV vaccination have highlighted the potential of viral replicon particles (RPs), which can efficiently express foreign proteins and induce robust cellular and humoral immune responses compared with the existing vaccines. In this study, we established a Semliki Forest virus (SFV) vector by producing replication-defective viral particles. This vector was used to deliver RPs expressing ASFV antigens. SFV-RPs expressing ASFV p32 (SFV-p32) and p54 (SFV-p54) were tested in baby hamster kidney (BHK-21) cells. Proteins expression was evaluated via western blotting and indirect immunofluorescence, while immunogenicity was evaluated in BALB/c mice. The resulting RPs exhibited high levels of protein expression and elicited robust humoral and cellular immune responses.