A VACCINE TO PROTECT A PREGNANT SOW AGAINST AFRICAN SWINE FEVER

Abstract

The invention pertains to a live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain for use in a vaccine for protecting a pregnant swine against an infection with African swine fever virus (ASFV) by administering the vaccine comprising the live attenuated ASFV-G-Δ9GL / ΔUK strain to the pregnant swine.

Description

1 / 18 A VACCINE TO PROTECT A PREGNANT SOW AGAINST AFRICAN SWINE FEVER FIELD OF THE INVENTION

[001] African swine fever virus (ASFV) is one of the most important disease-causing pathogens affecting the domestic swine population worldwide. The present invention relates to a vaccine to protect swine against ASFV infection, in particular the vulnerable group of pregnant sows. BACKGROUND OF THE INVENTION

[002] 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 severe and highly infectious viral hemorrhagic disease in pigs, endemic to Sub-Saharan Africa (24 genotypes based on the c-terminal sequence of the p72 surface antigen) and the island of Sardinia in Italy (p72 genotype I). Transcontinental spread of ASF has occurred on at least three separate occasions, most significantly to Georgia in 2007, where it spread from the Black Sea port of Poti, along the Caucasus region to the Russian Federation (RF) and Eastern Europe. In the following decade, the disease became epizootic in the Russian Federation and, in 2018, it spread westward to Belgium and eastward to the People's Republic of China, quickly taking over most of Southeast Asia and Oceania.All isolates discovered in these regions are related to the Georgia 2007 isolate, commonly referred to as ASFV Georgia 2007 or ASFV-G (US 9,808,520). Since 2018, the epidemiological situation of ASF has continued to deteriorate; in January 2022, the ASF virus reappeared on the Italian mainland. Since then, several recurrences have been reported in China, the Federal Republic of Mexico, Moldova, and Ukraine, and North Macedonia reported its first occurrence, as did Thailand, one of the... Petition 870250080553, dated 08 / 09 / 2025, page 88 / 110 2 / 18 Few countries in the region had remained unscathed. The virus also appeared in 2021 in the Dominican Republic and Haiti, constituting the first diagnosis of ASFV in over 40 years in the Western Hemisphere. These recent events highlight an extremely disconcerting pattern of ongoing spread, exacerbated by the fact that in many of these regions small-scale and semi-industrial farms account for the majority of pig production. As such, outbreaks bring serious socioeconomic consequences, devastating rural livelihoods dependent on livestock production and threatening overall market stability and food security, as well as severely affecting animal welfare.

[003] African swine fever virus (ASFV) is a large double-stranded nucleocytoplasmic DNA arbovirus, the only member of the Asfarviridae family. Virions are about 250 nm in diameter and consist of a central nucleoid surrounded by an icosahedral protein capsid (or core wall), an inner lipoprotein membrane (or inner envelope), an outer icosahedral protein capsid, and an outer lipoprotein envelope (or outer envelope) that is acquired when the virus buds through the plasma membrane. Both intracellular and extracellular viral forms are infectious. Its natural host range is limited to soft-bodied ticks of the genus Ornithodoros and members of the family Suidae, where it replicates primarily in cells of the mononuclear phagocytic system, resident macrophages, and specific reticular cells. The virus is endemic to African wild pigs.In domestic pigs and wild boars, however, clinical signs vary considerably, and the individual outcome can range from fatal to subclinical. Depending on the virulence of the strain involved, a graded series of forms occurs, with lethality ranging from 100% (for the acute form) to <30% (chronic form) of infected animals. The genotype II strain that currently affects... Petition 870250080553, dated 08 / 09 / 2025, pages 89 / 110 3 / 18 In Europe and Asia, it is highly virulent, causing the acute form of the disease, although there is evidence that some isolates of reduced virulence may be circulating among wild boars in the Baltic States and domestic pigs in China, with reports of both naturally mutated low-virulence genotype II strains and low-virulence epidemic genotype I strains detected in the field.

[004] Protective immunity against ASFV is still poorly understood. Several vaccine approaches are known in the state of the art, but this has not yet led to a commercially available vaccine. The first approach described in the art is vaccination with an inactivated vaccine. Virus inactivation is an established approach to vaccine production, relatively simple to achieve and, more importantly, with a higher safety profile when compared to live vaccines. The inactivation process negates reversion to a virulent phenotype and renders vaccine viruses non-transmissible, the two main disadvantages of attenuated vaccines. Furthermore, it is believed that safety for more vulnerable patient groups, such as very young animals and pregnant females, is of lesser risk. Animals in the latter group undergo significant physiological changes and are thus in a stressful metabolic state.Inactivation, however, does not necessarily produce a vaccine that elicits protective immunity. Attempts to immunize pigs with a variety of inactivated ASF antigens have not led to sufficient protection, although in some cases they have been able to induce a serological immune response (Cadenas-Fernández, et al. Vaccines, 2021, 9, 242; doi.org / 10.3390 / vaccines9030242).

[005] Another approach is the use of subunit, DNA, and viral vector vaccines. These vaccines are promising, and several candidates have shown to induce specific humoral and / or cellular immune responses that appear to confer Petition 870250080553, dated 08 / 09 / 2025, pages 90 / 110 4 / 18 partial to total protection. However, the different nature of the immunization protocols used in these studies, including the type of vaccine, the vaccination strategy, and the challenge model, makes the results difficult to compare. Further work will be needed to identify which immunological mechanisms need to be triggered to confer complete and lasting protection, which antigens (or combination thereof) should be included in a potential vaccine, and the most appropriate delivery method (Urbano, supra).

[006] Live attenuated vaccines, while inherently less safe than the vaccines mentioned above, are the most promising candidate vaccines against ASF. These vaccines circumvent a key problem presented by inactivated, subunit, vector, and DNA vaccines. Because they can successfully replicate within the host, they mimic natural infection, triggering humoral and cellular pathways, and typically do not require adjuvants. Additionally, some live attenuated vaccines have been shown to elicit mucosal IgA antibodies, an important feature for orally administered vaccines (oral immunization is a practical convenience for vaccines intended for wild boar populations). That said, these vaccines also pose a risk, as they can regain pathogenicity (i.e., revert to virulence), causing disease spread, and have the potential to cause post-vaccination reactions and side effects, particularly in vulnerable animals.Three main strategies were employed in the generation of live attenuated ASF vaccines: attenuation by cell passage, screening of naturally attenuated strains, and deletion of virulence-associated genes. To overcome some of the safety problems presented by live attenuated vaccines, particularly residual virulence, attempts were also made to further delete virulence-associated genes in naturally attenuated strains, or to adapt them to heterologous cell lines. Petition 870250080553, dated 08 / 09 / 2025, pages 91 / 110 5 / 18 viruses with deleted genes. Depending on the immunogenicity of the deleted genes, these candidates may also be suitable for a vaccination protocol known as 'DIVA': differentiation of naturally infected animals from vaccinated animals.

[007] As indicated in Urbano (above), meanwhile, about 10 promising live attenuated vaccines are available, developed between 2015 and 2022. In particular, NH / P68, OURT88 / 3, Lv17 / WB / Rie1, BA7^CD2v, HLJ / 18-7GD, ASFV-G-W177L, ASFV-G-W177L / âLVR, SY18W226R, ASFV-GΔA137R and ASFV-G^E184L are indicated to be available as live vaccines against ASF. Among these, the most promising live attenuated vaccine candidate to date is the ASFV-G^I177L strain, which was developed by the Agricultural Research Service (ARS) of the U.S. Department of Agriculture (USDA). It can be administered via intramuscular and oronasal routes, inducing robust sterile immunity against challenge with the virulent parental isolate ASFV Georgia 2007, involved in recent outbreaks throughout Europe and Asia, and in particular this vaccine strain has proven effective in follow-up field trials against the virulent Vietnamese strain TTKN / ASFV / DN / 2019.Since 2022, the ASFV-G^I177L strain has been on the market in Vietnam through a conditional license. The development of ASFV-G^I177L-derived strains is also in progress. For example, ASFV-G^I177LMLVR replicates efficiently in a stable porcine epithelial cell line and maintains the same level of attenuation, immunogenic characteristics, and protective efficacy in challenge studies. Other derivatives being tested as experimental vaccines are ASFV-G-α9GL, ASFV-G^MGF, ASFV-G-α9GLMUK, ASFV-GΔI177L, and ASFV-G^I177LMLVR, all developed by the Agricultural Research Service of the U.S. Department of Agriculture. OBJECTIVE OF THE INVENTION Petition 870250080553, dated 08 / 09 / 2025, pages 92 / 110 6 / 18

[008] One objective of the invention is to provide an ASFV vaccine that is safe for administration to pregnant sows, an animal that, due to its highly demanding metabolic status, is particularly vulnerable to infections by a pathogenic microorganism, leading, in particular, to the loss of viable offspring. This can be expressed as the loss of piglets that could have been born alive and are capable of reaching an age of more than two weeks, in particular: capable of reaching an age of at least 15-21 days. The objective is, in particular, to avoid losses in the number of piglets exceeding 50%: that is, more than 50% of the piglets born are stillborn or do not survive for more than two weeks due to the ASFV vaccine being administered to the sow. SUMMARY OF THE INVENTION

[009] In order to meet the objective of the invention, it has been found that a vaccine comprising a live attenuated Georgia 2007 African swine fever virus (ASFV-G) Δ9GL / ΔUK strain, which is known to be protective against ASFV infection, is safe for administration to pregnant sows. In particular, the loss of viable piglets can be very low, as low as 10%, which is within the typical (common natural) loss range of 5-25% for healthy pigs.

[010] For the inventors, this was a surprise, particularly because the loss of viable piglets for what is commonly considered the most promising ASF vaccine candidate, namely ASFV-G^I177L, can be as high as 90%. While a 50% loss of viable offspring might, under certain circumstances, still be acceptable for an ASF vaccine, particularly considering the fact that the survival of the dams is crucial for a swine facility, any value above 50% is considered unacceptable for a commercial ASF virus vaccine. Still, a lower loss, Petition 870250080553, dated 08 / 09 / 2025, pages 93 / 110 7 / 18 such as 40%, 30%, 25%, 20%, 15%, 10%, 5% or any loss that is not higher than a loss in negative control animals (i.e., an adequate control group of healthy pregnant sows that have not been given the ASF vaccine) is particularly preferable.

[011] In addition to a live attenuated Georgia 2007 African swine fever virus (ASFV-G) Δ9GL / ΔUK strain for use in a vaccine to protect pregnant sows against African swine fever virus (ASFV) infection by administering the vaccine comprising the live attenuated ASFV-G^9GLMUK strain to pregnant sows, the invention also relates to the use of a live attenuated Georgia 2007 African swine fever virus (ASFV-G) Δ9GL / ΔUK strain for the manufacture of a vaccine for the protection of pregnant sows against African swine fever virus infection, and to a method for protecting pregnant sows against African swine fever virus infection by administering to pregnant sows a vaccine comprising a live attenuated Georgia 2007 African swine fever virus (ASFV-G) Δ9GL / ΔUK strain. DEFINITIONS

[012] A pig is an animal that belongs to the Suidae family, in particular a porcine animal raised by man, for example, to be a fattening pig, raised for breeding stock or raised for slaughter, and wild boars.

[013] An ASFV strain is an ASFV Georgia 2007 strain (ASFV-G) if it is in the Georgia 2007 reference isolate (GenBank FR682468.2), as mentioned in US document 9,808,520 and in O'Donnel et al. in the Journal of Virology, January 2017, Volume 91, Issue 1, pp1-18; doi 10.1128 / JVI.01760-16), or a natural or recombinant variant of this reference isolate. Preferably, the nucleotide sequence identity of an ASFV Georgia 2007 strain for use in the invention is at least 99%, more preferably at least 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, Petition 870250080553, dated 09 / 08 / 2025, pp. 94 / 110 8 / 18 99.9% or even higher, when aligned with the total length of FR682468.2. Currently available molecular data, derived using standardized genotyping procedures, indicated that only this variant of ASFV is present in Central and Eastern Europe after the outbreak in Georgia in 2007 (Gallardo et al. Genetic Variation among African swine fever Genotype II Viruses, Eastern and Central Europe. Emerg Infect Dis. September 2014; 20(9): 1544-1547; doi: 10.3201 / eid2009.140554).

[014] To perform nucleotide sequence alignments, the NCBI Blast™ computer program is used (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), selecting the 'blastn' and “Align two or more sequences” options with default settings and parameters, and where the FR682468 sequence is selected as the objective (also known as “the target”).

[015] ASFV-G-Δ9GL / ΔUK is a variant of ASFV-G in which the 9GL and UK genes (viral gene numbers B119L and DP96R, respectively) have been functionally deactivated by deleting at least part of these genes, so that they can no longer be expressed at a normal level (i.e., at the level of the unaltered parental strain), in particular, they are no longer expressed at all. As is commonly known, this typically does not involve deleting the entire gene, as this also carries the risk of interfering with the transcription of the two flanking genes in the ASFV genome and thus deleting three genes instead of one. ASFV-G^9GLMUK does not exclude any additional mutations, spontaneous or recombinant, such as specific mutations intended to arrive at a strain suitable for differentiating between infected and vaccinated animals.

[016] A vaccine is a composition suitable for application to an animal, having acceptable safety, comprising one or more antigens. Petition 870250080553, dated 08 / 09 / 2025, pages 95 / 110 9 / 18 in an immunologically effective quantity, that is, capable of stimulating the immune system of the target animal sufficiently to induce an immune response, such as antibodies, against the antigens and thereby against the corresponding naturally occurring antigens and thus potentially the naturally occurring pathogen, typically combined with a pharmaceutically acceptable vehicle (i.e., a biocompatible medium, namely a medium that after administration does not induce significant adverse reactions in the individual animal, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine), such as a liquid containing water and / or any other biocompatible solvent or a solid vehicle such as commonly used to obtain lyophilized vaccines (based on sugars and / or proteins), optionally comprising immunostimulating agents (adjuvants),When administered to an animal, these vaccines induce an immune response capable of protecting the animal against infection (post-vaccination). For any vaccine, the requirement of acceptable safety is as important as efficacy in providing protection.

[017] Protecting an animal against ASFV infection means assisting in preventing, improving, or curing a pathogenic ASFV infection or assisting in preventing, improving, or curing a disorder resulting from the infection, for example preventing or reducing one or more clinical signs resulting from ASFV infection, preferably preventing the death of the animal as a result of ASFV infection. For a pregnant animal, fetuses carried by the animal (i.e., any unborn animal in the prenatal developmental stages) are considered part of the pregnant animal.

[018] Viable piglets are piglets born alive to a sow and capable of reaching an age of more than two weeks, in particular, reaching an age of at least 15-21 days. Petition 870250080553, dated 09 / 08 / 2025, pp. 96 / 110 10 / 18 ADDITIONAL EMBODIMENTS OF THE INVENTION

[019] In a first additional embodiment of the live attenuated ASFV-G^9GLMUK strain for use as a vaccine in protecting pregnant sows against African swine fever virus (ASFV) infection, when administering the vaccine comprising the live attenuated ASFV-G^9GLMUK strain to pregnant sows, the vaccine is administered to pregnant sows in the second half of gestation. Although early vaccination is feasible for protection, the second half of gestation is believed to be more critical for ASFV infection and for the safety of a vaccine. A live attenuated ASFV-G^GL / Δυκ strain has been found to be safe for administration at this stage of gestation and even in the last third of gestation, particularly in a period of 5-20 days before the expected date of farrowing of pregnant sows, for example, only 15 days before the expected date of farrowing.

[020] In another additional embodiment, the vaccine is administered once during gestation to pregnant sows, or it is administered in a primary and booster regimen during gestation. In a primary and booster regimen, the two administrations are given during the same gestation, but are separated in time by 1-12 weeks, typically by 2-10, 2-8, 2-6 or 2-4 weeks.

[021] In yet another additional method, the vaccine is administered intramuscularly, intradermally, or orally.

[022] In yet another additional embodiment, the vaccine is administered with a dose of the ASFV-G^9GLMUK strain of at least 102TCID50 per administration, for example, a dose above 103, 104 or 105TCID50 up to about 106TCID50 per administration.

[023] The invention will now be explained further using the following specific examples. EXAMPLES Petition 870250080553, dated 09 / 08 / 2025, pp. 97 / 110 11 / 18

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

[025] Example 2 is another experiment in which two other strains of additional live attenuated ASFV-G protective vaccine are tested for their safety in pregnant sows.

[026] Example 3 is an experiment to confirm the efficacy of the live attenuated ASFV-G vaccine strain. EXAMPLE 1

[027] Example 1 is an experiment to test the safety of the live attenuated ASFV-G protective vaccine strain ASFV-G^I177L in pregnant sows. As is known from the literature, this strain is highly effective in protecting swine against ASFV infection (Borca et al, Journal of Virology, April 2020, Volume 94, Issue 7, pages 1-18; doi 10.1128 / / JVI.02017-19) and safe for administration in swine (Tran et al. in Viruses 2022, 14: Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV-G-AI177L). Study objective

[028] The aim of this experiment was to establish the safety of the ASFV-G^I177L strain for administration in pregnant sows. Study project

[029] Four pregnant sows free of ASFV and without antibodies against ASFV were available for this study. At 100 days of gestation (i.e., approximately 2 weeks before farrowing), two pigs received 2 mL of a vaccine containing ASFV-G-ΔI177L intramuscularly (IM) in the right side of the neck at a dose of 1.5x102TCID50. Two sows served as unvaccinated controls. They received PBS similar to the vaccinated animals. The animals were monitored daily from the day before vaccination in Petition 870250080553, dated 08 / 09 / 2025, pages 98 / 110 12 / 18 regarding specific clinical signs of ASF. Blood samples (from the tail vein) were collected 4 days post-vaccination (dpv), 11 dpv, 23 dpv, and 32 dpv, while temperature was monitored daily from the day before inoculation onward. After birth, the health status of the piglets was monitored and recorded. All live piglets were weighed approximately 3-4 days after birth, as well as 16-17 days after birth. Piglets were monitored daily for specific clinical signs of ASF until the end of the study. Results Health status of the mothers

[030] Rectal temperatures were normal (between 37.5 °C and 39 °C) and not significantly different between vaccinated animals versus control animals. Control animals did not exhibit any clinical signs of ASF. In the second week after vaccination, one of the vaccinated animals showed mild signs of ASF, but these gradually decreased and disappeared. With PCR, no ASFV could be detected in blood samples obtained from control animals, while vaccinated animals showed virus in their blood at all times.

[031] Table 1 shows significant differences in the reproductive performance of vaccinated and control sows. In the case of vaccinated sows, 43% of piglets were stillborn, compared to 17% in control sows. During the experiment, while all live-born piglets from control sows survived to the third week of age, only 4 of the piglets born from vaccinated sows survived to that period. Furthermore, all surviving piglets from vaccinated sows showed specific clinical signs of ASF and were euthanized upon reaching the human endpoint (HEP). Petition 870250080553, dated 08 / 09 / 2025, pages 99 / 110 13 / 18 This means that, overall, there was a loss of more than 90% in viable piglets due to vaccination with ASFV-G^I177L. Table 1: Reproductive performance of the dams Vaccinated control Total number of piglets 41 46 Normal piglets at birth 14 36 Weak piglets at birth 9 2 Dead piglets at birth 18 8 Live piglets at the end of the test 4 38 Loss of viable piglets 90% 17% Health status of piglets

[032] The rectal temperatures of piglets born from control animals were normal throughout the experiment, ranging from 38.7 to 40 °C, while they reached 41 °C in piglets born from vaccinated animals. Regarding weight gain, piglets from control sows showed, on average, a 2.9-fold weight increase when weighed on days 3-4 and 17-18 after farrowing, while in the case of vaccinated sows, the surviving animals showed, on average, a 2.2-fold weight increase.

[033] The control piglets were all healthy throughout the experiment. Of the vaccinated animals, all live-born piglets showed clinical signs related to ASF, and ASFV infection was confirmed by PCR. A ΔI177L-specific PCR performed on DNA extracted from the blood of pregnant sows and their representative piglets indicated that the vaccine strain was transmitted vertically from pregnant sows to their piglets. Conclusion

[034] Comparing the health status of sows (and piglets born alive from those sows), in particular because the loss of viable offspring is (significantly) greater than 50%, it can be concluded that the ASFVG ΔI177L vaccine strain is not safe for administration in pregnant sows. Petition 870250080553, dated 08 / 09 / 2025, pages 100 / 110 14 / 18 EXAMPLE 2

[035] Example 2 is another experiment in which two additional live attenuated ASFV-G protective vaccine strains were tested for safety in pregnant sows. The first vaccine candidate was derived from the Lv17 / WB / Rie1 strain (WO 2020 / 049194). In particular, this first vaccine comprises a mutant designated as Lv17 / WB / Rie1^CD, which includes the additional genetic deletions ΔEP153R and ΔEP402R (see Petrovan et al, Journal of Virology, January 2022, Volume 96, Issue 1, pp 119). The parental strain Lv17 / WB / Rie1 is safe and effective for use in swine (Urbano, supra) and the double knockout mutant strain is also known to be (inherently safe and) effective for use in swine (European Patent Application No. EP22462011).1 for Attenuated African swine fever virus and use thereof in vaccine compositions, filed on behalf of Intervet International BV, Consejo Superior de Investigaciones Científicas (CSIC), Állatorvostudományi Kutatóintézet, Universidad Complutense de Madrid and Eurofins Ingenasa SA, on November 22, 2022 at the Hungarian Intellectual Property Office). The second strain is known in the literature (ia Urbano, supra) to be protective and safe for swine vaccination.

[036] The second vaccine candidate tested was ASFV-G^9GLMUK (US 9,808,520). This strain is also known in the literature (ia Urbano, supra) to be protective and safe for swine vaccination. Study objective

[037] The aim of this experiment was to establish the safety of these two additional ASFV-G strains for administration in pregnant sows. Study Project

[038] Six pregnant sows free of ASFV and free of antibodies against ASFV were available for this study. Around 100 days of gestation Petition 870250080553, dated 08 / 09 / 2025, pages 101 / 110 On 15 / 18 (i.e., approximately 2 weeks before farrowing), two sows received 2 mL of vaccine comprising the Lv17 / WB / Rie1^CD strain intramuscularly (IM) in the right side of the neck at a dose of 103 TCID50. Two sows received a corresponding vaccine containing the ASFV-G^9GL^UIK candidate vaccine strain IM in the right side of the neck at a dose of 104 TCID50. The two remaining sows served as uninoculated controls. The animals were monitored from vaccination onwards for specific clinical signs of ASF. Blood samples were collected 3 days post-vaccination (dpv), 10 dpv, 21 dpv, and 31 dpv, while temperatures were monitored daily from the day of inoculation onwards. After birth, the health status of the piglets was monitored and recorded. In the case of stillborn or dead piglets, a blood sample was collected, whenever possible, before the animal was disposed of.All live piglets were weighed 5 days after birth, as well as 19 days after birth. Piglets were monitored daily for specific clinical signs of ASF until the end of the study, 22 days after birth. Results Health status of the mothers

[039] Rectal temperatures were normal (between 37.5 °C and 39.5 °C) for the control animals and the animals that received ASFV-G^9GL^UIK, while the other animals vaccinated with Lv17 / WB / Rie1^CD had a continuous increase in temperature and showed signs of ASF from the 4th day after vaccination onwards. Both sows in this group were euthanized on the 10th day after vaccination. The piglets of these sows were born prematurely, as they were in the process of abortion, or were removed from the uterus for euthanasia. The control animals and one of the animals vaccinated with ASFV-G^9GLMUK did not exhibit any clinical signs of ASF. The other animal Petition 870250080553, dated 08 / 09 / 2025, pages 102 / 110 16 / 18 vaccinated with ASFV-G^9GLMUK showed mild signs of ASF. With PCR, no ASFV could be detected in the control animals, while the vaccinated animals showed virus in the blood at all times.

[040] Regarding reproductive performance, the sows used in the study had a history of healthy reproduction, with over 80% of viable piglets born from them. In this study, however, significant differences were observed in the reproductive performance of sows vaccinated with the two different candidate vaccine strains. Piglets were born prematurely 8 to 10 days before the expected date in the case of sows vaccinated with the Lv17 / WB / Rie1^CD strain. Both sows began to abort piglets, which were weak and underdeveloped, with no chance of survival. These piglets were sacrificed at birth due to reaching HEP. In total, three piglets were stillborn.

[041] In the case of sows vaccinated with ASFV-G^9GLMUK, all piglets born alive survived until the end of the study. One sow had 3 stillborn piglets, while the other had none. The farrowing was uneventful and under normal circumstances. For the control animals, farrowing was under normal circumstances and all animals born alive survived until the end of the study. The reproductive performance of the sows in this study is presented in Table 2 below. Table 2: Reproductive performance of the dams Lv17 / WB / Rie1^CD ASFV-GΔ9GL / ΔUK control Total number of piglets 26 41 43 Normal piglets at birth 0 37 39 Weak piglets at birth 23 1 0 Dead piglets at birth 3 3 4 Petition 870250080553, dated 08 / 09 / 2025, pages 103 / 110 17 / 18 Lv17 / WB / Rie1^CD ASFV-GΔ9GL / ΔUK control Live piglets born at the end of the test 0 34 28 Viable piglet loss 100% 10% 35%* * 8 of the 11 animals that did not reach the end of the study died in an accident (they got trapped under the sow). The corrected loss is therefore 16%. Health status of piglets

[042] As none of the piglets born to sows vaccinated with Lv17 / WB / Rie1^CD were alive for more than 1 day after farrowing, health status was monitored only for the other animals. Rectal temperatures of all piglets were normal throughout the experiment and were not different between control and vaccinated animals. Regarding weight gain, piglets from control sows showed, on average, a 3.7-fold increase in birth weight, while in the case of vaccinated sows, piglets showed, on average, a 2.7-fold increase in weight.

[043] The control piglets were all healthy throughout the experiment. Of the vaccinated animals, thirteen live-born piglets showed mild clinical signs related to ASFV and one animal had moderate signs. ASFV infection was confirmed by PCR. A specific PCR performed on DNA extracted from the blood of pregnant sows and their representative piglets indicates that the candidate vaccine strain was transmitted vertically from pregnant sows to their piglets. Five piglets were viremic 5 days after birth. Viremia peaked at 12 days, after which it gradually decreased. Notably, the viremic piglets remained healthy and alive until the end of the study. Petition 870250080553, dated 08 / 09 / 2025, pages 104 / 110 18 / 18 Conclusion

[044] Comparing the health status of sows (and piglets born alive from these sows) with control sows, particularly because the loss of viable offspring is greater than 50%, it can be concluded that the Lv17 / WB / Rie1^CD strain is not safe for administration in pregnant sows, while ASFV-G^9GLMUK, not even leading to a higher loss of viable piglets than the control (10% vs 35% / 16%), is considered safe for vaccination of pregnant sows. EXAMPLE 3

[045] The objective of this study was to provide confirmation that the ASF vaccine as used in Example 2, i.e., MLV ASFV-G^9GL / ÚUIK, was indeed protective for sows that received the vaccine during the third trimester of gestation. For this, three groups of animals were used: three sows from group 1, three sows from group 2, and 2 negative control animals.

[046] The sows in group 1, which were vaccinated during the third trimester of gestation, were viremic and in fact completely protected against a virulent challenge (data not provided), in accordance with what is known in the state of the art regarding the protective nature of ASFV-GΔ9GL / ΔUK. The sows in group 2 were not challenged and served as a negative control group, surviving until the end of the study. Petition 870250080553, dated 08 / 09 / 2025, pages 105 / 110

Claims

1 / 2 CLAIMS 1. Live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain, characterized in that it is for use in a vaccine to protect a pregnant sow against African swine fever virus (ASFV) infection by administering the vaccine comprising the live attenuated ASFV-G^9GLMUK strain to the pregnant sow.

2. Live attenuated ASFV-G^9GLMUK strain for use, according to claim 1, characterized in that the vaccine is administered to the pregnant sow in the second half of gestation.

3. Live attenuated ASFV-G^9GLMUK strain for use, according to claim 1 or 2, characterized in that the vaccine is administered to the pregnant sow in the last third of gestation.

4. Live attenuated ASFV-G^9GLMUK strain for use, according to any one of claims 1 to 3, characterized in that the vaccine is administered to the pregnant sow within a period of 5 to 20 days before the expected date of farrowing of the pregnant sow.

5. Live attenuated ASFV-G^9GLMUK strain for use, according to any one of claims 1 to 4, characterized in that the vaccine is administered once during gestation or in a primary and booster regimen to the pregnant sow.

6. Live attenuated ASFV-G^9GLMUK strain for use, according to any one of claims 1 to 5, characterized in that the vaccine is administered intramuscularly, intradermally or orally.

7. Live attenuated ASFV-G^9GLMUK strain for use, according to any one of claims 1 to 6, characterized in that the vaccine is administered with a dose of the ASFV-G^9GLMUK strain of at least 102 TCID50 per administration. Petition 870250080553, dated 09 / 08 / 2025, pp. 109 / 110 2 / 2 8. Live attenuated ASFV-G^9GLMUK strain for use, according to any one of claims 1 to 7, characterized in that the vaccine is administered with a dose of the ASFV-G^9GLMUK strain between 102 TCID50 and 106 TCID50 per administration.

9. Use of a live attenuated Georgia 2007 African swine fever virus (ASFV-G) Δ9GL / ΔUK strain, characterized in that it is for the manufacture of a vaccine for the protection of pregnant sows against infection by African swine fever virus.

10. Product, process, system, kit, means or use, characterized in that it comprises one or more elements described in the descriptive report, claims, drawings, sequence listing, or summary of this application, when applicable. Petition 870250080553, dated 08 / 09 / 2025, page 110 / 110