A vaccine for protecting a pregnant swine against african swine

Abstract

The present invention pertains to the use of a live attenuated African swine fever virus Georgia 2007-Δ9GL / ΔUK / ΔEP153R (ASFV-G-Δ9GL / ΔUK / ΔEP153R) strain for protecting a pregnant swine against an infection with African swine fever virus (ASFV) by administering a vaccine comprising the live attenuated ASFV-G-Δ9GL / ΔUK / ΔEP153R strain to the pregnant swine.

Description

[0001] A VACCINE FOR PROTECTING A PREGNANT SWINE AGAINST AFRICAN SWINE FEVER

[0002] TECHNICAL FIELD

[0003] African swine fever virus (ASFV) is one of the most important disease-causing pathogen affecting the domestic swine population globally. The present invention pertains to a vaccine to protect swine against an infection with ASFV, in particular the vulnerable group of pregnant swine.

[0004] BACKGROUND

[0005] As described in the review article by A.C. Urbano et al. (Emerging Microbes & Infections, 2022, Vol. 11 , pp 2021 - 2033), African swine fever (ASF) is a highly infectious and severe haemorrhagic viral disease of pigs, endemic to South- Saharan Africa (24 genotypes based on the sequence of the c-terminus of the p72 surface antigen) and the island of Sardinia in Italy (p72 genotype I). The transcontinental spread of ASF occurred on at least three separate occasions, most significantly to Georgia, in 2007, where it spread from the Black Sea port of Poti across the Caucasus region into the Russian Federation (RF) and Eastern Europe. The following decade saw the disease become epizootic in the RF, and by 2018 it had spread as far West as

[0006] Belgium and East to the People’s Republic of China, quickly taking over most of Southeast Asia and Oceania. All 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; in January 2022 ASF virus reappeared on the Italian mainland.

[0007] Several reoccurrences have since been reported in China, the RF, Moldova, and Ukraine, and North Macedonia reported their first occurrence, as did Thailand, one of the few countries in the region that had remained unscathed. The virus also appeared in 2021 in the Dominican Republic and Haiti, constituting the first diagnosis of ASFV in more than 40 years in the Western hemisphere. These recent events highlight an extremely disconcerting pattern of continuous spread, exacerbated by the fact that in many of these regions small-scale and semiindustrial farms account for the majority of pig production. As such, outbreaks carry severe socio-economic consequences, causing devastation of rural livelihoods dependent on livestock production, and threatening overall market stability and food security, as well as severely affecting animal welfare.

[0008] The African swine fever virus (ASFV) is a large, double-stranded nucleocytoplasmic DNA arbovirus, the only member of the Asfarviridae family. Virions have a diameter of around 250 nm and consist of a central nucleoid enclosed by an icosahedral protein capsid (or core shell), an internal lipoprotein membrane (or inner envelope), an icosahedral protein outer capsid, and an external lipoprotein envelope (or outer envelope) that is obtained when the virus buds out 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 mainly in cells of the mononuclear phagocytic system, resident macrophages, and specific reticular cells. The virus is endemic in African wild pigs. In domestic pigs and wild boar, 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% (per- acute form) to <30% (chronic form) of the infected animals. The genotype II strain currently affecting Europe and Asia is highly virulent, causing the acute form of the disease, although there is evidence that some reduced-virulence isolates may be circulating among wild boar in the Baltic States and domestic pigs in China, with reports of both naturally mutated genotype II low virulent strains and genotype I low virulent epidemic strains detected in the field.

[0009] Protective immunity against ASFV is still poorly understood. There are several vaccine approaches known in the art. The first approach described in the art is vaccinating with an inactivated vaccine. Virus inactivation is an established approach to vaccine production, relatively straightforward to achieve and importantly, with a higher safety profile when compared to live vaccines. The inactivation process negates reversion to a virulent phenotype and renders vaccine viruses nontransmissible, the two major drawbacks of attenuated vaccines. Also, it is believed that the safety for more vulnerable patient groups such as very young animals and pregnant animals is less of a risk. The animals in the latter group go through significant physiological changes and are thus in a stressful metabolic status. Inactivation however, does not necessarily produce a vaccine that elicits protective immunity. Attempts at immunization of pigs with a variety of inactivated ASF antigens, did not lead to sufficient protection even though in some cases it was capable of inducing a serological immune response (Cadenas-Fernandez, et al. Vaccines, 2021 , 9, 242; doi.org / 10.3390 / vaccines9030242).

[0010] Another approach is the use of subunit, DNA and virus vectored vaccines. These vaccines show promise and several candidates have been shown to induce specific humoral and / or cellular immune responses which appear to confer partial to full protection. However, the different nature of the immunization protocols used in these studies, including the type of vaccine, vaccination strategy and challenge model, makes results difficult to compare. Further work will be needed to identify which immune mechanisms need to be triggered to confer complete, lasting protection, which antigens (or combination of) should be included in a potential vaccine, and the most appropriate delivery method (Urbano, supra).

[0011] Live attenuated vaccines, although inherently less safe than the above mentioned vaccines, are the most promising ASF vaccine candidates. These vaccines circumvent a key issue presented by both inactivated, subunit, vector and DNA vaccines. Because they can successfully replicate within the host, they mimic natural infection thereby triggering both 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 vaccines administered via the oral route (oral immunisation is a practical convenience for vaccines aimed at the wild boar population). That being said, these vaccines also pose a risk, as they may regain pathogenicity (i.e. revert to virulence), causing the spread of disease, and they have the potential to cause post-vaccination reactions and side effects, in particular in vulnerable animals. Three main strategies have been employed in the generation 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 presented by live attenuated vaccines, in particular residual virulence, attempts have also been aimed at further deletion of virulence- associated genes in naturally attenuated strains, or adaptation to heterologous cell lines of gene deleted viruses. 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.

[0012] As indicated in Urbano (supra), in the meantime about 10 promising live attenuated vaccines are available, developed in 2015-2022. In particular NH / P68, OURT88 / 3, Lv17 / WB / Rie1 , BA71ACD2v, HLJ / 18-7GD, ASFV-G-AI177L, ASFV-G- AI177L / ALVR, SY18AI226R, ASFV-G-AA137R and ASFV-G-AE184L are indicated to be available as live ASF vaccines. Of these, the most promising live attenuated vaccine candidate to date is the ASFV-G-AI177L strain that was engineered by the U.S. Department of Agriculture’s (USDA) Agricultural Research Service (ARS). It can be administered by the intramuscular and oronasal route, inducing robust sterile immunity against challenge with the virulent parental ASFV Georgia 2007 isolate, involved in recent outbreaks throughout Europe and Asia, and in particular this vaccine strain proved effective in follow up field trials against the virulent Vietnamese strain TTKN / ASFV / DN / 2019. Since 2022 the ASFV-G-AI177L stain is on the market in Vietnam through a conditional license. The development of derivative strains of ASFV-G-AI177L is also in progress. For example, ASFV-G- AI177L / ALVR 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 that are tested as experimental vaccines are ASFV-G-A9GL, ASFV-G-AMGF, ASFV-G-A9GL / AUK and ASFV-G-AI177L / ALVR, all engineered by the U.S. Department of Agriculture’s Agricultural Research Service. SUMMARY OF INVENTION

[0013] It is an object of the invention to provide for alive attenuated ASFV vaccine that is safe for administration to a pregnant swine, an animal that due to its highly demanding metabolic status is particularly vulnerable for infections with a pathogenic microorganism, in particular leading to a loss of viable offspring. This can be expressed as the loss of piglets that could have been bom alive and are able to reach an age of more than two weeks, in particular: able to reach an age of at least 15-21 days of age. The object is in particular to avoid any losses in the number of piglets of over 50%: i.e. more than 50% of the piglets carried are bom still, or do not survive more than two weeks due to the ASFV vaccine being administered to the mother animal during gestation.

[0014] Auxiliary, it is an object that the vaccine, i.e. the live attenuated ASFV strain in the vaccine, does not revert to virulence when passaged serially in animals, and that the strain does not spread to animals that are in the neighbourhood of the vaccinated animal.

[0015] In order to meet the objects of the invention, it was found that a vaccine comprising a live attenuated African swine fever virus Georgia 2007- A9GL / AUK / AEP153R (ASFV-G-A9GL / AUK / AEP153R) strain, is protective and safe for administration to a pregnant swine. Next to this, it has been shown that in a conventional 5 passage model the strain does not revert to virulence and that the strain does not spread to sentinel animals.

[0016] To the inventors this came as a surprise, in particular since the loss of viable piglets for what is commonly regarded as the most promising ASF vaccine candidate, viz. ASFV-G-AI177L, can be as high as 90%. Although a loss of up to 50% of viable offspring might, under certain circumstances, still be acceptable for an ASF vaccine, in particular given the fact that the survival of the mother animals is crucial for a swine facility, any value above 50% is deemed unacceptable for a commercial ASF virus vaccine. Still, a lower loss 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. a suitable control group of healthy pregnant swine that has not been administered the ASF vaccine) is particularly preferred. Also, the surprise was further supported by the fact that it was recently published that the deletion of the EP153R gene from an ASFV-G-A9GL / ACD2v strain abrogates its effectiveness as a vaccine (Gladue et al., Viruses, 2020, 12, 1185). Similarly, it was described that the deletion of the EP153R gene from the NH / P68 strain resulted in a vaccine strain that was not able to protect pigs against ASFV challenge (Gallardo et al., Vaccine, 2018, vol 36, issue 19, pp 2694-2704; doi.org / 10.1016 / j. vaccine.

[0017] 2018.03.040). Also, it is inherently uncertain what the effect of one or more gene deletions is on the reversion to virulence, and the capacity of the virus to spread to other animals after infecting a first animal. Therefore, the combined effect of being safe and protective, while showing no (effective) reversion to virulence of spreading to other animals was totally unexpected for the strain according to the invention.

[0018] Next to a live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use in a vaccine for protecting a pregnant swine against an infection with African swine fever virus by administering the vaccine comprising the live attenuated ASFV-G- A9GL / AUK / AEP153R strain to the pregnant swine, the invention also pertains to the use of a live attenuated ASFV-G-A9GL / AUK / AEP153R strain for the manufacture of a vaccine for the protection of a pregnant swine against an infection with African swine fever virus, and to a method for the protection of a pregnant swine against an infection with African swine fever virus by administering to the pregnant swine a vaccine comprising a live attenuated ASFV-G- A9GL / AUK / AEP153R strain.

[0019] The present invention pertains to a mutant having (at least) the 9GL, UK and EP153R genes functionally disabled. It is noted than an encoding mutant to arrive at a Georgia 2007 (ASFV-G) A9GL / AUK strain is known from US 9,808,520, in which mutant the amino acids 11 -68 of the wild type 9GL protein and amino acids respectively from the corresponding genes), hence functionally disabling the corresponding genes. However, it is within the common knowledge that the exact deletions are not essential for functionally disabling a gene per se. The 9GL and UK genes can be functionally disabled through deletion of a somewhat shorter, longer or shifted part of these genes, as long as they can no longer be expressed at a normal level (i.e. at the level of the unaltered parent strain), in particular no longer expressed at all. The same is true for the EP153R gene.

[0020] DEFINITIONS

[0021] A swine is an animal that belongs to the family of Suidae, in particular a porcine animal raised by man, for example to be a feeder pig, raised for seedstock, or raised for slaughter, and wild boars.

[0022] An ASFV strain is an ASFV Georgia 2007 (ASFV-G) strain if it is the reference isolate Georgia 2007 (GenBank FR682468.2), as referred to in US 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, 99.9% or even higher, when aligned to the full length of the sequence FR682468.2. Current available molecular data derived by using standardised genotyping procedures have indicated that only this ASFV variant is present in Eastern and Central 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. 2014 Sep; 20(9): 1544- 1547; doi: 10.3201 / eid2009.140554).

[0023] For making the nucleotide sequence alignments, the NCBI’s Blast™ computer program is used (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), selecting the options ‘blastn’ and “Align two or more sequences” with standard settings and default parameters, and whereby the sequence of FR682468 is selected as the subject (also known as “the target”).

[0024] ASFV-G-A9G / _ / Al / / is a variant of ASFV-G in which the 9GL and UK genes (viral gene numbers B119L and DP96R, respectively), have been functionally disabled through deletion of at least part of these genes, such that they can no longer be expressed at a normal level (i.e. at the level of the unaltered parent strain), in particular no longer expressed at all. As is commonly known, for this typically not the complete gene is deleted, since this also brings with it the risk of interfering with the transcription of the two flanking genes in the ASFV genome, and therewith actually deleting three genes instead of one. ASFV-G-A9GL / AUK does not exclude any further mutations, either spontaneously or recombinantly. This strain is known i.a. from US 9,808,520 and from literature (i.a. Urbano, supra).

[0025] ASFV-G-A9GL / AUK / AEP153R is a variant of ASFV-G-A9GL / AUK wherein the EP153R gene is functionally disabled corresponding to the functional disabling of the 9GL and UK genes as described directly here above. The EP153R gene and ASFV mutants wherein this gene is functionally disabled is commonly known in the art, i.a. from Gladue et al. in Viruses, 2020, 12, 1185 and from Petrovan et al 2022 in J Virol, Volume 96, No 1 , as well as Gallardo et al in Vaccine, 2018; 36:2694- 2704. ASFV-G-A9GL / AUK / AEP153R does not exclude any further mutations, either spontaneously or recombinantly.

[0026] A vaccine is a constitution suitable for application to an animal, having an acceptable safety, comprising one or more antigens in an immunologically effective amount, i.e. capable of stimulating the immune system of the target animal sufficiently to induce an immune response, such as antibodies, against the antigens and therewith against the corresponding naturally occurring antigens and therewith potentially the naturally occurring pathogen, typically combined with a pharmaceutically acceptable carrier (i.e. a biocompatible medium, viz. a medium that after administration does not induce significant adverse reactions in the subject 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 carrier such as commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), optionally comprising immunostimulating agents (adjuvants), which upon administration to the animal induces an immune response that is able to protect the animal against a (post-vaccinating) infection. For any vaccine, the requirement of an acceptable safety is as important as efficacy regarding protection.

[0027] To administer a vaccine once means that for arriving at protection the vaccine only needs to be administered at one single occasion, as compared to a common prime-boost scheme needing a second dose, typically within 6 weeks, preferably within 4 weeks such as at 2 weeks, before protection is arrived at. Since protection typically decreases over time, administering a vaccine once does not exclude that the vaccination is repeated after 6-12 months or later, in order to bring back the level of protection to an adequate level again.

[0028] To protect an animal against an infection with ASFV means aiding in preventing, ameliorating or curing a pathogenic infection with ASFV, or aiding in preventing, ameliorating or curing a disorder arising from that infection, for example to prevent or reduce one or more clinical signs resulting from the infection with ASFV, preferably preventing death of the animal as a result of the infection with ASFV. For a pregnant animal, the foetuses carried by the animal (i.e. any unborn animal in the stages of prenatal development) are regarded a part of the pregnant animal.

[0029] Viable piglets are piglets bom alive from a sow and able to reach an age of more than two weeks, in particular reach an age of at least 15-21 days.

[0030] DESCRIPTION OF EMBODIMENTS

[0031] In a first further embodiment of the live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use as a vaccine in protecting a pregnant swine against an infection with African swine fever virus by administering the vaccine comprising the live attenuated ASFV-G-A9GL / AUK / AEP153R strain to the pregnant swine, the vaccine is administered to the pregnant swine in the second half of gestation. Although earlier vaccination is feasible for protection, the second half of gestation is believed to be more critical for an ASFV infection and safety of a vaccine. It was found that a live attenuated ASFV-G-A9GL / AUK / AEP153R strain is safe for administration at this stage of gestation and even in the last third of gestation, most notably in a period of 5-20 days before the expected date of farrowing of the pregnant swine, for example at only 15 days before the expected date of farrowing.

[0032] In another further embodiment the vaccine is administered either once during gestation to the pregnant swine, or is administered in a prime- and boost regimen during gestation. In a prime- and boost regimen, the two administrations are provided during the same gestation, but are separated in time by 1 -12 weeks, typically by 2-10, 2-8, 2-6 or by 2-4 weeks, such as 3 weeks.

[0033] In again another further embodiment the vaccine is administered intramuscularly (meaning that the muscle tissue is the target tissue for depositing the vaccine), intradermally (in this case the dermis being the target tissue for depositing the vaccine) or orally (wherein the vaccine is deposited in the oral cavity of the subject animal).

[0034] In yet again another further embodiment the vaccine is administered with a dose of the ASFV-G-A9GL / AUK / AEP153R strain of at least 102TCID50 per administration, for example a dose above 103, 104or 105TCID50 up to about 106TCID50 per administration.

[0035] The invention will now be further explained using the following specific examples.

[0036] EXAMPLES

[0037] Example 1 shows that the new strain is safe and protective in pigs.

[0038] Example 2 shows that the new strains does not revert to virulence.

[0039] Example 3 shows the safety of the new strain in pregnant sows. EXAMPLE 1

[0040] Object of the study

[0041] The object of this study was to assess the safety and protective effect of the new strain and also, whether or not the strain after administration to an animal spreads to sentinel animals.

[0042] Producing the ASFV-G-A9GL7AUK / AEP153R strain

[0043] To delete 333 nucleotides of the EP153R gene of ASFV-G-A9GL / AUK (nucleotide 73892 to 74225; GenBank no. NC_044959.2) and create the ASFV-G- A9GL / AUK / AEP153R mutant virus, CRISPR / Cas9-mediated homologous recombination in porcine alveolar macrophages (PAMs) was used similar to what was described (Borca et al., 2018, Sci Rep 8, 3154, https: / / doi.org / 10.1038 / s41598-018-21575-8: CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses). For this, a transfer vector was used harboring a reporter gene cassette containing the eGFP gene with the ASFV p72 late gene promoter in between the left 1 -kb and right 1 -kb flanking genomic region of the EP153R gene. The resulting recombinant virus was purified by six consecutive PAM cell pick-up / infection steps, before virus stocks were created. The full-length genomic sequence of ASFV-G-A9GL / AUK / AEP153R in the stocks was determined by using the Illumina NGS platform as described (Olasz et al., Viruses, 2019 Dec 6;11 (12): A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus), to confirm the successful creation of the triple-gene-deleted virus.

[0044] Study design

[0045] Twenty-three (23) ASFV-free and ASFV antibody-free 5-week-old pigs were used for this study. Group 1 with 5 animals were vaccinated with 2 ml of ASFV-G- A9GL / AUK / AEP153R at 3.3 logw TCIDso / dose via the IM route. Group 2a comprised 5 animals. Each of the animals was vaccinated via the IM route with 2 ml of ASFV-G- A9GL / AUK / AEP153R at 4.3 log TCIDso / dose. Three animals were unvaccinated and used as sentinel animals (group 2b). In group 3a with 5 animals, each animal was vaccinated via the IM route with 2 ml of ASFV-G- A9GL / AUK / AEP153R at 5.3 logw TCIDso / dose. Three animals remained unvaccinated and were used as sentinel animals (group 3b). Group 4, having 2 animals, served as the unvaccinated challenge control group. Three weeks post vaccination, all animals were challenged with ASFV Armenia / 07 strain (Arm07) at 2.0 logw TCID50 via the IM route. The animals were assessed for 22 days after challenge.

[0046] EDTA blood samples were taken just before vaccination, 7 and 14 days post vaccination and just before challenge and 6, 12, 16 and 23 days post challenge. At 7, 14 and 21 days post vaccination, nasal and rectal swabs were taken. Blood samples, nasal swabs and rectal swabs were investigated for the presence of ASFV by qPCR.

[0047] TCID50 values were determined by making ten-fold serial dilutions of samples in microtiter plates containing monolayers of Porcine alveolar macrophages (PAMs). Subsequently the plates were incubated at 37°C. After incubation, the presence of ASFV was detected by fluorescent signal in titration plates (the vaccine strain harbours fluorescent reporter genes, such as green fluorescent protein (GFP) and m-Cherry). The viral titer is calculated according to the method of Spearman and Karber and is expressed in log TCID50 per ml.

[0048] Resu / ts

[0049] Vaccination did not lead to any unacceptable local and systemic reactions. The animals were all assessed for the presence of ASF specific clinical signs. In the weeks post vaccination, less than half of the vaccinated animals had an elevated rectal temperature with a maximum of three consecutive days. No to very mild ASF-clinical signs were observed in the vaccinated animals post immunization.

[0050] In the weeks post challenge multiple ASF-specific clinical signs were observed in all groups. In group 1 one animal reached the human end point (HEP) due to ASF- specific clinical signs at 11 dpc. Three animals showed some clinical signs but survived until the end of the study. The remaining animal of group 1 did not show any clinical signs and survived until the end of the study.

[0051] In group 2a one animal reached a HEP due to ASF-specific clinical signs. Two animals showed some clinical signs but survived until the end of the study. The two remaining animals of group 2a did not show any clinical signs and survived until the end of the study. In group 2a sentinel animals were included (group 2b), two of these animals were found dead after they had some ASF-specific clinical signs at 6 and 7 dpc. The other animal reached the HEP due to ASF-specific clinical signs at 7 dpc. In group 3a two animals reached the HEP due to ASF- specific clinical signs at 7 dpc. The three remaining animals did not show any clinical signs and survived until the end of the study. In group 3a sentinel animals were included (group 3b), one animal reached the HEP due to ASF- specific clinical signs at 6 dpc. The other two animals were found dead at 6 and 7 dpc after they had some ASF-specific clinical signs. In group 4, the challenge control animals were found dead at 6 dpc after they had some ASF-specific clinical signs.

[0052] The above data and other data (viraemia, nasal and faecal secretion and protection against death) are depicted in the table below as a percentage of the animals in the respective group.

[0053] Table 1 Post vaccination (pv) and post challenge (pc) data

[0054] The data shows that the novel strain is able to protect 60-80% of the animals against death due to an ASFV infection, even after only one shot of the vaccine. The lowest dose already seems to induce this level of protection. Also, the vaccine strain does not spread to sentinel animals, even though a very high percentage of the co-housed vaccinated animals gets viraemic.

[0055] Conclusion

[0056] The novel ASFV mutant when used as an antigen in a vaccine is able to protect vaccinated animals against ASFV, is safe for these animals and does not spread to sentinel animals.

[0057] EXAMPLE 2

[0058] Object of the study

[0059] The object of the study was to assess whether or not the new strain shows any reversion to virulence in a conventional five passage in vivo model.

[0060] Study design

[0061] A five passage model was used to assess reversion to virulence. For each passage 4-week-old pigs were transported to the animal facility. After an acclimatization period of 7 days, the animals were inoculated as described below.

[0062] Passage 1 : Three pigs were inoculated with 2 ml of the virus at 4.54 logw TCIDso / dose via the intramuscular route in the left side of the neck. Just before inoculation, a blood sample was taken. The animals were observed daily for clinical signs. At 10 days post inoculation the animals were euthanised, and a blood sample was taken. The ASFV titre was estimated by qPCR based on a serial dilution with known titres of ASFV in the ASFV qPCR. The sample with the highest amount of ASFV DNA was used for inoculation of the next passage. If the viral load of the material was higher than 4.54 log TCIDso / dose, then the material was diluted with PBS such that the diluted material was at this titre.

[0063] Passage 2, 3 and 4: Three pigs were inoculated with 1 ml of (diluted) material obtained in the previous passage via the intramuscular route. Just before inoculation, a blood sample was taken. The animals were observed daily for clinical signs. At 10 days post inoculation the animals were euthanised, and a blood sample was taken. The ASFV titre was estimated by qPCR based on a serial dilution with known titres of ASFV in the ASFV qPCR. The sample with the highest amount of ASFV DNA was used for inoculation of the next passage. If the viral load of the material was higher than the titre used in passage 1 , then the material was diluted with PBS such that the diluted material was at this titre.

[0064] Passage 5: Ten pigs were inoculated with 1 ml of material obtained in the previous passage via the intramuscular route. Just before inoculation, a blood sample was taken. The animals were observed for clinical signs, daily. At 21 days post inoculation the animals were euthanised, and a blood sample was taken. The ASFV titre was estimated by qPCR.

[0065] Results

[0066] No reversion to virulence could be detected at any passage for the novel strain. This was in contrast with strain ASFV-G-AI177L which in a corresponding test showed reversion to full virulence in passage 3. The results are summarised in table 2.

[0067] Table 2 Reversion to virulence in a five passage model Conclusion

[0068] Strain ASFV-G-A9GL / AUK / AEP153R shows no reversion to virulence in a five passage in vivo model.

[0069] EXAMPLE 3

[0070] In this example the safety of the novel strain for pregnant swine is evaluated. Safety for a particular strain in this subgroup of swine is not straightforward, not even if the strain has been established to be safe for non pregnant swine. In order to show this, in two comparative examples 3.1 and 3.2, the safety of two known protective and safe strains is tested in pregnant swine.

[0071] Example 3. 1

[0072] Object of the study

[0073] The goal of this experiment was to establish the safety of strain ASFV-G-AI177L for administration to pregnant swine. As is known from literature, this strain is highly effective in protecting swine against an infection with ASFV (Borca et al, Journal of Virology, April 2020, Volume 94, Issue 7, pages 1-18; doi

[0074] 10.1128 / / JVI.02017-19) and safe for administration to swine (Tran et al. in Viruses 2022, 14: Evaluation of the Safety Profile of the ASFV Vaccine Candidate ASFV- G-AI177L).

[0075] Study design

[0076] Four ASFV-free and ASFV antibody-free pregnant sows were available for this study. At 100 days in gestation (i.e. approximately 2 weeks before farrowing) two pigs received 2ml of a vaccine containing ASFV-G-AI177L 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. Animals were monitored daily from the day before vaccination onwards for ASF- specific clinical signs. Blood samples (from tail vein) were collected at 4 days post vaccination (dpv),11 dpv, 23 dpv, 32 dpv, while temperature was monitored daily from the day prior to inoculation onwards. Upon birth, the health status of the piglets was monitored and recorded. All the live piglets were weighed around 3-4 days after birth as well as at 16-17 days after birth. Piglets were monitored daily for ASF-specific clinical signs until the end of the study.

[0077] Results

[0078] Health status of the mother animals

[0079] Rectal temperatures were normal (between 37.5°C and 39°C) and not significantly different for vaccinated versus 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 slight signs of ASF, but these subsided gradually and disappeared. With qPCR, no ASFV could be detected in blood samples obtained from the control animals, whereas the vaccinated animals showed virus in their blood at all timepoints.

[0080] Table 3 shows significant differences in the reproductive performance of vaccinated and control sows. In case of vaccinated sows, 43% of the piglets were bom dead as compared to 17% for control sows. During the experiment, while all the live born piglets of the control sows survived into the third week of age, only 4 of the piglets born to the vaccinated sows survived this period. In addition, all surviving piglets of the vaccinated sows presented ASF-specific clinical signs and were euthanised on reaching the humane endpoint (HEP). This means that overall, there was a loss of more than 90% in viable piglets due to the vaccination with ASFV-G-AI177L. Table 3 reproductive performance of mother animals

[0081] Health status of the piglets

[0082] The rectal temperatures of the piglets bom from control animals were normal throughout the experiment, ranging from 38.7 to 40°C, whereas this was up to 41 °C in the piglets bom from vaccinated animals. With regard to weight gain, piglets of the control sows showed on average a 2.9-fold increase in weight when weighed at days 3-4 and day 17-18 post farrowing, while in case of the vaccinated sows surviving animals showed on average a 2.2-fold increase in weight.

[0083] The control piglets were all healthy throughout the experiment. Of the vaccinated animals, all piglets bom alive showed ASF-related clinical signs, and ASFV infection was confirmed by qPCR. A AI177L-specific PCR performed on DNA extracted from blood of pregnant sows and their representative piglets indicated that the vaccine strain was transmitted vertically from the pregnant sows to their piglets.

[0084] Conclusion

[0085] By comparison of the health status of the sows (and piglets bom alive from these sows), in particular since the loss of viable offspring is (significantly) over 50%, it can be concluded that the vaccine strain ASFV-G AI177L is not safe for administration to pregnant swine. Example 3.2

[0086] Object of the study

[0087] Example 3.2 is another experiment wherein a further protective live attenuated ASFV-G vaccine strains was tested for its safety in pregnant sows. The vaccine candidate is derived from the Lv17 / WB / Rie1 strain (WO 2020 / 049194). In particular, this vaccine comprises a mutant indicated as Lv17 / WB / Rie1 -ACD, which mutant comprises the additional gene deletions AEP153R and AEP402R (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 efficacious for use in swine (Urbano, supra) and the double knock out mutant strain is also known to be (inherently safe and) efficacious for the use in swine (European Patent Application No. EP22462011.1 for 'Attenuated African swine fever virus and use thereof in vaccine compositions”, filed in the name of Intervet International BV, Consejo Superior de Investigaciones Cientificas (CSIC), Allatorvostudomanyi Kutatointezet, Universidad Complutense de Madrid, and Eurofins Ingenasa SA, on 22 November 2022 at the Hungarian Intellectual Property Office).

[0088] Study Design

[0089] Four ASFV-free and ASFV antibody-free pregnant sows were available for this study. At around 100 days in gestation (i.e. approximately 2 weeks before farrowing) two sows received 2ml of vaccine comprising strain Lv17 / WB / Rie1 -ACD intramuscularly (IM) in the right side of the neck at a dose of 103TCID50. The other two sows served as non-inoculated controls. Animals were monitored from vaccination onwards for ASF specific clinical signs. Blood samples were collected at 3 days post vaccination (dpv), 10 dpv, 21 dpv and 31 dpv, while temperatures were monitored daily from day of inoculation onwards. Upon birth, the health status of the piglets was monitored and recorded. In case of any still bom or dead piglets, a blood sample was taken, whenever possible before the animal was disposed. All the live piglets were weighed 5 days after birth as well as 19 days after birth. Piglets were monitored daily for ASF-specific clinical signs till the end of the study at 22 days after birth.

[0090] Results

[0091] Health status of the mother animals

[0092] Rectal temperatures were normal (between 37.5°C and 39.5°C) for the control animals. The animals vaccinated with Lv17 / WB / Rie1-ACD had a continuous increase in temperature and showed signs of ASF from day 4 post vaccination and onwards. Both the sows in this group were euthanised on the 10thday post vaccination. The piglets from these sows were either bom prematurely as they were in the process of abortion or were taken from the womb at euthanasia. The control animals did not exhibit any clinical signs of ASF. With qPCR, no ASFV could be detected in the control animals, whereas the vaccinated animals showed virus in their blood at all timepoints.

[0093] Regarding reproductive performance, the sows used in the study had a healthy breeding history with overall more than 80% of viable piglets bom to them. In this study, however, significant differences were observed in the reproductive performance of sows vaccinated with the vaccine strain. Piglets were bom prematurely 8 to 10 days before the expected due date in case of sows vaccinated with the Lv17 / WB / Rie1-ACD strain. Both these sows began to abort piglets, which were weak, and underdeveloped with no chance of survival. These piglets were euthanised upon birth due to reaching HEP. In total three piglets were bom still.

[0094] For the control animals, the delivery was under normal circumstances and all the live bom animals survived until end of the study. The reproductive performance of sows in this study is presented in Table 4 here beneath. Table 4 reproductive performance of mother animals

[0095] (they got trapped under the sow). A corrected loss is 16%.

[0096] Health status of the piglets

[0097] None of the piglets bom from sows vaccinated with Lv17 / WB / Rie1 -ACD were alive more than 1 day post farrow, the health status was only monitored for the control animals. The rectal temperatures of all piglets were normal throughout the experiment. With regard to weight gain, piglets of the control sows showed on average a 3.7-fold increase from weight at birth, which is a normal figure. Thus, the control piglets were all healthy throughout the experiment.

[0098] Conclusion

[0099] By comparison of the health status of the sows (and piglets bom alive from these sows) with control sows, in particular since the loss of viable offspring is over 50%, it can be concluded that the Lv17 / WB / Rie1 -ACD strain is not safe for administration to pregnant swine.

[0100] Example 3.3

[0101] Object of the study

[0102] Example 3.3 is an experiment wherein the live attenuated ASFV-G- A9GL / AUK / AEP153R vaccine strains was tested for its safety in pregnant sows.

[0103] Study Design

[0104] Sixteen ASFV-free and ASFV antibody-free pregnant sows were available for this study. At around 100 days in gestation (i.e. approximately 2 weeks before farrowing) eight sows received 2ml of vaccine comprising strain ASFV-G- A9GL / AUK / AEP153R intramuscularly (IM) in the right side of the neck at a dose of 4.5log TCID50. The other eight sows served as non-inoculated controls. Animals were monitored from vaccination onwards for ASF specific clinical signs. Blood samples were collected at 3 days post vaccination (dpv), 10 dpv, 21 dpv and 31 dpv, while temperatures were monitored daily from day of inoculation onwards.

[0105] Upon birth, the health status of the piglets was monitored and recorded. In case of any still bom or dead piglets, a blood sample was taken, whenever possible before the animal was disposed. Piglets were monitored daily for ASF-specific clinical signs till the end of the study at approximately 22 days after birth (not all piglets were born on the same day).

[0106] Results

[0107] Health status of the mother animals

[0108] Average rectal temperatures were normal (between 37.3°C and 38.3°C) for the control animals as well as the animals vaccinated with ASFV-G- A9GL / AUK / AEP153R. None of the animals exhibited any clinical signs typical of ASF (such as neurological signs or haemorrhages on the skin). With qPCR, no ASFV could be detected in the control animals, whereas the vaccinated animals showed virus in their blood at all timepoints.

[0109] Regarding reproductive performance, the sows used in the study had a healthy breeding history. Overall, no substantial differences were observed in the reproductive performance of sows vaccinated with the vaccine strain. In case of one vaccinated sow, piglets were bom prematurely. These piglets were weak, underdeveloped and had an inherent low chance of survival. This event with this one sow reduced the overall survival rate of the piglets in the vaccinated group substantially.

[0110] For the control animals, the delivery was under normal circumstances and a large part of the live bom animals survived until end of the study. The reproductive performance of sows in this study is presented in Table 5 here beneath.

[0111] Table 5 reproductive performance of mother animals

[0112] * an unknown number of animals of this group that did not reach the end of the study died of an accident (they got trapped under the sow). A corrected loss could not be calculated since the number of trapped piglets was not recorded.

[0113] Health status of the piglets

[0114] None of the piglets bom from sows vaccinated were infected with ASFV at one day of age. At approximately one week of age, some piglets became infected. The rectal temperatures and weight gain of all piglets were normal throughout the experiment. No typical ASF clinical signs were observed.

[0115] Conclusion

[0116] By comparison of the health status of the sows (and piglets bom alive from these sows) with control sows, in particular since the loss of viable offspring due to vaccination is well below 50%, it can be concluded that the ASFV-G- A9GL / AUK / AEP153R strain is safe for administration to pregnant swine.

Claims

CLAIMS1 . A live attenuated African swine fever virus Georgia 2007-A9GL / AUK / AEP153R (ASFV-G-A9GL / AUK / AEP153R) 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- A9GL / AUK / AEP153R strain to the pregnant swine.

2. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to claim 1 , characterised in that the vaccine is administered to the pregnant swine in the second half of gestation.

3. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to any of the preceding claims, characterised in that the vaccine is administered to the pregnant swine in the last third of gestation.

4. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to any of the preceding claims, characterised in that the vaccine is administered to the pregnant swine in a period of 5-20 days before the expected date of farrowing of the pregnant swine.

5. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to any of the preceding claims, characterised in that the vaccine is administered once or in a prime and boost regimen to the pregnant swine.

6. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to any of the preceding claims, characterised in that the vaccine is administered intramuscularly, intradermally or orally.

7. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to any of the preceding claims, characterised in that the vaccine is administered with a dose of the ASFV-G-A9GL / AUK / AEP153R strain of at least 102TCIDso per administration.

8. A live attenuated ASFV-G-A9GL / AUK / AEP153R strain for use according to any of the preceding claims, characterised in that the vaccine is administered with a dose of the ASFV-G-A9GL / AUK / AEP153R strain between 102TCID50 and 106TCID50 per administration.

9. Use of a live attenuated ASFV-G-A9GL / AUK / AEP153R strain for the manufacture of a vaccine for the protection of a pregnant swine against an infection with African swine fever virus.

10. Method for the protection of a pregnant swine against an infection with African swine fever virus by administering to the pregnant swine a vaccine comprising a live attenuated ASFV-G-A9GL / AUK / AEP153R strain.

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