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BR-112018076633-B1 - Recombinant mutant of ASFV-G (African Swine Fever Virus Isolate Georgia 2007), mutant cDNA, vaccine composition, method for protecting pigs against African Swine Fever Virus Isolate Georgia 2007 (ASFV-G), method for differentiating a mammal vaccinated with a live attenuated ASFV-G vaccine. DELTA-9GL/DELTA-UK

BR112018076633B1BR 112018076633 B1BR112018076633 B1BR 112018076633B1BR-112018076633-B1

Abstract

African swine fever virus (ASFV) is the causative agent of a frequently fatal contagious viral disease in domestic pigs. Control of African swine fever is hampered by the unavailability of vaccines. Experimental vaccines have been derived from naturally occurring live attenuated ASFVs, adapted to cell culture, or genetically modified; however, these vaccines are only successful in protecting against homologous viruses. We constructed a recombinant 9GL/UK virus derived from the Georgia 2007 isolate (ASFV-G) of highly virulent ASFV by deleting the specific 9GL (B119L) and UK (DP96R) genes associated with virulence. In vivo, the 9GL/UK ASFV-G, administered intramuscularly to pigs, even at relatively high doses (10⁶ HAD₅₀), does not induce disease. Substantially, animals infected with 104 or 106 HAD50 are strongly protected against exposure to clinical disease when challenged 28 days after infection with the virulent parental Georgia 2007 strain.

Inventors

  • MANUEL V. BORCA
  • DOUGLAS P. GLADUE
  • LAUREN G. HOLINKA-PATTERSON
  • GUILLERMO R. RISATTI
  • VIVIAN K. O ' DONNELL

Assignees

  • THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF AGRICULTURE
  • THE UNIVERSITY OF CONNECTICUT

Dates

Publication Date
20260317
Application Date
20170626
Priority Date
20160701

Claims (5)

  1. 1. Recombinant mutant of ASFV-G (African Swine Fever Virus Georgia 2007 isolate), characterized in that the ASFV-G Δ9GL/ΔUK mutant virus comprises cDNA encoding the ASFV-G Δ9GL/ΔUK mutant polypeptides, wherein the mutant cDNA comprises two deletions: a 173-nucleotide deletion resulting in a 9GL mutant protein comprising 58 fewer amino acids than the unchanged, wild-type ASFV-G 9GL protein, with amino acids #11 to #68 deleted; and a second 255-nucleotide deletion resulting in a UK mutant protein comprising 85 fewer amino acids than the non-mutated, wild-type ASFV-G UK protein, with amino acids #1 to #85 deleted.
  2. 2. Mutant cDNA, as defined in claim 1, characterized in that said cDNA has SEQ ID NO: 3.
  3. 3. Vaccine composition, characterized by comprising the recombinant mutant virus ASFV-G Δ9GL/ΔUK as defined in claim 1 or claim 2.
  4. 4. Use of a live attenuated ASFV-G (African Swine Fever Virus Isolate Georgia 2007), characterized in that it is for preparing a Δ9GL/ΔUK vaccine comprising a recombinant mutant ASFV-G Δ9GL/ΔUK virus as defined in claim 1 or claim 2 in an amount effective to protect said swine from clinical ASF-G.
  5. 5. Use of a live attenuated ASFV-G (African Swine Fever Virus Isolate Georgia 2007), according to claim 4, characterized in that the effective amount to protect said swine from clinical ASF-G is a vaccine comprising 10⁴ HAD₅₀ to 10⁶ HAD₅₀ of the ASFV-G Δ9GL/ΔUK virus.

Description

Field of Invention [0001] This invention relates to the construction of a candidate live attenuated African Swine Fever Virus (ASFV) strain vaccine recombinant to the highly virulent Georgia 2007 isolate of ASFV-G. The vaccine comprises the modified ASFV-G Δ9GLΔUK virus, a recombinant ASFV-G modified by the deletion of a large portion of the 9GL gene (B119L) and the UK gene (DP96R). History of the Invention [0002] African Swine Fever (ASF) is a contagious viral disease of swine. The causative agent, ASF virus (ASFV), is a large enveloped virus containing a double-stranded genome of approximately 190 kilobase pairs. ASFV shares aspects of genomic structure and replication strategy with other large double-stranded DNA viruses, including Poxviridae, Iridoviridae, and Phycodnaviridae (Costard et al. 2009. Phil. Trans. Royal Soc. B 364:2683-2696). ASFV infections in domestic pigs are often fatal and are characterized by fever, hemorrhage, ataxia, and severe depression. However, the course of the infection varies, ranging from highly lethal to subclinical, depending on the characteristics of the host and the particular strain of the virus (Tulman et al. 2009. Curr. Top. Microbiol. Immunol. 328:43-87). [0003] Currently, the disease is endemic in more than twenty countries in sub-Saharan Africa. In Europe, ASFV is still endemic on the island of Sardinia (Italy), and new outbreaks have been reported in the Caucasus region since 2007, affecting Georgia, Azerbaijan, and Russia. Isolated outbreaks have recently been reported in Ukraine, Belarus, Lithuania, Latvia, and Poland, representing the risk of further spread to neighboring countries. The epidemic virus, ASFV Georgia 2007/1, is a highly virulent isolate belonging to genotype II (Chapman et al. 2011. Emerging Infect. Dis. 17:599-605). [0004] Currently, there is no vaccine available for ASF and outbreaks of the disease are controlled by quarantining and culling the animals. Attempts to vaccinate animals using infected cell extracts, peripheral blood leukocyte supernatants from infected pigs, inactivated and purified virions, infected macrophages fixed with glutaraldehyde, or infected alveolar macrophages treated with detergent have failed to induce protective immunity (Coggins, L. 1974. Prog. Med. Virol. 18:48-63; Forman et al. 1982. Arch. Virol. 74:91-100; Kihm et al. 1987. In: African Swine Fever, Becker, Y. (ed), Martinus Nijhoff, Boston, pp 127-144; Mebus, C. A. 1988. Adv. Virus Res. 35:251-269). Homologous protective immunity develops in pigs that survive viral infection. Pigs that survive acute infection with moderately virulent or attenuated variants of ASFV develop long-lasting resistance to homologous viral challenge, but rarely to heterologous challenge (Hamdy and Dardiri, 1984. Am. J. Vet. Res. 45:711-714; Ruiz-Gonzalvo et al., 1981. In: FAO/CEC Expert Consultation in ASF Research, Wilkinson, P. J. (ed), Rome, pp. 206-216). Pigs immunized with live attenuated ASF viruses containing engineered deletions of specific genes associated with ASFV virulence were protected when challenged with the homologous parental virus. Specifically, individual deletions of UK (DP69R), 23-NL (DP71L), TK (A240L), or 9GL (B119L) genes originating from pathogenic ASF virus genomes (Malawi Lil-20/1, Pretoriuskop/96/4, E70, and Georgia 2007) markedly attenuated the virus in swine, and animals immunized with these attenuated viruses were protected against challenge with homologous viruses (Moore et al. 1998. J. Virol. 72:10310-10315; Lewis et al. 2000. J. Virol. 74:1275-1285; Zsak et al. 1996. J. Virol. 70:8865-8871; Zsak et al. 1998. J. Virol. 72:1028- 1035). These observations constitute the only experimental evidence describing the rational development of a live attenuated virus effective against ASFV. [0005] In particular, the deletion of 9GL (B119L) in highly virulent ASFV isolates Malawi Lil-20/1, Pretoriuskop/96/4 and Georgia2007 (Lewis et al., supra; Neilan et al. 2004. Virol. 319:337-342; O’Donnell et al. 2015. J. Virol. 89: 8556-8566) resulted in complete attenuation of these viruses in swine. Administration of Malawi Lil-20/1 Δ9GL or Pretoriuskop/96/4 Δ9GL or the E70 UK mutants in pigs via IM injection at a relatively high viral dose did not induce clinical signs, and all animals survived the infection. Furthermore, IM inoculation with these viruses in pigs induced protection against challenge with virulent parental viruses (Zsak et al. 1998, supra; Lewis et al., supra; O’Donnell et al., supra). These observations constitute the only experimental evidence describing the rational development of a live attenuated virus effective against ASFV. [0006] As there are currently no ASFV vaccines available, the development of any experimental vaccine that can induce any type of protection against a lethal outbreak of the disease is of great interest. Brief Description of the Invention [0007] We have developed the new recombinant mutant virus ASFV-G Δ9GL/ΔUK which is a modificatio