Evaluation of the Deletion of MGF110-5L-6L on Swine Virulence from the Pandemic Strain of African Swine Fever Virus and Use as a DIVA Marker in Vaccine Candidate ASFV-G-ΔI177L.

African swine fever virus (ASFV) is responsible for an ongoing pandemic that is affecting central Europe, Asia, and recently the Dominican Republic, the first report of the disease in the Western Hemisphere in over 40 years. ASFV is a large, complex virus with a double-stranded DNA (dsDNA) genome that carries more than 150 genes, most of which have not been studied. Here, we assessed the role of the MGF110-5L-6L gene during virus replication in cell cultures and experimental infection in swine. A recombinant virus with MGF110-5L-6L deleted (ASFV-G-ΔMGF110-5L-6L) was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. ASFV-G-ΔMGF110-5L-6L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the MGF110-5L-6L gene is nonessential for virus replication. Similarly, domestic pigs inoculated with ASFV-G-ΔMGF110-5L-6L presented with a clinical disease undistinguishable from that caused by the parental ASFV-G, confirming that the MGF110-5L-6L gene is not involved in producing disease in swine. Sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognized the protein encoded by the MGF110-5L-6L gene as a potential target for the development of an antigenic marker differentiation of infected from vaccinated animals (DIVA) vaccine. To test this hypothesis, the MGF110-5L-6L gene was deleted from the highly efficacious ASFV vaccine candidate ASFV-G-ΔI177L, generating the recombinant ASFV-G-ΔI177L/ΔMGF110-5L-6L. Animals inoculated with ASFV-G-ΔI177L/ΔMGF110-5L-6L developed an ASFV-specific antibody response detected by enzyme-linked immunosorbent assay (ELISA). The sera strongly recognized ASFV p30 expressed in eukaryotic cells but did not recognize ASFV MGF110-5L-6L protein, demonstrating that deletion of the MGF110-5L-6L gene can enable DIVA capabilities in preexisting vaccine candidates. IMPORTANCE Currently, there are no African swine fever (ASF) commercial vaccines that can be used to prevent or control the spread of ASF. The only effective experimental vaccines against ASF are live-attenuated vaccines. However, these experimental vaccines, which rely on a deletion of a specific gene of the current circulating strain of ASF, make it hard to tell the difference between a vaccinated and an infected animal. In our search for a serological marker, we identified that the virus protein encoded by the MGF110-5L-6L gene induced an immune response, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that deletion of MGF110-5L-6L does not affect virulence or virus replication. However, when the deletion of MGF110-5L-6L was added to vaccine candidate ASFV-G-ΔI177L, a reduction in the effectiveness of the vaccine occurred.

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

Genotype II Live-Attenuated ASFV Vaccine Strains Unable to Completely Protect Pigs against the Emerging Recombinant ASFV Genotype I/II Strain in Vietnam.

Background: African swine fever virus (ASFV) continues to spread globally, causing severe economic losses to pig farmers. Vietnam licensed two live attenuated vaccines based on the ASFV strains ASFV-G-ΔI177L and ASFV-G-ΔMGF to control the ongoing ASF outbreaks. In 2023, newly emerging highly virulent recombinant ASF viruses (rASFV I/II) containing genetic elements from both p72 genotype I and II ASF viruses were reported from Northern Vietnam. Objective: This study evaluated whether the two vaccine strains were able to protect the pigs against the emerging rASFV I/II strain VNUA/rASFV/TN1/23. Results: Pigs vaccinated with ASFV-G-ΔMGF or ASFV-G-ΔI177L, when challenged with rASFV I/II, succumbed to the infection, or developed signs of chronic ASF. Conclusions: The findings from this study show that both vaccine strains that are licensed and used in Vietnam are unlikely to protect pigs from the emerging highly virulent rASFV I/II. This complicates the ongoing efforts to control ASF in Asia and globally and emphasizes the urgent need for a novel vaccine that can effectively protect pigs from the rASFV I/II.

African Swine Fever Vaccine Candidate ASFV-G-ΔI177L Produced in the Swine Macrophage-Derived Cell Line IPKM Remains Genetically Stable and Protective against Homologous Virulent Challenge.

ASFV vaccine candidate ASFV-G-ΔI177L has been shown to be highly efficacious in inducing protection against challenges with the parental virus, the Georgia 2010 isolate, as well as against field strains isolated from Vietnam. ASFV-G-ΔI177L has been shown to produce protection even when used at low doses (102 HAD50) and shows no residual virulence even when administered at high doses (106 HAD50) or evaluated for a relatively long period of time (6 months). ASFV-G-ΔI177L stocks can only be massively produced in primary cell macrophages. Alternatively, its modified version (ASFV-G-ΔI177L/ΔLVR) grows in a swine-derived cell line (PIPEC), acquiring significant genomic modifications. We present here the development of ASFV-G-ΔI177L stocks in a swine macrophage cell line, IPKM, and its protective efficacy when evaluated in domestic pigs. Successive passing of ASFV-G-ΔI177L in IPKM cells produces minimal genomic changes. Interestingly, a stock of ASFV-G-ΔI177L obtained after 10 passages (ASFV-G-ΔI177Lp10) in IPKM cells showed very small genomic changes when compared with the original virus stock. ASFV-G-ΔI177Lp10 conserves similar growth kinetics in primary swine macrophage cultures than the original parental virus ASFV-G-ΔI177L. Pigs infected with 103 HAD50 of ASFV-G-ΔI177Lp10 developed a strong virus-specific antibody response and were completely protected against the challenge with the parental virulent field isolate Georgia 2010. Therefore, IPKM cells could be an effective alternative for the production of ASFV vaccine stocks for those vaccine candidates exclusively growing in swine macrophages.

ASF Vaccine Candidate ASFV-G-∆I177L Does Not Exhibit Residual Virulence in Long-Term Clinical Studies.

African swine fever (ASF) is an important disease in swine currently producing a pandemic affecting pig production worldwide. Except in Vietnam, where two vaccines were recently approved for controlled use in the field, no vaccine is commercially available for disease control. Up to now, the most effective vaccines developed are based on the use of live-attenuated viruses. Most of these promising vaccine candidates were developed by deleting virus genes involved in the process of viral pathogenesis and disease production. Therefore, these vaccine candidates were developed via the genomic modification of parental virus field strains, producing recombinant viruses and reducing or eliminating their residual virulence. In this scenario, it is critical to confirm the absence of any residual virulence in the vaccine candidate. This report describes the assessment of the presence of residual virulence in the ASFV vaccine candidate ASFV-G-∆I177L in clinical studies conducted under high virus loads and long-term observation periods. The results demonstrated that domestic pigs intramuscularly inoculated with 106 HAD50 of ASFV-G-∆I177L did not show the presence of any clinical sign associated with ASF when observed daily either 90 or 180 days after vaccination. In addition, necropsies conducted at the end of the experiment confirmed the absence of macroscopic internal lesions associated with the disease. These results corroborate the safety of using ASFV-G-∆I177L as a vaccine candidate.

Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV-G-ΔI177L.

African swine fever (ASF) is the cause of a recent pandemic that is posing a threat to much of the world swine production. The etiological agent, ASF virus (ASFV), infects domestic and wild swine, producing a variety of clinical presentations depending on the virus strain and the genetic background of the pigs infected. No commercial vaccine is currently available, although recombinant live attenuated vaccine candidates have been shown to be efficacious. In addition to determining efficacy, it is paramount to evaluate the safety profile of a live attenuated vaccine. The presence of residual virulence and the possibility of reversion to virulence are two of the concerns that must be evaluated in the development of live attenuated vaccines. Here we evaluate the safety profile of an efficacious live attenuated vaccine candidate, ASFV-G-ΔI177L. Results from safety studies showed that ASFV-G-ΔI177L remains genetically stable and phenotypically attenuated during a five-passage reversion to virulence study in domestic swine. In addition, large-scale experiments to detect virus shedding and transmission confirmed that even under varying conditions, ASFV-G-ΔI177L is a safe live attenuated vaccine.

African swine fever virus vaccine candidate ASFV-G-ΔI177L efficiently protects European and native pig breeds against circulating Vietnamese field strain.

The recent spread of African swine fever (ASF) in the People's Republic of China and neighbouring countries in Asia has had significant economic consequences with an estimated direct cost of $55-$130 billion. This pandemic, originally detected in Republic of Georgia in 2007, has devastated the swine industry in large geographical areas of Southeast Asia with 14 countries reporting ASF outbreaks since the first documented case was confirmed in the city of Shenyang, Liaoning Province, China, on 3 August 2018. In the absence of any available vaccines, the control of ASF relies on the detection and culling of infected animals. The United States Department of Agriculture recently developed a recombinant experimental vaccine candidate, ASFV-G-ΔI177L, by deleting the I177L gene from the genome of the highly virulent pandemic ASFV strain Georgia, which efficaciouly protects pigs from the parental virus. Here, the initial studies were extended demonstrating that ASFV-G-ΔI177L is able to protect pigs against the virulent ASFV isolate currently circulating and producing disease in Vietnam with similar efficacy as reported against the Georgia strain. Comparative studies performed using a large number of pigs of European and Vietnamese origin demonstrated that a minimum protective dose of 102 HAD50 of ASFV-G-ΔI177L equally protects animals of both breeds. In concurrence with those results, the onset of immunity in these animal breed showed appearance of protection in approximately one-third of the animals by the second week post vaccination, with full protection achieved by the fourth week post vaccination. Therefore, results presented here demonstrated that ASFV-G-ΔI177L is able to induce protection against virulent Vietnameese ASFV field strains and is effective in protecting local breeds of pigs as efficiently as previously shown for European cross-bred pigs. To our knowledge, this is the first report showing the efficacy of a Georgia 2007 based vaccine candidate in Asian breed of pigs or challenged with an Asian ASFV strain.

A Highly Effective African Swine Fever Virus Vaccine Elicits a Memory T Cell Response in Vaccinated Swine.

African Swine Fever Virus (ASFV) is the causative agent of a highly contagious and lethal vector-borne disease in suids. Recently, a live attenuated virus strain, developed using the currently circulating, virulent Georgia strain (ASFV-G) with a single gene deletion (ASFV-G-ΔI177L), resulted in an effective vaccine. Nevertheless, protective immune response mechanisms induced by this candidate are poorly understood. In this study, Yorkshire crossbred swine intramuscularly vaccinated with 106 50% hemadsorption dose (HAD50) of ASFV-G-ΔI177L or a vehicle control were challenged at 28 days post-inoculation (dpi) with 102 HAD50 of ASFV-G. Analysis of purified peripheral blood mononuclear cells following inoculation and challenge revealed that CD4+, CD8+ and CD4+CD8+ central memory T cells (CD44+CD25-CD27-CD62L+CCR7+, Tcm) decreased significantly by 28 dpi in ASFV-G-ΔI177L-vaccinated swine compared to baseline and time-matched controls. Conversely, CD4+, CD8+ and CD4+CD8+ effector memory T cells (CD44+CD25-CD27-CD62-CCR7-, Tem) increased significantly among ASFV-G-ΔI177L-vaccined swine by 28 dpi compared to baseline and time-matched controls. Additionally, the percentage of natural killer (NK), CD4+ and CD4+CD8+ Tem and CD8+ Tcm and Tem positive for IFNγ increased significantly following inoculation, surpassing that of controls by 28 dpi or earlier. These results suggest that NK and memory T cells play a role in protective immunity and suggest that studying these cell populations may be a surrogate immunity marker in ASF vaccination.

Expounding the role of tick in Africa swine fever virus transmission and seeking effective prevention measures: A review.

African swine fever (ASF), a highly contagious, deadly infectious disease, has caused huge economic losses to animal husbandry with a 100% mortality rate of the most acute and acute infection, which is listed as a legally reported animal disease by the World Organization for Animal Health (OIE). African swine fever virus (ASFV) is the causative agent of ASF, which is the only member of the Asfarviridae family. Ornithodoros soft ticks play an important role in ASFV transmission by active biological or mechanical transmission or by passive transport or ingestion, particularly in Africa, Europe, and the United States. First, this review summarized recent reports on (1) tick species capable of transmitting ASFV, (2) the importance of ticks in the transmission and epidemiological cycle of ASFV, and (3) the ASFV strains of tick transmission, to provide a detailed description of tick-borne ASFV. Second, the dynamics of tick infection with ASFV and the tick-induced immune suppression were further elaborated to explain how ticks spread ASFV. Third, the development of the anti-tick vaccine was summarized, and the prospect of the anti-tick vaccine was recapitulated. Then, the marked attenuated vaccine, ASFV-G-ΔI177L, was compared with those of the anti-tick vaccine to represent potential therapeutic or strategies to combat ASF.

ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever.

The African swine fever virus (ASFV) is currently causing a pandemic affecting wild and domestic swine from Western Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in overwhelming economic losses to the swine industry. We recently developed a recombinant vaccine candidate, ASFVG-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV strain Georgia (ASFV-G). ASFV-G-ΔI177L has been proven safe and highly efficacious in challenge studies using parental ASFV-G. Here, we present data demonstrating that ASFV-G-ΔI177L can be administered by the oronasal (ON) route to achieve a similar efficacy to that of intramuscular (IM) administration. Animals receiving ON ASFV-G-ΔI177L were completely protected against virulent ASFV-G challenge. As previously described, similar results were obtained when ASFV-G-ΔI177L was given intramuscularly. Interestingly, viremias induced in animals inoculated oronasally were lower than those measured in IM-inoculated animals. ASFV-specific antibody responses, mediated by IgG1, IgG2 and IgM, do not differ in animals inoculated by the ON route from that had IM inoculations. Therefore, the ASFV-G-ΔI177L vaccine candidate can be administered oronasally, a critical attribute for potential vaccination of wild swine populations.