United States of America, As Represented by the Secretary of Agriculture

University of Connecticut

Universidad Del Pais Vasco/euskal Herriko Univertsitatea (upv-ehu)

Manuel V Borca

Guillermo R Risatti

Douglas P Gladue

Vivian O'Donnell

Lauren G Holinka-Patterson

Luis Leandro Rodriguez

Peter William Krug

James J Zhu

Daniel L Rock

Jose Nieva

Guillermo S Risatti

Ayushi Rai

Douglas Gladu

Vivian K O'donnell

Vivian K O'DONNELL

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate ('ASFV-G'). An exemplary vaccine comprises the ASFV-G&#x394;I1771 modified virus, a recombinant ASFV-G modified by deleting a portion of the I177L ORF rendering the I177L gene nonfunctional.

Development of a novel live attenuated African Swine Fever vaccine based in the deletion of gene I177L

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate ('ASFV-G'). An exemplary vaccine comprises the ASFV-G&#x394;A137R modified virus, a recombinant ASFV-G modified by deleting a portion of the A137R ORF rendering the A137R gene nonfunctional.

Development of a novel live attenuated African swine fever vaccine based in the deletion of gene A137R

Methods are provided herein utilizing a stable cell line capable of efficient infection by African swine fever virus (ASFV) and also provides for the detection of the presence of virus in samples applied to the cells. Detection of the virus by means such as red blood cell rosetting is a surprising result given that the cell line is derived from African green monkeys. This cell line provides a marked improvement over the currently available testing strategies.

Continuous stable cell line for identification of infectious African swine fever virus in clinical samples

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate ('ASFV-G'). An exemplary vaccine comprises a deletion of multiple genes allowing for industrial-scale growth in stable cell lines.

Genomic deletion in African swine fever vaccine allowing efficient growth in stable cell lines

The role of a specific E2 region containing a putative fusion peptide (FP) sequence was evaluated. FPs critically contribute to the interaction between proteins and the membrane system of the host cell. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within this region of E2 may affect replication of BICv in cell cultures and affect virus virulence in swine. Interestingly, mutated virus FPi.c was completely attenuated when inoculated intranasally at a dose of 10TCID50 in swine. Importantly, animals infected with FPi.c virus were protected against the virulent challenge with Brescia virus at 3 and 28 days after vaccination. Protection was evidenced by absence of clinical signs related with CSF as well as the absence of viremia produced by the challenge virulent virus.

Live attenuated classical swine fever vaccine based in genetic manipulation of a putative fusion peptide area in the virus structural glycoprotein E2

African swine fever virus (ASFV) is the etiological agent of a contagious, often lethal viral disease of domestic pigs. The control of African Swine Fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFVs; however, these vaccines are only successful when protecting against homologous viruses. We have constructed a recombinant &#x394;9GL/&#x394;UK virus derived from the highly virulent ASFV Georgia 2007 (ASFV-G) isolate by deleting the specific virulence-associated 9GL (B119L) and the UK (DP96R) genes. In vivo, ASFV-G &#x394;9GL/&#x394;UK administered intramuscularly to swine even at relatively high doses (10HAD) does not induce disease. Importantly, animals infected with 10or 10HADare solidly protected against the presentation of clinical disease when challenged at 28 days post infection with the virulent parental strain Georgia 2007.

Rationally developed african swine fever attenuated virus strain protects against challenge with parental virus georgia 2007 isolate

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. Control of ASF has been hampered by the unavailability of vaccines. Experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFVs; however, these vaccines are only successful when protecting against homologous viruses. Among viral genes reported to be involved in virulence are components of the multi gene family (MGF). Here we report the construction of a recombinant &#x394;MGF virus derived from the highly virulent ASFV Georgia 2007 (ASFV-G) isolate. In vivo, ASFV-G &#x394;MGF administered intramuscularly (IM) to swine at either 10or 10HADare completely attenuated; the inoculated animals are completely asymptomatic. Animals infected with 10or 10HAD50 of ASFV-G &#x394;MGF are protected against the presentation of clinical disease when challenged at 28 days post infection with the virulent parental strain Georgia 2007.

Attenuated African swine fever virus vaccine based in the deletion of MGF genes

We have developed an ASFV Georgia strain adapted to grow in Vero cell line. The resulting virus, ASF-GVAV, efficiently grows in Vero cells although it still is able to significantly replicate in primary cell cultures of swine macrophages. ASF-GVAV virus was successfully used as parental virus to develop several recombinant ASF viruses. The development of an ASFV adapted to grow in an established cell line is a significant advance for research and development of vaccine candidate strains using genetic manipulation based in the process of homologous recombination. The GVAVS can be utilized as a basis for large scale production of ASF vaccines.

African swine fever virus georgia strain adapted to efficiently grow in the vero cell line

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for swine breeding. Control of ASF has been hampered by the unavailability of vaccines. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against the challenge with parental viruses. Here we report the construction of a recombinant &#x394;9GL virus derived from the highly virulent ASFV Georgia 2007 (ASFV-G) isolate. In vivo, ASFV-G &#x394;9GL administered intramuscularly (IM) to swine at relatively high doses (10HAD) retains a virulent phenotype practically indistinguishable from the parental virus. Conversely, at low IM doses (10or 10HAD), ASFV-G &#x394;9GL does not induce disease. Importantly, animals infected with 10HADare protected against the presentation of clinical disease when challenge at 28 days post infection with the virulent parental strain Georgia 2007.

Attenuated african swine fever virus strain induces protection against challenge with homologous virulent parental virus georgia 2007 isolate

Controlling Classical Swine Fever Virus (CSFV) involves either prophylactic vaccination or non-vaccination and elimination of infected herds depending on the epidemiological situation. Marker vaccines allowing distinction between naturally infected from vaccinated swine could complement 'stamping out' measures. Previously, we reported the development of FlagT4v, a double antigenic marker live attenuated CSFV strain. FlagT4v was later shown as not to be completely stable in terms of its attenuation when assessed in a reversion to virulence protocol. We have developed a modified version of the FlagT4v where changes in the codon usage of genomic areas encoding for Flag and T4 were introduced to rectify the reversion to the virulent genotype. The new virus, FlagT4-mFT-Gv, possesses the same amino acid sequence as FlagT4v except for one substitution, Asparagine is replaced by Glycine at position 852 of the CSFV polypeptide. FlagT4-mFT-Gv protected swine against challenge with Brescia virulent virus at 21 days post vaccination.

Live attenuated antigenically marked classical swine fever vaccine

E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E2 is involved in several functions including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Seven putative glycosylation sites in E2 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2, but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection suggesting that glycosylation of E2 could be modified for development of CSF live-attenuated vaccines. Additionally, a new developed virus, contained deletions of putative glycosylation sites N1 in E2 and N1 in E0 (6b), called N1E0/2v, induce a solid protection against the challenge at 3 and 28 days post-inoculation.

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Classical Swine Fever Virus (CSFV) E2 glycoprotein is a major inducer of neutralizing antibodies and protective immunity in swine. E2 mediates virus adsorption to the target cell, and harbors genetic determinants associated with virus virulence. CSFV E2 also contains between residues 829 and 837 a discrete epitope (TAVSPTTLR) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related Pestiviruses Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV). In this report, a CSFV infectious clone of the virulent Brescia isolate (BICv) was used to progressively mutate the mAb WH303 epitope of CSFV E2 to the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). While the resulting virus mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) demonstrated in vitro growth characteristics similar to those of parental BICv, mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and a significant decrease in plaque size relative to parental BICv. Immunohistochemical reactivity with WH303 was lost only in T3v, T4v and T5v. Interestingly, progressive mutation of the WH303 epitope had an additive effect on attenuation for the virus in swine, with mutants T1v, T2v or T3v inducing progressively milder but invariably lethal CSF, T4v inducing only mild and transient clinical disease, and T5v inducing no disease. Swine infected with either T4v or T5v showed decreased virus replication in tonsils, draining lymph node, spleen and kidney and a significant reduction in virus shedding. Finally, T5v-infected animals were protected from clinical disease when challenged with virulent Brescia virus at 3 or 21 days post T5v inoculation. These results indicate that amino acid residues 830 to 834 of E2 are critical for virulence of CSFV in swine and that engineering at this locus may provide basis for a rationally designed live attenuated CSF vaccine.

Virulence determinant within the E2 structural glycoprotein of classical swine fever virus

E1, along with Erns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Our previous studies indicated that glycosylation status of either E2 or Erns strongly influence viral virulence in swine. Here, we have investigated the role of E1 glycosylation of highly virulent CSFV strain Brescia during infection in the natural host. The three putative glycosylation sites in E1 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E1 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all three putative glycosylation sites in E1. Single mutations of each of the E1 glycosylation sites showed that CSFV amino acid N594 (E1.N3 virus), as well the combined mutation of N500 and N513 (E1.N1N2 virus) resulted in BICv attenuation. Infection of either E1.N1N2 or E1.N3 viruses were able to efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. These results, along with those demonstrating the role of glycosylation of Eand E2, suggest that manipulation of the pattern of glycosylation could be a useful tool for development of CSF live-attenuated vaccines.

N-linked glycosylation alteration in E1 glycoprotein of classical swine fever virus and novel classical swine fever virus vaccine

Classical swine fever virus is a world-wide distributed highly-contagious disease affecting swine. The two main strategies for diseases control are prophylactic vaccination and non-vaccination stamping out policies. Marker vaccines are a promising strategy. Here we report the rational development of a doubly antigenic marker CSFV experimental live attenuated candidate strain vaccine (Flag/T4 virus). Flag/T virus (Flag/T4v) is based in the combination of two Brescia derived recombinant attenuated viruses: RB-C22 and T4. RB-C22v contains a 19mer insertion in the structural glycoprotein E1, while T4v posses mutated CSFV amino acid residues 830 to 834 in the structural glycoprotein E2, deleting the highly conserved epitope recognized by monoclonal antibody (mAb) WH303. Flag/T4 virus contains a positive foreign antigenic marker, due to the insertion of the highly antigenic epitope Flag in the 19mer insertion of E1, as well as a negative antigenic marker, the lack of reactivity with mAb WH303. Immunized with Flag/T4v induced a complete protection against the challenge with virulent strain Brescia both at 3 and 28 days post infection when nasally administered and since the second day post infection when intramuscularly administered. These results constitute an example of rational design of a CSFV antigenically marked LAV.

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Westbrook, CT, United States of America

Manuel V Borca