Vaccines for protecting pregnant pigs against African swine fever

Abstract

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

Description

[Technical Field]

[0001] FIELD OF THE INVENTION African swine fever virus (ASFV) is one of the most important pathogens affecting domestic pig populations worldwide. The present invention relates to a vaccine for protecting pigs, particularly vulnerable pregnant pig herds, against infection with ASFV. [Background technology]

[0002] Background of the Invention As reviewed by ACUrbano et al. (Emerging Microbes & Infections, 2022, Vol. 11, pp. 2021-2033), African swine fever (ASF) is a highly infectious, severely hemorrhagic, viral disease of pigs endemic to the southern Sahara of Africa (24 genotypes based on the C-terminal sequence of the p72 surface antigen) and the Italian island of Sardinia (p72 genotype I). Transcontinental spread of ASF has occurred on at least three separate occasions, most notably in Georgia in 2007, when it spread from the Black Sea port city of Poti across the Caucasus to the Russian Federation (RF) and Eastern Europe. Over the following decade, the disease became endemic in the RF, and by 2018, it had spread west to Belgium and east to the People's Republic of China, rapidly sweeping much of Southeast Asia and Oceania. All of the isolates found 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, with the re-emergence of ASF virus in mainland Italy in January 2022. Several re-emergences have since been reported in China, the Republic of Korea, Moldova, and Ukraine, with North Macedonia reporting its first outbreak, as well as Thailand, one of several countries in previously unaffected regions. The virus also emerged in the Dominican Republic and Haiti in 2021, marking the first diagnosis of ASFV in the Western Hemisphere in over 40 years. These recent events highlight a highly alarming pattern of ongoing spread, exacerbated by the fact that small-scale and semi-industrial farms account for the majority of pig production in many of these regions. Outbreaks therefore have severe socio-economic impacts, causing devastation to rural livelihoods dependent on livestock production, threatening overall market stability and food security, and severely affecting animal welfare.

[0003] African swine fever virus (ASFV) is a large, double-stranded nucleoplasmic DNA arbovirus and the only member of the Asfarviridae family. The virion is approximately 250 nm in diameter and consists of a central nucleoid surrounded by an icosahedral protein capsid (or core shell), an inner lipoprotein membrane (or inner envelope), an icosahedral protein outer capsid, and an outer lipoprotein envelope (or outer envelope) (obtained when the virus buddings from the cellular membrane). Both intracellular and extracellular viral forms are infectious. Its natural host range is restricted to members of the Ornithodoros genus of ticks and the Suidae family, where it replicates primarily in cells of the mononuclear phagocytic system, resident macrophages, and specialized reticulocytes. The virus is endemic to African wild pigs (Sus scrofa). However, in domestic pigs and wild boars, clinical symptoms are highly variable, and individual outcomes can range from fatal to asymptomatic. Depending on the virulence of the strain involved, a series of morphological stages occur, with mortality ranging from 100% (super-acute form) to less than 30% (chronic form) of infected animals. The genotype II strains currently occurring in Europe and Asia are highly virulent and cause the acute form of the disease, but there is evidence that some hypovirulent isolates may be circulating among wild boars in the Baltic countries and domestic pigs in China, with reports of both naturally mutated hypovirulent genotype II strains and hypovirulent epidemic genotype I strains being detected in the field.

[0004] Protective immunity against ASFV is not yet fully understood. Although several vaccine approaches are known in the art, they have not yet resulted in a commercially available vaccine. The first approach described in the art is inactivated vaccination. Virus inactivation is an established approach to vaccine production, is relatively easy to achieve, and importantly, has a high safety profile compared to live vaccines. The inactivation process prevents reversion to a virulent phenotype and renders the vaccine virus incapable of infection, avoiding two major drawbacks of attenuated vaccines. It is also believed to pose a lower risk to more vulnerable patient groups, such as very young animals and pregnant animals. Animals in the latter group experience significant physiological changes and are 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 in some cases been able to induce serological immune responses but have not provided sufficient protection (Cadenas-Fernandez et al. Vaccines, 2021, 9, 242; doi.org / 10.3390 / vaccines9030242).

[0005] Another approach is the use of subunit, DNA, and viral vector vaccines. These vaccines have shown promise, with several candidates shown to induce specific humoral and / or cellular immune responses that appear to confer partial to complete protection. However, the nature of the immunization protocols used in these studies (vaccine type, vaccination strategy, and challenge model) varies, making it difficult to compare results. Further research is needed to determine which immune mechanisms need to be triggered to confer complete and durable protection, to identify which antigens (or combinations thereof) should be included in potential vaccines, and to identify the most appropriate administration methods (Urbano, supra).

[0006] Live attenuated vaccines are the most promising ASF vaccine candidates, despite their inherently lower safety profile than the vaccines mentioned above. These vaccines avoid key problems presented by inactivated, subunit, vectored, and DNA vaccines. They mimic natural infection because they can successfully replicate within the host, thereby triggering both humoral and cellular pathways and typically do not require adjuvants. Some live attenuated vaccines have also been shown to induce mucosal IgA antibodies, an important feature for vaccines administered orally (oral immunization is practically convenient for vaccines targeting wild boar populations). However, these vaccines also carry risks because they may reacquire virulence (i.e., revert to a virulent form), causing disease outbreaks, and potentially causing post-vaccination reactions and side effects, especially in vulnerable animals. Three main strategies have been employed in the production of live attenuated ASF vaccines: attenuation by cell passage, screening for naturally attenuated strains, and deletion of virulence-associated genes. To overcome some of the safety issues associated with live attenuated vaccines, particularly residual virulence, attempts have also been made to further delete virulence-associated genes in naturally attenuated strains or adapt gene-deleted viruses to heterologous cell lines. Depending on the immunogenicity of the deleted genes, these candidates may also be suitable for a vaccination protocol known as "DIVA" (differentiating naturally infected from vaccinated animals).

[0007] As shown in Urbano (ibid.), approximately 10 promising live-attenuated vaccines developed between 2015 and 2022 are currently available. Among them, NH / P68, OURT88 / 3, Lv17 / WB / Rie1, BA71ΔCD2v, HLJ / 18-7GD, ASFV-G-ΔI177L, ASFV-G-ΔI177L / ΔLVR, SY18ΔI226R, ASFV-G-ΔA137R, and ASFV-G-ΔE184L have been shown to be viable live ASF vaccines. Of these, the most promising live-attenuated vaccine candidate to date is the ASFV-G-ΔI177L strain, designed by the United States Department of Agriculture's (USDA) Agricultural Research Service (ARS). It can be administered intramuscularly and oronasally and induces robust sterile immunity against challenge with the virulent ASFV Georgia 2007 isolate, which has been implicated in recent outbreaks throughout Europe and Asia. Notably, this vaccine strain has proven effective in follow-up field trials against the virulent Vietnam strain TTKN / ASFV / DN / 2019. Since 2022, the ASFV-G-ΔI177L strain has been marketed in Vietnam under conditional approval. Development of derivatives of ASFV-G-ΔI177L is also underway. For example, ASFV-G-ΔI177L / ΔLVR replicates efficiently in stable porcine epithelial cell lines and maintains the same levels of attenuation, immunogenicity, and protective efficacy in challenge studies. Other derivatives being tested as experimental vaccines include ASFV-G-Δ9GL, ASFV-G-ΔMGF, ASFV-G-Δ9GL / ΔUK, ASFV-G-ΔI177L, and ASFV-G-ΔI177L / ΔLVR, all designed by the USDA Agricultural Research Service. Summary of the Invention

[0008] Object of the invention It is an object of the present invention to provide an ASFV vaccine that is safe when administered to pregnant pigs. Due to their demanding metabolic state, pregnant pigs are particularly vulnerable to infection with pathogenic microorganisms, which in particular leads to the loss of viable offspring. This can be expressed as the loss of piglets that would have been able to be born alive and that would have reached the age of 2 weeks or more, in particular at least 15-21 days. The object is in particular to avoid a loss in the number of piglets of more than 50% (i.e., more than 50%) (i.e., more than 50% of the gestational piglets are stillborn or do not survive for more than 2 weeks) due to ASFV vaccine administered to dams.

[0009] Overview of the invention In accordance with the objectives of the present invention, a vaccine containing the live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain, known to be protective against ASFV infection, has been found to be safe when administered to pregnant sows. In particular, it can result in a very low loss of viable piglets, as low as 10%, which is within the typical (spontaneous) loss rate of 5-25% in healthy pigs.

[0010] This was surprising to the inventors, especially since the loss of viable piglets can be as high as 90% for what is generally considered the most promising ASF vaccine candidate, i.e., ASFV-G-ΔI177L. Given the critical importance of maternal survival to pig farms, a loss of 50% of viable piglets may still be acceptable for an ASF vaccine under certain circumstances, but values ​​above 50% are considered unacceptable for a commercially available ASF virus vaccine. Furthermore, lower loss rates, such as 40%, 30%, 25%, 20%, 15%, 10%, 5%, or loss rates no higher than the mortality rate in negative control animals (i.e., an appropriate control group of healthy pregnant pigs not administered the ASF vaccine), are particularly preferred.

[0011] In addition to the live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain for use in a vaccine for protecting pregnant pigs against African swine fever virus (ASFV) infection by administering to the pregnant pigs a vaccine comprising the live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain, the present invention also relates to the use of the live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain for the manufacture of a vaccine for the protection of pregnant pigs against African swine fever virus infection, and to a method for the protection of pregnant pigs against African swine fever virus infection by administering to the pregnant pigs a vaccine comprising the live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain.

[0012] definition Pigs are animals of the family Suidae, in particular pigs kept by humans as feeder pigs, breeding pigs or those kept for slaughter, and wild boars.

[0013] An ASFV strain is ASFV Georgia 2007 (ASFV-G) if it is the reference isolate Georgia 2007 (GenBank FR682468.2) described in U.S. Pat. No. 9,808,520 and O'Donnel et al., Journal of Virology, January 2017, Volume 91, Issue 1, pp. 1-18; doi 10.1128 / JVI.01760-16, or a natural or recombinant variant of this reference isolate. The nucleotide sequence identity of the ASFV Georgia 2007 strain for use in the present invention 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, when aligned to the full length of FR682468.2. Currently available molecular data derived using standardized genotyping methods indicate that only this ASFV variant is present in Eastern and Central Europe after the 2007 outbreak in Georgia (Gallardo et al., Genetic Variation among African swine fever Genotype II Viruses, Eastern and Central Europe. Emerg Infect Dis. 2014 Sep;20(9):1544-1547; doi:10.3201 / eid2009.140554). To generate a nucleotide sequence alignment, NCBI's Blast™ computer program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) is used with standard settings and default parameters, selecting the options "blastn" and "Align two or more sequences," thereby selecting the sequence of FR682468 as the subject (also known as the "target").

[0014] ASFV-G-Δ9GL / ΔUK is a mutant of ASFV-G in which the 9GL and UK genes (viral gene numbers B119L and DP96R, respectively) have been functionally disabled by deletion of at least part of these genes, so that they are no longer expressed at normal levels (i.e., at the level of the unmodified parent strain), and in particular no longer expressed at all. As is generally known, in this case, the entire gene is typically not deleted, because this would also pose the risk of inhibiting transcription of two adjacent genes in the ASFV genome, thereby effectively deleting three genes instead of one. ASFV-G-Δ9GL / ΔUK does not exclude further mutations, either spontaneous or due to recombination (e.g., specific mutations aimed at obtaining a strain suitable for distinguishing infected from vaccinated animals).

[0015] A vaccine is a composition suitable for application to animals, with acceptable safety, containing an immunologically effective amount of one or more antigens (i.e., an amount capable of stimulating the target animal's immune system sufficiently to induce an immune response (e.g., antibodies) against the antigen, the corresponding native antigen, and potentially against the native pathogen), typically combined with a pharmaceutically acceptable carrier (i.e., a biocompatible medium, i.e., a medium capable of presenting the antigen to the host animal's immune system after administration of the vaccine and which does not induce significant adverse reactions in the target animal after administration), such as a liquid containing water and / or any other biocompatible solvent, or a solid carrier, such as those commonly used to obtain lyophilized vaccines (based on sugars and / or proteins), optionally containing an immunostimulant (adjuvant), which, upon administration to an animal, induces an immune response capable of protecting the animal against infection (after vaccination). For any vaccine, the requirement of acceptable safety is as important as efficacy in terms of protection.

[0016] "Protecting an animal against infection with ASFV (ASFV infection)" means preventing, ameliorating, or assisting in the cure of a pathogenic infection with ASFV, or preventing, ameliorating, or curing a disorder resulting from the infection, for example, preventing or reducing one or more clinical symptoms resulting from infection with ASFV, preferably preventing the death of the animal as a result of infection with ASFV. In the case of a pregnant animal, the fetus that the animal is carrying (i.e., any unborn animal at a prenatal developmental stage) is considered part of the pregnant animal.

[0017] A viable piglet is one that is born alive to its sow and that can reach an age of more than two weeks, particularly at least 15-21 days.

[0018] Further detailed embodiments of the present invention In a first more detailed embodiment of the live-attenuated ASFV-G-Δ9GL / ΔUK strain for use as a vaccine for protecting pregnant pigs against infection with African swine fever virus (ASFV) by administering a vaccine comprising the live-attenuated ASFV-G-Δ9GL / ΔUK strain to the pregnant pigs, the vaccine is administered to pregnant pigs in the late stages of gestation. While earlier vaccination is possible for protection, the late stages of gestation are considered more critical for ASFV infection and vaccine safety. The live-attenuated ASFV-G-Δ9GL / ΔUK strain has been found to be safe during this stage of gestation, and even during the last third of gestation, particularly when administered between 5 and 20 days before the expected farrowing date of the pregnant pig, for example, as little as 15 days before the expected farrowing date.

[0019] In another more particular embodiment, the vaccine is administered to pregnant sows once during gestation, or in a prime-boost regime during gestation, in which two doses are administered during the same gestation period but separated by 1 to 12 weeks, typically 2 to 10 weeks, 2 to 8 weeks, 2 to 6 weeks, or 2 to 4 weeks.

[0020] In yet another more particular embodiment, the vaccine is administered intramuscularly, intradermally, or orally.

[0021] In yet another more particular embodiment, the vaccine contains at least 10 2 TCID 50 A dose of ASFV-G-Δ9GL / ΔUK strain, e.g., 10 per administration 3 , 10 4 or 10 5 TCID 50 ~about 10 6 TCID 50 is administered at a dose of

[0022] The present invention will now be described in more detail by way of the following specific examples.

[0023] Example Example 1 is an experiment to test the safety of a protective live attenuated ASFV-G vaccine strain in pregnant sows.

[0024] Example 2 is another experiment testing two additional protective live attenuated ASFV-G vaccine strains for their safety in pregnant sows.

[0025] Example 3 is an experiment to confirm the effectiveness of the live attenuated ASFV-G vaccine strain.

[0026] Example 1 Example 1 is an experiment to test the safety of the live attenuated ASFV-G vaccine strain ASFV-G-ΔI177L for protection in pregnant sows. As is known from the literature, this strain is highly effective in protecting pigs against infection with ASFV (Borca et al., Journal of Virology, April 2020, Volume 94, Issue 7, pp. 1-18; doi 10.1128 / / JVI.02017-19) and is safe when administered to pigs (Tran et al., Viruses 2022, 14: Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV-G-ΔI177L).

[0027] Research objectives The aim of this study was to establish the safety of the ASFV-G-ΔI177L strain when administered to pregnant pigs.

[0028] Study design Four ASFV-free and ASFV antibody-free pregnant pigs were available for this study. On day 100 of gestation (i.e., approximately 2 weeks before farrowing), two pigs were vaccinated with 2 ml of vaccine containing ASFV-G-ΔI177L at 1.5 × 10 2 TCID 50 The vaccine was administered intramuscularly (IM) to the right side of the neck at a dose of 0.01 mg / kg / day. Two pigs served as unvaccinated controls. They received PBS in the same manner as the vaccinated animals. Animals were monitored daily for clinical signs specific to ASF starting from the day before vaccination. Blood samples (from the tail vein) were collected at 4 days post-vaccination (dpv), 11 dpv, 23 dpv, and 32 dpv, while body temperatures were monitored daily starting from the day before vaccination. At birth, the health status of the piglets was monitored and recorded. All surviving piglets were weighed on postnatal days 3-4 and 16-17. Piglets were monitored daily for clinical signs specific to ASF until the end of the study.

[0029] result maternal health Rectal temperatures were normal (37.5°C-39°C) and did not differ significantly between vaccinated and control animals. Control animals did not show any clinical signs of ASF. Two weeks after vaccination, one of the vaccinated animals showed mild symptoms of ASF, which gradually subsided and disappeared. PCR could not detect ASFV in blood samples obtained from control animals, but vaccinated animals showed virus in their blood at all time points.

[0030] Table 1 shows significant differences in reproductive performance between vaccinated and control sows. 43% of piglets were stillborn in the vaccinated sows, compared with 17% in the control sows. During the experimental period, all live-born piglets from the control sows survived to 3 weeks of age, whereas only four piglets from the vaccinated sows survived this period. Furthermore, all surviving piglets from the vaccinated sows showed clinical signs typical of ASF and were euthanized upon reaching the humane endpoint (HEP). Overall, this means that vaccination with ASFV-G-ΔI177L resulted in a greater than 90% loss of viable piglets. [Table 1] Piglet health Rectal temperatures in piglets born to control animals remained normal throughout the study, ranging from 38.7 to 40°C, whereas in piglets born to vaccinated animals this was up to 41°C. With regard to weight gain, piglets from control sows gained an average of 2.9 times more weight when weighed on days 3-4 and 17-18 post-partum, whereas in vaccinated sows, survivors gained an average of 2.2 times more weight.

[0031] All control piglets remained healthy throughout the experimental period. All live-born piglets from vaccinated animals showed ASF-associated clinical signs and ASFV infection was confirmed by PCR. ΔI177L-specific PCR performed on DNA extracted from the blood of pregnant sows and their representative piglets showed that the vaccine strain was vertically transmitted from pregnant sows to their piglets.

[0032] conclusion By comparing the health status of the sows (and the piglets born alive from these sows), it can be concluded that the vaccine strain ASFV-G ΔI177L is not safe for administration to pregnant sows, especially since the loss rate of viable piglets was (significantly) above 50%.

[0033] Example 2 Example 2 is another experiment in which two additional protective live-attenuated ASFV-G vaccine strains were tested for their safety in pregnant sows. The first vaccine candidate was derived from the Lv17 / WB / Rie1 strain (WO 2020 / 049194). In particular, this first vaccine contains a mutant designated Lv17 / WB / Rie1-ΔCD, which contains the additional gene deletions ΔEP153R and ΔEP402R (see Petrovan et al., Journal of Virology, January 2022, Volume 96, Issue 1, pp 1-19). The parent strain Lv17 / WB / Rie1 is safe and effective for use in pigs (Urbano, supra), and the double knockout mutant strain is also known to be (essentially safe and) effective for use in pigs (European Patent Application No. EP22462011.1 (“Attenuated African swine fever virus and use thereof in vaccine compositions”; filed November 22, 2022, with the Hungarian Intellectual Property Office in the name of Intervet International BV (Consejo Superior de Investigaciones Cientificas (CSIC), Allatorvostudomanyi Kutatointezet, Universidad Complutense de Madrid, and Eurofins Ingenasa SA). The second strain is known from the literature (inter alia, Urbano, supra) to be protective and safe for vaccination of pigs.

[0034] The second vaccine candidate tested was ASFV-G-Δ9GL / ΔUK (US 9,808,520), a strain known from the literature (see, inter alia, Urbano, supra) to be protective and safe for vaccination of pigs.

[0035] Research objectives The aim of this study was to establish the safety of these two additional ASFV-G strains when administered to pregnant pigs.

[0036] Study design Six ASFV-free and ASFV antibody-free pregnant sows were available for this study. Approximately 100 days into gestation (i.e., approximately 2 weeks before farrowing), 10 pigs were vaccinated with 2 ml of vaccine containing the Lv17 / WB / Rie1-ΔCD strain. 3 TCID 50 The corresponding vaccine containing the candidate vaccine strain ASFV-G-Δ9GL / ΔUK was administered intramuscularly (IM) to two pigs in the right side of the neck at a dose of 10 4 TCID 50 Two pigs were administered IM into the right side of the neck at a dose of 0.01 mg / kg / day. The remaining two pigs served as unvaccinated controls. After vaccination, the animals were monitored for clinical signs specific to ASF. Blood samples were collected at 3 days post-vaccination (dpv), 10 dpv, 21 dpv, and 31 dpv, while body temperatures were monitored daily from the day before vaccination. At birth, the health of the piglets was monitored and recorded. If there were stillborn or dead piglets, blood samples were collected, if possible, before the animals were culled. All surviving piglets were weighed on postnatal days 5 and 19. The piglets were monitored daily for clinical signs specific to ASF until the end of the study at postnatal day 22.

[0037] result maternal health The rectal temperatures of control animals and animals receiving ASFV-G-Δ9GL / ΔUK were normal (37.5°C to 39.5°C), whereas the other animals vaccinated with Lv17 / WB / Rie1-ΔCD showed a continuing increase in temperature from day 4 postvaccination onward, indicating symptoms of ASF. Both sows in this group were euthanized on day 10 postvaccination. Piglets from these sows were either born prematurely during abortion or removed from the uterus at the time of euthanasia. Neither the control animals nor one of the animals vaccinated with ASFV-G-Δ9GL / ΔUK showed clinical signs of ASF. The other ASFV-G-Δ9GL / ΔUK-vaccinated animal showed mild ASF symptoms. ASFV was not detectable in control animals by PCR, whereas vaccinated animals showed virus in their blood at all time points.

[0038] Regarding reproductive performance, the sows used in this study had a healthy reproductive history, giving birth to over 80% of viable piglets overall. However, significant differences in reproductive performance were observed between sows vaccinated with the two different candidate vaccine strains. Sows vaccinated with the Lv17 / WB / Rie1-ΔCD strain delivered piglets prematurely, 8 to 10 days before the expected delivery date. Both sows began aborting piglets that were weak, stunted, and unlikely to survive. These piglets reached HEP and were euthanized at birth. A total of three piglets were stillborn.

[0039] In the case of sows vaccinated with ASFV-G-Δ9GL / ΔUK, all of the piglets born alive survived to the end of the study. One sow stillborn three piglets, while the other sow did not. Parturition was uneventful and under normal circumstances. In the case of control animals, parturition was under normal circumstances, and all of the piglets born alive survived to the end of the study. The reproductive performance of the sows in this study is shown in Table 2 below. [Table 2] Piglet health None of the piglets born to sows vaccinated with Lv17 / WB / Rie1-ΔCD survived more than one day after farrowing, so their health was monitored only for the other animals. Rectal temperatures of all piglets were normal throughout the experimental period and did not differ between control and vaccinated animals. With regard to weight gain, piglets from control sows showed an average of 3.7-fold increase from birth weight, while piglets from vaccinated sows showed an average of 2.7-fold increase in weight.

[0040] All control piglets remained healthy throughout the experimental period. Thirteen live-born piglets from vaccinated animals showed mild ASF-associated clinical signs, and one showed moderate signs. ASFV infection was confirmed by PCR. Specific PCR performed on DNA extracted from the blood of pregnant sows and their representative piglets indicated that the candidate vaccine strain was vertically transmitted from pregnant sows to their piglets. Five piglets developed viremia 5 days after birth. Viremia peaked on day 12, after which it gradually decreased. Remarkably, the viremic piglets remained healthy and alive until the end of the study.

[0041] conclusion By comparing the health status of the sows (and the piglets born alive from these sows) with control sows, it can be concluded that the Lv17 / WB / Rie1-ΔCD strain is not safe for administration to pregnant pigs, in particular since the loss rate of viable piglets was greater than 50%, whereas ASFV-G-Δ9GL / ΔUK did not result in a higher loss of viable piglets than the control (10% vs. 35% / 16%) and is considered safe for vaccination of pregnant pigs.

[0042] Example 3 The aim of this study was to obtain confirmation that the ASF vaccine used in Example 2, i.e., MLV ASFV-G-Δ9GL / ΔUK, was indeed protective in sows vaccinated in the third trimester of pregnancy. For this purpose, three groups of animals were used: three sows in group 1, three sows in group 2, and two negative control animals.

[0043] Group 1 sows vaccinated during the third trimester of gestation developed viremia and were completely protected against virulent challenge (data not shown). This is consistent with what is known in the art regarding the protective properties of ASFV-G-Δ9GL / ΔUK. Group 2 sows were not challenged and served as a negative control group; they survived to the end of the study.

Claims

1. A live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain for use in a vaccine to protect pregnant pigs against African swine fever virus (ASFV) infection by administering to the pregnant pigs a vaccine comprising the live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain.

2. The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to claim 1, wherein the vaccine is administered to pregnant pigs in the late stages of gestation.

3. The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to any one of the preceding claims, wherein the vaccine is administered to pregnant pigs in the last third of gestation (third trimester).

4. The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to any one of the preceding claims, wherein the vaccine is administered to pregnant pigs in the period between 5 and 20 days before the expected farrowing date of the pregnant pigs.

5. The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to any one of the preceding claims, wherein the vaccine is administered to pregnant pigs once during pregnancy or in a prime and boost regime.

6. The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to any one of the preceding claims, wherein the vaccine is administered intramuscularly, intradermally or orally.

7. The vaccine is administered in at least 10 ２ T.C.I.D. ５０ The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to any one of the preceding claims, wherein the live attenuated ASFV-G-Δ9GL / ΔUK strain is administered in a dose of 0.05 mg / mL.

8. The vaccine was administered at 10 ２ T.C.I.D. ５０ ~10 ６ T.C.I.D. ５０ The live attenuated ASFV-G-Δ9GL / ΔUK strain for use according to any one of the preceding claims, wherein the live attenuated ASFV-G-Δ9GL / ΔUK strain is administered in a dose of 0.05 mg / mL.

9. Use of a live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain for the manufacture of a vaccine for the protection of pregnant pigs against African swine fever virus infection.

10. A method for the protection of pregnant pigs against African swine fever virus infection by administering to the pigs a vaccine comprising a live attenuated African swine fever virus Georgia 2007 (ASFV-G) Δ9GL / ΔUK strain.

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