African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses
Introduction
African swine fever virus (ASFV), the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size that encodes more than 150 proteins, including structural and host immunomodulatory proteins. ASFV is the etiological agent of African swine fever (ASF), a devastating disease affecting wild boar and domestic pigs. An outbreak in the Caucasus in 2007 [1] initiated the spread of ASFV across Russia and Eastern Europe, currently also affecting Ukraine, Belarus, Poland, the Baltic States, the Czech Republic, Moldova and Romania, posing a serious risk for the global swine industry. Due to the absence of an effective vaccine, prevention, control and eradication measures are mostly based on early laboratory diagnostic detection and the implementation of strict sanitary measures.
Approaches for development of ASFV vaccines have included inactivated viruses, recombinant proteins/peptides, viral vectors for antigen delivery and live-attenuated vaccines [2], [3]. Attempts to protect animals using inactivated vaccines have failed, even in the presence of adjuvants [4], [5], [6], [7], whereas subunit vaccines have provided partial protection against lethal challenge [8], [9], [10], [11], [12]. Immunization of pigs with DNA encoding a fusion protein able to induce a strong CTL response generated partial protection against homologous ASFV virulent challenge but without an antibody response [13]. Further studies immunizing with a DNA expression library containing several other viral ORFs fused to ubiquitin also conferred partial protection against virulent challenge [14]. In contrast, immunization with baculovirus-expressed ASFV proteins p30, p54, p72 and p22, although capable of inducing neutralizing antibodies in vivo, failed to be protective [15]. Finally, a very recent strategy combining recombinant proteins and DNA vectors was also able to generate neutralizing antibodies and CTL, but protection was still lacking (Sunwoo S-Y, Pérez-Núñez D, Sánchez EG, Revilla Y et al., personal communication).
Given the challenging vaccination scenario, a so-called “live-attenuated vaccine” (LAV) emerges as a putative strategy in protection. Recombinant ASFVs containing specific deletions within virulence genes such as DP148R [16], EP402R [17], 9GL (B119L) genes or members of multigene families 360 and 505 (MGF 360/505) [18], [19], [20], [21], resulted in attenuation of virulent ASFV isolates and induction of protective immune responses against homologous and heterologous virus challenge [17].
On the other side, the attenuated ASFV strains OURT88/3 and NH/P68 have been shown to induce good levels of protection against lethal challenge by related virulent viruses [22], [23], [24], [25], [26], [27]. The use of attenuated strains as LAV candidates should be safer due to their low virulence, although adverse reactions including fever and joint swelling are observed [2], [25].
In the current study, we investigated the effect of deletion of genes (A238L [28], [29], [30]; A224L [31], [32]; EP153R [33], [34] and A276R [35]), involved in virus-host interaction and immune system control on protection and virulence of the naturally-attenuated ASFV strain NH/P68.
Section snippets
Cells and viruses
The low virulence, non-hemadsorbing ASFV isolate NH/P68 and the virulent, hemadsorbing isolate Lisbon 60 were both isolated in Portugal, and both are p72 genotype I [25], [36]. For challenge experiments, the virulent, hemadsorbing genotype II ASFV Arm07 was used [37]. Viruses were grown in porcine alveolar macrophages (PAM) [cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% pig serum (PS) (Sigma)] or in porcine blood monocytes (PBM) as previously described [38]. Viral
ASFV deletion mutants NH/P68ΔA238L, NH/P68ΔA224L, NHV/P68ΔA276R and NH/P68ΔEP153R are viable and infectious in vitro
In an attempt to attenuate NH/P68, viral genes involved in virus/host interactions and virulence were deleted. Genes included: A238L, which encodes a protein that modulates factors related to the immune and inflammatory response during infection [28], [29], [39], [42], [43], [44]; A224L, homologous to IAP apoptosis inhibitors [31]; EP153R, homologous to C-type lectins [40]; and A276R, described to inhibit IFN responses [35].
After isolation of recombinant mutants from plaques (Fig. 1A),
Discussion
Currently ASF is one of the most devastating viral diseases of both domestic pigs and wild boars. The present worrisome situation in Europe, as well as its endemic status in Africa, highlights the capacity of ASFV to spread and emphasize the urgent need for effective vaccines.
So far, inactivated ASFV and/or subunit vaccines have not been shown to fully protect against virulent challenge (reviewed in [2], [3]), thus LAVs may be the best strategy for protection. Experiments immunizing pigs with
Acknowledgements
This work was partly supported under the EU project ASFRISK, Evaluating and Controlling the Risk of African Swine Fever in the EU, (DG-Research, EC, 7FP Grant Agreement, KBBE211691), the EU project ASFORCE, Targeted Research Effort on African Swine Fever (DG-Research, EC, 7FP Grant Agreement, KBBE.2012.1.3-02, 311931) and the EU Reference laboratory for ASF (grant no UE-LR PPA/03). We would like to thank all the animal facilities staff at the INIA-CISA especially Miguel Angel Sánchez and Victor
References (48)
Challenges for African swine fever vaccine development – “… perhaps the end of the beginning.”
Vet Microbiol
(2017)- et al.
Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation
Vaccine
(2014) African swine fever
Adv Virus Res
(1988)- et al.
African swine fever virus structural protein p72 contains a conformational neutralizing epitope
Virology
(1994) - et al.
The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response
Virology
(1998) - et al.
Virulent African swine fever virus isolates are neutralized by swine immune serum and by monoclonal antibodies recognizing a 72-kDa viral protein
Virology
(1993) - et al.
Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection
Virology
(2004) - et al.
African swine fever virus Georgia isolate harboring deletions of 9GL and MGF360/505 genes is highly attenuated in swine but does not confer protection against parental virus challenge
Virus Res
(2016) - et al.
Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response
Vaccine
(2016) - et al.
Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus
Virology
(2013)
Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation
Vaccine
Different routes and doses influence protection in pigs immunised with the naturally attenuated African swine fever virus isolate OURT88/3
Antiviral Res
Identification and utility of innate immune system evasion mechanisms of ASFV
Virus Res
African swine fever virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells
Virology
The viral protein A238L inhibits cyclooxygenase-2 expression through a nuclear factor of activated T cell-dependent transactivation pathway
J Biol Chem
Inhibition of nuclear factor kappaB activation by a virus-encoded IkappaB-like protein
J Biol Chem
African swine fever virus isolate, Georgia, 2007
Emerg Infect Dis
The immunological response of pigs and guinea pigs to antigens of African swine fever virus
Arch Virol
Antibody response to inactivated preparations of African swine fever virus in pigs
Am J Veter Res
Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization
J Virol
Inhibition of African swine fever infection in the presence of immune sera in vivo and in vitro
Am J Veter Res
DNA vaccination partially protects against African swine fever virus lethal challenge in the absence of antibodies
PloS One
Expression library immunization can confer protection against lethal challenge with African swine fever virus
J Virol
Cited by (99)
ASFV epitope mapping by high density peptides microarrays
2024, Virus ResearchASFV proteins presented at the surface of T7 phages induce strong antibody responses in mice
2023, Journal of Virological MethodsRecent progress and major gaps in the vaccine development for African swine fever
2024, Brazilian Journal of MicrobiologyIdentification of L11L and L7L as virulence-related genes in the African swine fever virus genome
2024, Frontiers in MicrobiologyThe attenuated African swine fever vaccine HLJ/18-7GD provides protection against emerging prevalent genotype II variants in China
2024, Emerging Microbes and Infections
- 1
Both authors contributed equally to the work.