1. Introduction
African swine fever (ASF) is a devastating viral disease that only affects suids. ASF is asymptomatic in African wild suids, such as bush pigs or warthogs, but depending on the strain virulence, it can cause a hemorrhagic syndrome with a high lethality rate, up to 95–100%, in domestic pigs and wild boars (
Sus scrofa sp.). Pigs infected with a highly virulent strain can display diverse symptoms such as fever, loss of appetite, depression, hemorrhages, skin lesions, vomiting and coughing. Chronic disease characterized by reduced growth and joint swelling can be seen when pigs are infected with a less virulent strain [
1].
The disease is due to the African swine fever virus (ASFV), a large, enveloped, linear double-stranded DNA virus that is the only representative of the
Asfarviridae family. Its genome of 170–194 kbp encodes for 150 to 200 proteins, of which the functions for a number of them remain unknown [
2]. Based on the sequencing of C-terminus of the p72 gene, 24 genotypes of ASFV have been identified [
3].
ASF was first described in Africa in 1910 [
4] and remains endemic in many sub-Saharan countries. After the eradication of outbreaks due to an ASFV strain of genotype I in Europe and the Americas in the 1960–1990s, all countries outside of Africa remained free of the virus, with the exception of Sardinia, Italy, where the disease has been present since 1978 [
5]. However, in 2007, a new ASFV strain of genotype II was introduced on the European continent in Georgia [
6], which has further spread to the Caucasus and the Russian Federation [
7], entering the European Union in 2014 [
8], China in 2018 [
9], and then further affecting East Asia [
10]. In 2021, ASF crossed the Atlantic ocean to reach the Dominican Republic and Haiti [
11], and it is still spreading among the European Union member states, as is seen with the emergence of the disease in Germany in 2021 [
12] and in mainland Italy in 2022 [
13].
As there is no vaccine or treatment available, the only control measures that can be applied are the culling of affected pig herds and the restriction of movement around the outbreak to contain viral spread. Regarding wild boar populations, a strategy relying on the suspension of hunting in the heart of the infected zone and any forestry activities that could disperse the animals, the division of the landscape with fences, and the drastic reduction in the number of wild boar present in the area bordering the infected zone (the so-called “white zone”), has enabled the Czech Republic and Belgium to regain their ASF-free status in two years [
14]. However, this strategy could prove more difficult to apply when the affected wild boar population lives in an area that is more difficult to delimit due to its geography and landscape (mountains, wetlands, etc.), or in a region where the population undergoes multiple introductions of the virus [
15,
16].
Since ASF has become a global threat for pig producers, the demand for the development of a vaccine for either domestic pigs or wild boars has considerably increased over these last five years.
As recently reviewed, different vaccine development strategies have been attempted with varying success [
17,
18,
19,
20,
21,
22]. The administration of the inactivated virus in the absence or presence of modern adjuvants did not induce any protection, even if the candidate vaccines induced seroconversion [
23,
24]. Subunit vaccine-type approaches, based on recombinant proteins or plasmid DNAs, have induced little or no protection. Partial protection with a delay in clinical expression was obtained through the inoculation of ASFV DNA plasmid in the absence of seroconversion [
25]. More complete protection with a marked reduction in clinical signs in 60% of the immunized pigs was obtained following immunization with a DNA library [
26]. The vector-based approaches developed have also failed to protect pigs, regardless of the vector used, even if they were immunogenic [
27,
28], with the exception of a more recent study using a prime with a cocktail of eight recombinant adenoviruses, followed by a boost with the recombinant modified vaccinia Ankara that codes for the same eight genes [
29].
Currently, it is still the live attenuated strains that induce the best protection, as already demonstrated with the naturally attenuated strains of genotype I that allow homologous or cross-protection against some other genotypes [
30]. However, these attenuated strains can induce some inflammation and edema at the joint level, with negative impacts on the growth of pigs. Tentatively increasing the attenuation by introducing genetic deletion into the genomes of these naturally attenuated strains has led, depending on the case, either to a reduction in pig protection [
31], or to a better protection [
32], highlighting the extreme complexity of the host–virus interactions. Because of our better knowledge of the ASF virus genome, the development of virulence gene-deleted strains has emerged as a promising approach for vaccine development. As such, the deletion of multigene family (MGF) genes (MGF 505 and MGF 360) has been conducted to attenuate the Georgia strain with a good protection of immunized pigs against a Georgia virulent challenge [
33]. In contrast, the deletion of other genes such as CD2v/EP402R induces inconsistent attenuation according to the parental strain [
34,
35]. The last gene-deleted strain developed by the USDA (ASFV-G-ΔI177L) showed very promising results, and its safety has been concluded to be satisfactory in experimental and field conditions in Vietnam [
36].
Here, we report the results of experimental studies conducted on specific-pathogen-free (SPF) pigs to assess the potency and safety of the “ASFV-989”, a new live vaccine candidate generated by thermo-attenuation of the Georgia 2007/1 strain. Animals inoculated either by intramuscular or oronasal routes were fully clinically protected when challenged with the parental Georgia 2007/1 strain as soon as 2 weeks after immunization. Moreover, the Georgia genome was never detected in the blood of the immunized pigs after the challenge.
4. Discussion
We did not expect to generate the ASFV-989 strain when we carried out the protocol of thermo-inactivation by incubating a tube of ASFV Georgia 2007/1 in a wet bath for two hours at 60 °C. Even if it was not possible to isolate the virus on PAMs after this heat treatment, one of the two inoculated SPF pigs developed moderate hyperthermia, ASFV viremia, and an antibody response. From a blood sample collected from pig 989 at 7 dpi, we could isolate a new attenuated strain that we named “ASFV-989”.
Thermo-attenuation of ASFV has never been reported before. The only other virus for which we found reports of heat treatment attenuation is the infectious bronchitis virus (IBV) [
42,
43]. In this case, the thermo-attenuation process consists of repeated combinations of heat treatment at 56 °C, followed by inoculation of embryonated eggs. For IBV, the mechanism underlying the thermo-attenuation would be related to a higher thermal resistance of low-virulence isolates.
In the case of the thermo-attenuation of ASFV we reported here, we speculate that the underlying mechanism may be different as the PCR 989 performed on the initial Georgia 2007/1 viral suspension submitted to heat treatment gave negative results, indicating that the ASFV-989 strain was not present before heat treatment. Even if highly speculative, we could hypothesize that the heat treatment may have induced a fragility of the viral genome that conducted to the deletion in the MGF 505/360 genes. Considering that the genetic alteration of the virus due to heat treatment is certainly random, we can assume that reproducing the procedure by applying the same heat treatment to the same ASFV isolate would probably not induce the same results.
Through full-genome sequencing, we identified in the ASFV-989 strain a deletion of 7458 nucleotides corresponding to the partial deletion of MGF 505-1R and 505-4R and the complete deletion of MGF 360-12L, 360-13L, 360-14L, 505-2R, 505-3R, and ASFV_G_ACD_00520. It is worth mentioning that the deleted region in the ASFV-989 strain is very close to the one deleted in the ASFV-G-ΔMGF attenuated strain previously described by O’Donnel et al. [
33], which encompasses MGF 505-1R, 360-12L, 360-13L, 360-14L, 505-2R, and 505-3R. The nonessential genes deleted in both strains are known to be involved in ASFV immune evasion, especially in the suppression of the type I interferon response [
44,
45]. The kinetics of multiplication of this new strain were then compared to Georgia 2007/1 on PAMs, and we showed that the ASFV-989 strain demonstrated the same capacity to replicate in the host cells as the parental strain.
As ASF has become a global threat to pig production, a vaccine that could be applied via an intramuscular route in commercial pig farms or by an oral route to backyard or wild boar populations is of paramount interest for the pork industry as a complementary tool to control the disease. For this reason, we directly compared the two routes of inoculation, intramuscular (IM) and oronasal (ON), in our SPF pigs.
Only three out of seventeen ASFV-989 IM-inoculated pigs displayed some severe clinical signs and died or had to be euthanized. All the other pigs developed lower fever, limited symptoms, and a drop in growth performance in the two first weeks post-inoculation, and then they recovered. Even if genetically close, the virulence of the ASFV-989 strain seems slightly higher than the one of the ASFV-G-ΔMGF strain, as O’Donnel et al. did not detect any clinical signs after IM inoculation of 10
4 HAD
50 of the ASFV-G-ΔMGF strain [
33]. This difference in virulence level could be due to the slight genetic differences between the two deleted strains. Indeed, as highlighted by Rathakrishnan and al. [
45], the differential deletion of only a few MGF genes can significantly modify the virulence level of ASFV strains.
Another explanation for the relatively high level of residual virulence measured for the ASFV-989 strain could be linked to our SPF pig infection model. Our SPF pigs were previously shown to be highly sensitive to ASFV when they were inoculated with attenuated strains such as OURT88/3 [
30] or E75CV1 [
26], possibly in relation to their relatively poor microbiota diversity compared to conventional farm pigs [
46].
Pigs inoculated oronasally with the ASFV-989 strain displayed a drop in growth performance during the first week post-inoculation, and then recovered and remained clinically normal until the end of the experiment, with no pig mortality in this group. IM- and ON-inoculated pigs showed a peak of ASFV-989 viremia at 7 days post-inoculation, but this peak was lower in ON-inoculated compared to IM-inoculated pigs. The duration of the viremia was also shorter for ON-inoculated than for IM-inoculated pigs. Here, a link might be made between the lower virulence of the ASFV-989 strain in the pigs inoculated via the ON route and the lower ASFV viremia measured in these animals. Interestingly, we also measured a higher level of a cell-mediated immune response in pigs inoculated through the ON route, which could explain the better control of the ASFV-989 viremia in these animals.
After the challenge with the Georgia 2007/1 strain at 4 weeks post-immunization, all the pigs remained clinically healthy no matter the inoculation route. No Georgia 2007/1 viremia was detected in any pigs, even among the pigs immunized through the ON route that were further challenged intramuscularly, which suggests that the strong vaccine efficacy is unrelated to the immunization or the challenge route. This high level of clinical and virological protection against the Georgia 2007/1 challenge was also reached when reducing the delay between the immunization and challenge to 2 weeks, suggesting a rapid onset of immunity that could be linked to the observed rapid seroconversion, but also to the induction of an ASFV-specific, cell-mediated immune response in less than 14 days post-immunization. Indeed, as previously described by Takamatsu et al. [
47], cellular immune responses are probably involved in the strong clinical and virological protection induced by the ASFV-989 immunization.
In terms of vaccine efficacy, the ASFV-989 strain seems to show a higher efficiency compared to the ASFV-G-ΔMGF strain since the Georgia strain was detected after the challenge in 30 to 40% of the pigs immunized with the ASFV-G-ΔMGF strain [
33]. Regarding the safety/efficacy balance, we can consider the ASFV-G-ΔMGF strain to have very good safety but imperfect efficacy, whereas the ASFV-989 strain has perfectible safety but very strong efficacy. As mentioned previously, these apparent differences between the phenotype of the two strains could be also due to differences between the pig models used to assess each strain.
The next step will be to adapt the ASFV-989 strain to grow on a permanent cell line, as it is difficult to scale-up to the industrial production a strain cultivated on primary cells such as macrophages. However, the adaptation of the strain to a cell line can further attenuate it and modify its efficacy; thus, other in vivo experiments will be necessary to check if the adaptation has led to better attenuation while keeping its efficacy, as performed by Borca et al. for the ASFV-G-ΔI177L vaccine candidate [
48].
Based on the results presented in this report, the ASFV-989 strain can be considered as a good vaccine candidate because of its high efficacy in inducing protection, even by the oronasal route, opening up a way for its potential usage through oral vaccination in wild-boar-affected populations, as carried out successfully in the past in Western Europe to eradicate classic swine fever [
49]. Moreover, the differential PCR that we have developed would allow for differentiating between vaccinated and infected animals, which is an important step in eradication plans for wild fauna [
50].
Although there is currently one vaccine candidate that has already been approved in Vietnam [
36], we believe that ASFV-989 is a promising vaccine, as it has not been obtained by genetic manipulation, which makes it easier to be applied through open distribution in the wild. Moreover, more than one gene was deleted, which might confer less risk of this strain reverting to virulence.