Vaccine for protecting piglets against African swine fever

Abstract

The present invention relates to a live attenuated African swine fever virus Georgia 2007 (ASFV-G) [delta] 9GL / [delta] UK strain for use in a vaccine for protecting a piglet against infection of African swine fever virus (ASFV) by administering to a pregnant pig a vaccine comprising the live attenuated ASFV-G-[delta] 9GL / [delta] UK strain, said piglet being part of the offspring of the pregnant pig.

Description

Technical Field

[0001] African swine fever virus (ASFV) is one of the most important pathogens affecting domestic pig populations worldwide. This invention relates to vaccines that protect pigs against ASFV infection, particularly susceptible piglets. Background Technology

[0002] As described in the review article by AC Urbano et al. (Emerging Microbes & Infections, 2022, Vol. 11, pp 2021-2033), African swine fever (ASF) is a highly contagious and severe hemorrhagic viral disease of pigs, prevalent in sub-Saharan Africa (24 genotypes based on the C-terminal sequence of the p72 surface protein) and Sardinia, Italy (p72 genotype I). Transcontinental transmission of ASF has occurred on at least three separate occasions, most notably in Georgia in 2007, where ASF spread from the Black Sea port of Poti across the Caucasus region to the Russian Federation (RF) and Eastern Europe. In the following decade, the disease was observed to become an animal epidemic in the RF, and by 2018, it had spread westward to Belgium and eastward to the People's Republic of China, rapidly covering much of Southeast Asia and Oceania. All isolates found in these regions are associated with the Georgia 2007 isolate, commonly referred to as ASFV Georgia 2007 or ASFV-G (US 9,808,520). The prevalence of ASF has continued to worsen since 2018; in January 2022, ASF virus re-emerged in mainland Italy. Since then, several resurgences have been reported in China, RF, Moldova, and Ukraine, with its first occurrence reported in North Macedonia, as well as Thailand, one of the few countries in the region to date unaffected. The virus also appeared in the Dominican Republic and Haiti in 2021, leading to the first diagnosis of ASFV in the Western Hemisphere in over 40 years. These recent events underscore a deeply disturbing pattern of continued transmission, exacerbated by the fact that small-scale and semi-industrial farms constitute the majority of pig production in many of these regions. Consequently, outbreaks have severe socioeconomic consequences, devastating rural livelihoods dependent on livestock production, threatening overall market stability and food security, and severely impacting animal welfare.

[0003] ASFV is a large double-stranded nucleoplasmic DNA arbovirus. AsfarviridaeThe sole member of the Viridae family. The virion has a diameter of approximately 250 nm and consists of a central nucleoid surrounded by an icosahedral protein capsid (or nucleocapsid), an internal lipoprotein membrane (or inner envelope), an icosahedral protein outer capsid, and an external lipoprotein envelope (or outer envelope) acquired during budding through the plasma membrane. Both intracellular and extracellular viral forms are infectious. Its natural host range is limited to ticks (Clathrus rubrum). Ornithodoros Soft ticks of the genus *Phragmites* and pig ticks ( Suidae The virus is a member of the family genotype II, which primarily replicates in mononuclear phagocytes, resident macrophages, and specific reticular cells. It is prevalent in African wild boars. However, clinical signs vary considerably in both domestic and wild boars, and individual outcomes can range from fatal to subclinical. A series of graded forms emerge based on the virulence of the strains involved, with mortality rates in infected animals ranging from 100% (peracute form) to <30% (chronic form). Currently, the genotype II strain affecting Europe and Asia is highly virulent, causing acute forms of disease, although there is evidence that some reduced-virulence isolates may circulate in wild boars in Baltic countries and domestic pigs in China, with reports of naturally mutated genotype II low-virulence strains and genotype I low-virulence circulating strains detected in the region.

[0004] Protective immunization against ASFV remains poorly understood. Several vaccine routes are known in the art, but no fully licensed commercially available vaccine has yet been developed. The first route described in the art is vaccination with inactivated virus. Virus inactivation is an established method of vaccine production, which is relatively straightforward when compared to live vaccines and, importantly, has a higher safety profile. The inactivation process eliminates the reversion of the toxic phenotype and renders the vaccine virus non-transmissible, two major drawbacks of live attenuated vaccines. Moreover, a lower safety risk is believed to exist for more vulnerable patient groups such as very young animals and pregnant animals. The latter group of animals experiences significant physiological changes and is therefore in a stressful metabolic state. However, inactivation does not necessarily produce a vaccine that induces protective immunity. Attempts to immunize pigs with various inactivated ASF antigens have not produced sufficient protection, even though in some cases it has been able to induce a serological immune response (Cadenas-Fernández et al.). Vaccines , 2021, 9, 242; doi.org / 10.3390 / vaccines9030242).

[0005] Another approach involves the use of subunit, DNA, and viral vector vaccines. These vaccines have shown promise, and several candidates have been shown to induce specific humoral and / or cellular immune responses that appear to confer partial to complete protection. However, the different nature of the immunization protocols used in these studies (including vaccine type, administration strategy, and challenge mode) makes it difficult to compare results. Further work is needed to identify which immune mechanisms need to be triggered to confer complete, durable protection, which antigens (or combinations thereof) should be included in potential vaccines, and the most suitable delivery method (Urbano, ibid.).

[0006] Although live attenuated vaccines are inherently less safe than the aforementioned vaccines, they are the most promising candidates for ASF vaccines. These vaccines avoid the key problems associated with inactivated vaccines and subunit, vector, and DNA vaccines. Because they can replicate successfully within the host, they mimic natural infection, triggering both humoral and cellular pathways, and generally do not require adjuvants. Furthermore, some live attenuated vaccines have shown the ability to induce mucosal IgA antibodies, a key characteristic of vaccines administered orally (oral immunization is a practical convenience for vaccines targeting wild boar populations). Nevertheless, these vaccines still carry risks, as they may revert to pathogenicity (i.e., virulence), potentially leading to disease transmission, and they have the potential to cause post-vaccination reactions and side effects, particularly in vulnerable animals. Three main strategies have been employed in the development of live attenuated ASF vaccines: attenuation through cell passage, screening of naturally attenuated strains, and deletion of virulence-related genes. To overcome some safety concerns associated with live attenuated vaccines, particularly residual virulence, attempts have been made to further delete virulence-related genes from naturally attenuated strains or to adapt heterologous cell lines to gene-deleted viruses.

[0007] As Urbano (ibid.) stated, several promising live attenuated vaccines developed between 2015 and 2022 are also available. Specifically, Lv17 / WB / Rie1, BA71ΔCD2v, HLJ / 18-7GD, ASFV-G-ΔI177L, ASFV-G-ΔI177L / ΔLVR, SY18ΔI226R, ASFV-G-ΔA137R, and ASFV-G-ΔE184L have shown promise as live ASF vaccines. Among these, the most promising live attenuated vaccine candidate to date is the ASFV-G-Δ1177L strain, engineered by the US Department of Agriculture's (USDA) Agricultural Research Service (ARS). It can be administered via intramuscular and oral-nasal routes to induce potent aseptic immunity against the virulent parent ASFV Georgia 2007 isolate (involved in recent outbreaks throughout Europe and Asia), and this vaccine strain has demonstrated efficacy in subsequent field trials against the virulent Vietnamese strain TTKN / ASFV / DN / 2019. The ASFV-G-Δ1177L strain has been available in Vietnam since 2022. Development of derivatives of ASFV-G-Δ1177L is also possible. For example, ASFV-G-Δ1177L / ΔLVR replicates efficiently in stable porcine epithelial cell lines and maintains the same levels of attenuation, immunogenicity, and protective potency in challenge studies. Other derivatives tested as experimental vaccines include ASFV-G-Δ9GL, ASFV-G-ΔMGF, ASFV-G-Δ9GL / ΔUK, ASFV-G-ΔI177L, and ASFV-G-ΔI177L / ΔLVR, all of which were engineered by the US Department of Agriculture's Agricultural Research Service. Summary of the Invention

[0008] The purpose of this invention is to provide an ASFV vaccine that can protect piglets against ASF virus.

[0009] To achieve the objectives of this invention, it has been found that vaccines comprising the attenuated live African swine fever virus Georgia 2007 Δ9GL / ΔUK (ASFV-G-Δ9GL / ΔUK) strain (known to be protective against ASFV infection in animals actually vaccinated) are safe for administration to pregnant sows (also known as swine) and can protect the offspring of sows vaccinated against ASFV, for example, through ingestion of colostrum from vaccinated sows. For this concept of protecting piglets through vaccination of sows, two things are crucial: the safety in pregnant sows (including their unborn offspring) and the efficacy in piglets.

[0010] Regarding safety, it is important to recognize that pregnant sows are particularly vulnerable to pathogens due to their high-demand metabolic state, especially leading to the loss of surviving offspring. This can be expressed as the loss of piglets that may have been born live and reached more than two weeks of age, specifically: piglets that reached at least 15-21 days of age. The objective, in particular, is to avoid any loss of more than 50% of the piglet population due to ASFV vaccination of the mother animal: that is, more than 50% of pregnant piglets stillborn or surviving for no more than two weeks.

[0011] To achieve the objectives of this invention, it has been found that a vaccine comprising the attenuated live African swine fever virus (ASFV) strain G-Δ9GL / ΔUK (known to be protective against ASFV infection in vaccinated animals) is safe for administration to pregnant sows and simultaneously induces protection in the offspring of vaccinated pregnant sows. In particular, the loss of surviving piglets can be very low, and can be as low as 10%, which is in the typical (naturally common) range of 5-25% loss in healthy pigs.

[0012] The above results were surprising to the inventors, particularly because known, supposedly safe vaccine strains appear to be unsafe for pregnant sows, and because no method for protecting offspring against ASF by vaccinating pregnant sows has previously been established in the art. Regarding safety, it has been found that for the ASF vaccine candidate generally considered the most promising, ASFV-G-Δ1177L, the loss of surviving piglets can be as high as 90%. While a loss of 50% of surviving offspring may still be acceptable for ASF vaccines in some cases, especially considering the criticality of maternal survival for pig farms, any value higher than 50% is considered unacceptable for commercial ASF virus vaccines. Nevertheless, lower losses, such as 40%, 30%, 25%, 20%, 15%, 10%, 5%, or any loss not exceeding that in negative control animals (i.e., a suitable control group of healthy pregnant sows not vaccinated against ASF), are particularly preferred.

[0013] In addition to the use of the attenuated live African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain in vaccines to protect piglets against African swine fever virus (ASFV) infection by administering a vaccine containing the attenuated live African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain to pregnant sows, the present invention also relates to the use of the attenuated live African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain in the preparation of vaccines to protect piglets against African swine fever virus infection, and to a method of protecting piglets against African swine fever virus infection by administering a vaccine containing the attenuated live African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain to pregnant sows, the piglets being part of the offspring of the pregnant sows.

[0014] It should be noted that the coding mutant of Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain is known in US 9,808,520, in which amino acids 11-68 of the wild-type 9GL protein and amino acids 1-85 of the wild-type UK protein are deleted (by deleting 173 and 255 nucleotides from the corresponding genes, respectively), thus functionally disabling the corresponding genes. However, exact deletions are not necessary for the genes themselves to become dysfunctional, which is common knowledge. The 9GL and UK genes can become functionally disabled by deleting slightly shorter, slightly longer, or slightly altered portions of these genes, as long as they are no longer expressed at normal levels (i.e., at the level of the unaltered parental strain), and especially no longer expressed at all.

[0015] definition Pigs are animals belonging to the family Suidae, especially domesticated pigs such as feeder pigs, which are raised for breeding or slaughter, and wild boars.

[0016] piglets Piglets are piglets aged 0 (immediately after birth) to at most 10 weeks old.

[0017] ASFV strain is ASFV Georgia 2007 (ASFV-G) strain, if it is the reference isolate Georgia 2007 (GenBank FR682468.2), as mentioned in US 9,808,520 and O'Donnel et al., Journal of Virology, January 2017, Volume 91, Issue 1, pp1-18; doi 10.1128 / JVI.01760-16), or a natural or recombinant variant of that reference isolate. When compared with the full-length of FR682468.2, it is used in this invention. ASFV Georgia 2007 The nucleotide sequence identity of the strain is preferably at least 99%, more preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher. Currently available molecular data obtained using standardized genotyping methods indicate that this ASFV variant exists only in Eastern and Central Europe after the 2007 outbreak in Georgia (Gallardo et al., Genetic Variation among African swinefever Genotype II Viruses, Eastern and Central Europe. Emerg Infect Dis. 2014Sep; 20(9): 1544-1547; doi: 10.3201 / eid2009.140554).

[0018] To perform nucleotide sequence alignment, NCBI's BLAST tool was used. TM The computer program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) selects the options "blastn" and "Align two or more sequences" with standard settings and default parameters, thereby selecting the sequence FR682468 as the target (also known as the "target").

[0019] ASFV-G- Δ9GL / ΔUK It is a variant of ASFV-G in which the 9GL and UK genes (viral gene IDs B119L and DP96R, respectively) are functionally disabled by the deletion of at least a portion of these genes, so that they may no longer be expressed at normal levels (i.e., at the level of the unaltered parental strain), and in particular, no longer expressed at all. As is generally known, the complete genes are not usually deleted for this purpose, as this would also introduce the risk of interfering with the transcription of two flanking genes in the ASFV genome, thus effectively resulting in the deletion of three genes instead of one. ASFV-G-Δ9GL / ΔUK does not exclude any further spontaneous or recombination mutations, such as those aimed at obtaining specific mutations suitable for distinguishing between infected and inoculated animals.

[0020] vaccineA vaccine is a suitable constitution for animal administration with an acceptable safety profile, containing an immunologically effective amount of one or more antigens, i.e., capable of adequately stimulating the target animal's immune system to induce an immune response against the antigen, and thereby against the corresponding naturally occurring antigen and potentially against naturally occurring pathogens, such as antibodies. It is typically combined with a pharmaceutically acceptable carrier (i.e., a biocompatible medium that, upon administration, does not induce significant adverse reactions in test animals and can present the antigen to the host animal's immune system after vaccination), such as a liquid containing water and / or any other biocompatible solvent, or a solid carrier such as that commonly used to obtain lyophilized vaccines (sugar- and / or protein-based), optionally containing an immunostimulant (adjuvant) that, upon administration to animals, induces an immune response capable of protecting the animal against (post-vaccination) infection. For any vaccine, acceptable safety is as important as efficacy in terms of protection.

[0021] Protecting animals against ASFV infection This refers to helping prevent, improve, or cure ASFV pathogenic infection, or helping to prevent, improve, or cure symptoms caused by the infection, such as preventing or alleviating one or more clinical signs caused by ASFV infection, preferably preventing animal death due to ASFV infection. For pregnant animals, the fetus carried by the animal (i.e., any unborn animal in the prenatal development stage) is considered part of the pregnant animal.

[0022] Survival The piglets are born live to sows and are able to reach more than two weeks of age, especially piglets that are at least 15-21 days old.

[0023] Pregnant pigs descendants A group (including one or more) of piglets produced from that pig, also known as the offspring of that pig.

[0024] Pregnant pigs Expected delivery date This is the estimated date of the end of gestation, which is set at approximately 115 days after fertilization for most pigs. Detailed Implementation

[0025] Other embodiments of the present invention In a first further embodiment of the attenuated live ASFV-G-Δ9GL / ΔUK strain used according to the invention, the vaccine is administered to pregnant pigs during the second half of gestation. Although early vaccination is feasible for protection, the second half of gestation is considered more critical for ASFV infection and vaccine safety. It has been found that administration of the attenuated live ASFV-G-Δ9GL / ΔUK strain during this stage of gestation and even in the latter third of gestation, most notably 5–20 days before the expected farrowing date of the pregnant pig, e.g., only 15 days before the expected farrowing date, is safe.

[0026] In another further implementation, the vaccine is administered to pregnant pigs once during pregnancy, or during pregnancy in a priming and booster regimen. In the priming and booster regimen, two administrations are provided during the same period of pregnancy, but at intervals of 1–12 weeks, typically 2–10 weeks, 2–8 weeks, 2–6 weeks, or 2–4 weeks.

[0027] In yet another further implementation, the vaccine is administered intramuscularly, intradermally, or orally.

[0028] In yet another further implementation plan, the vaccine is administered at least 10 doses per application. 2 TCID 50 The dosage of ASFV-G-Δ9GL / ΔUK strain should be, for example, higher than 10 mg per application. 3 10 4 Or 10 5 TCID 50 Until about 10 6 TCID 50 The dosage.

[0029] The invention will now be further explained using the following specific embodiments.

[0030] Example Example 1 is an experiment used to test the safety of a protective attenuated live ASFV-G vaccine strain in pregnant sows.

[0031] Example 2 is another experiment in which the safety of two additional protective attenuated live ASFV-G vaccine strains was tested in pregnant sows.

[0032] Example 3 is an experiment used to test the protection of offspring of vaccinated sows against ASFV. Example 1 Example 1 is an experiment testing the safety of the protective attenuated live ASFV-G vaccine strain ASFV-G-ΔI177L in pregnant sows. As is known from the literature, this strain is highly effective in protecting pigs against ASFV infection (Borca et al., Journal of Virology, April 2020, Volume 94, Issue 7, pages 1-18; doi10.1128 / / JVI.02017-19) and is safe for administration to pigs (Tran et al., Viruses 2022, 14: Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV-G-Δ I177L ). Research Objective The purpose of this experiment was to determine the safety of the ASFV-G-ΔI177L strain for use in pregnant sows. Research Design Four pregnant sows without ASFV or ASFV antibodies were used in this study. On day 100 of gestation (approximately two weeks before farrowing), two sows received 2 ml of vaccine containing ASFV-G-Δ1177L intramuscularly (IM) on the right side of the neck, at a dose of 1.5 × 10⁻⁶ ml. 2 TCID 50 Two sows served as unvaccinated controls. They received PBS similarly to the vaccinated animals. ASF-specific clinical signs in the animals were monitored daily starting the day before vaccination. Blood samples (from the tail vein) were collected at 4, 11, 23, and 32 days post-vaccination (dpv), and temperature was monitored daily starting the day before vaccination. The health status of piglets was monitored and recorded post-birth. All live piglets were weighed approximately 3–4 days after birth and again at 16–17 days post-birth. ASF-specific clinical signs in piglets were monitored daily until the end of the study.

[0036] result health status of the mother animal Rectal temperatures were normal (between 37.5°C and 39°C) and there was no significant difference between the vaccinated and control animals. The control animals did not exhibit any clinical signs of ASF. In the second week post-vaccination, one of the vaccinated animals showed mild signs of ASF, but these gradually subsided and disappeared. ASFV was not detectable in blood samples obtained from the control animals by PCR, while the vaccinated animals showed viral load in their blood at all time points.

[0037] Table 1 shows the significant difference in reproductive performance between inoculated and control sows. In the case of inoculated sows, 43% of piglets died at birth, compared to 17% in control sows. During the experiment, although all live-born piglets from control sows survived to the third week of age, only 4 piglets from inoculated sows survived to that time. Furthermore, all surviving piglets from inoculated sows exhibited ASF-specific clinical signs and were euthanized upon reaching the humane endpoint (HEP). This means that overall, the loss of surviving piglets due to inoculation with ASFV-G-Δ1177L exceeded 90%.

[0038] Table 1 Reproductive performance of the mother animal

[0039] piglet health status Throughout the experiment, the rectal temperature of piglets born to control animals was normal, ranging from 38.7°C to 40°C, while that of piglets born to inoculated animals reached as high as 41°C. Regarding weight gain, when weighed on days 3-4 and 17-18 postpartum, piglets from control sows showed an average weight gain of 2.9 times, while in the case of inoculated sows, surviving animals showed an average weight gain of 2.2 times.

[0040] Control piglets remained healthy throughout the experiment. In the inoculated animals, all live-born piglets exhibited ASF-related clinical signs, and ASFV infection was confirmed by PCR. Δ1177L-specific PCR of DNA extracted from the blood of pregnant sows and their representative piglets indicated vertical transmission of the vaccine strain from pregnant sows to their piglets.

[0041] in conclusion By comparing the health status of sows (and piglets born from these sows), especially given the (significant) loss of surviving offspring exceeding 50%, it can be concluded that the vaccine strain ASFV-G Δ1177L is unsafe for use in pregnant sows.

[0042] Example 2 Example 2 is another experiment in which the safety of two additional protective attenuated live ASFV-G vaccine strains was tested in pregnant sows. The first vaccine candidate was the ASFV-G strain, which contained additional gene deletions ΔEP153R and ΔEP402R (see Petrovan et al., Journal of Virology, January 2022, Volume 96, Issue 1, pp 1-19 for details on deletions). The parental strain X (which is a proprietary ASFV-G strain) was safe and effective for use in pigs (data available, but not shown here). Therefore, the double knockout mutant strain (denoted here as X-ΔCD) was expected to be safe and effective for use in pigs.

[0043] The second vaccine candidate tested was ASFV-G Δ9GL / ΔUK (US9,808,520). This strain is also known to be protective and safe for swine vaccination, according to literature (iaUrbano, ibid.). Research Objective The purpose of this experiment was to determine the safety of these two additional ASFV-G strains for use in pregnant pigs.

[0045] Research Design Six pregnant sows without ASFV or ASFV antibodies were used in this study. At approximately 100 days of gestation (about 2 weeks before farrowing), two sows received 2 ml of vaccine containing strain X-ΔCD intramuscularly (IM) on the right side of the neck, at a dose of 10... 3 TCID 50 Two sows received the corresponding vaccine containing the candidate vaccine strain ASFV-G-Δ9GL / ΔUK at a dose of 10 mg / L on the right side of their neck (IM). 4 TCID 50 The remaining two sows served as unvaccinated controls. ASF-specific clinical signs were monitored in the animals from the start of vaccination. Blood samples were collected at 3, 10, 21, and 31 days post-vaccination (dpv), and temperature was monitored daily from the day of vaccination. The health status of piglets was monitored and recorded post-birth. In the case of any stillbirth or death, blood samples were collected before disposal of the animal whenever possible. All live piglets were weighed at 5 and 19 days post-birth. ASF-specific clinical signs were monitored daily in the piglets until the end of the study at 22 days post-birth.

[0046] result health status of the mother animal The rectal temperatures of the control animals and those receiving ASFV-G-Δ9GL / ΔUK were normal (between 37.5°C and 39.5°C), while the temperatures of the other animals inoculated with X-ΔCD remained elevated, and ASF signs began to appear on day 4 post-inoculation and persisted. Two sows in this group were euthanized on day 10 post-inoculation. Piglets from these sows were either prematurely born during abortion or removed from the uterus at the time of euthanasia. One of the control animals and the animals inoculated with ASFV-G-Δ9GL / ΔUK did not show any clinical signs of ASF. The other animal inoculated with ASFV-G-Δ9GL / ΔUK showed mild signs of ASF. ASFV was undetectable in the control animals by PCR, while the inoculated animals showed the virus in their blood at all time points.

[0047] Regarding reproductive performance, the sows used in the study had a healthy breeding history, with a total piglet survival rate exceeding 80%. However, significant differences in reproductive performance were observed in sows vaccinated with two different candidate vaccine strains in this study. In the case of sows vaccinated with the X-ΔCD strain, piglets were born prematurely 8 to 10 days before their expected expiration date. Both sows began aborting piglets, which were weak, underdeveloped, and had no chance of survival. These piglets were euthanized at birth due to reaching HEP (Heat Emission Precipitation). A total of three piglets were stillborn.

[0048] In sows vaccinated with ASFV-G-Δ9GL / ΔUK, all live-born piglets survived until the end of the study. One sow had three stillborn piglets, while another had none. Parturition was successful and under normal conditions. In the control animals, parturition was under normal conditions, and all live-born animals survived until the end of the study. The reproductive performance of the sows in this study is shown in Table 2 below.

[0049] Table 2 Reproductive performance of the mother animal

[0050] Of the 11 animals that did not reach the end of the study, 8 died in the accident (they were trapped under the sow). Therefore, the adjusted loss was 16%. piglet health status Since none of the piglets born to sows inoculated with X-ΔCD survived beyond one day postpartum, only the health status of other animals was monitored. Rectal temperatures of all piglets were normal throughout the experiment and showed no difference between control and inoculated animals. Regarding weight gain, piglets from control sows showed an average weight gain of 3.7 times from birth, while those from inoculated sows showed an average weight gain of 2.7 times.

[0052] Control piglets remained healthy throughout the experiment. Of the inoculated animals, 13 live-born piglets exhibited mild ASF-related clinical signs, and one animal had moderate signs. ASFV infection was confirmed by PCR. Specific PCR of DNA extracted from the blood of pregnant sows and their representative piglets indicated vertical transmission of the candidate vaccine strain from pregnant sows to their piglets. Five piglets were viremic at 5 days of age. Viremia peaked at 12 days and then gradually declined. Notably, the viremic piglets remained healthy and survived to the end of the study.

[0053] in conclusion By comparing the health status of sows (and piglets born from these sows) with that of control sows, particularly given the loss of more than 50% of surviving offspring, it can be concluded that the X-ΔCD strain is unsafe for use in pregnant sows, while ASFV-G-Δ9GL / ΔUK, which does not even result in a higher loss of surviving piglets than the control (10% vs. 35% / 16%), is considered safe for use in pregnant sows.

[0054] Example 3 The aim of this study was to evaluate whether 2-week-old offspring of pregnant sows were protected against African swine fever virus challenge by inoculating their mothers with the modified live virus ASFV-G-Δ9GL / ΔUK of Example 2 in late gestation. For this study, six sows were inoculated and divided into two groups. The study setup was essentially the same as in Example 2, although protection against ASF was now actually tested on the offspring of the inoculated sows. Study details are shown in Table 3.

[0055] Following the challenge, animals in the unvaccinated control group (Group 3) developed ASF-related clinical signs and did not survive the challenge infection. Offspring of Group 1, who were viremic on the day of challenge, were protected against the challenge. Sows and their offspring in Group 2 were not challenged and served as a negative control group; they survived until the end of the study.

[0056] Table 3 Setup of a sow vaccination study

[0057] This study demonstrates that vaccine virus viremia in offspring is an important parameter for protection against challenge infections: vaccine strains received by piglets from their mothers during gestation (via vertical transmission of the virus) or post-gestation (e.g., via colostrum) induce a protective immune response in offspring. Although not all piglets are likely to become viremic, vaccine strain viremia may substantially contribute to protection against ASF virus in the piglet herd.

Claims

1. A live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain for use in a vaccine, for protecting piglets against infection with African swine fever virus (ASFV) by administering a vaccine containing the live attenuated ASFV-G-Δ9GL / ΔUK strain to pregnant sows, said piglets being part of the offspring of said pregnant sows.

2. The attenuated live ASFV-G-Δ9G1 / ΔUK strain used according to claim 1, characterized in that... The vaccine is administered to the pregnant pigs during the second half of pregnancy.

3. The attenuated live ASFV-G-Δ9GL / ΔUK strain used according to any one of the preceding claims, characterized in that... The vaccine was administered to the pregnant pigs during the second third of their gestation period.

4. The attenuated live ASFV-G-Δ9GL / ΔUK strain used according to any one of the preceding claims, characterized in that... The vaccine is administered to the pregnant sows during a period of 5-20 days prior to their expected farrowing date.

5. The attenuated live ASFV-G-Δ9GL / ΔUK strain used according to any one of the preceding claims, characterized in that... The vaccine is administered to the pregnant pigs once during pregnancy or in a primary and booster regimen.

6. The attenuated live ASFV-G-Δ9GL / ΔUK strain used according to any one of the preceding claims, characterized in that... The vaccine can be administered intramuscularly, intradermally, or orally.

7. The attenuated live ASFV-G-Δ9GL / ΔUK strain used according to any one of the preceding claims, characterized in that... The vaccine is administered at least 10 doses per administration. 2 TCID 50 Dosage of ASFV-G-Δ9GL / ΔUK strain.

8. The attenuated live ASFV-G-Δ9GL / ΔUK strain used according to any one of the preceding claims, characterized in that... The vaccine is administered in doses of 10 mg per administration. 2 TCID 50 Up to 10 6 TCID 50 Dosage of ASFV-G-Δ9GL / ΔUK strain.

9. Use of attenuated live African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain in the preparation of a vaccine for protecting piglets against infection with African swine fever virus by administering a vaccine containing the attenuated live ASFV-G-Δ9GL / ΔUK strain to pregnant sows, said piglets being part of the offspring of said pregnant sows.

10. A method for protecting piglets against infection with African swine fever virus, comprising administering a vaccine to pregnant sows containing a live attenuated African swine fever virus strain Georgia 2007 (ASFV-G) Δ9GL / ΔUK, the piglets being part of the offspring of the pregnant sows.

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