Engineering topics

Spinard, Edward

Publications and source records attributed to Spinard, Edward.

5 records

DOE OSTI2024

Lyophilization of ASFV vaccine candidate ASFV-G-ΔI177L offers long term stability

Abstract For over a century African swine fever (ASF) has been causing outbreaks leading to devastating losses for the swine industry. The current pandemic of ASF has shown no signs of stopping and continues to spread causing outbreaks in additional countries. Currently control relies mostly on culling infected farms, and strict biosecurity procedures. Recently a vaccine, ASFV-G-ΔI177L was approved for use in Vietnam. In this study we evaluate the long-term stability of lyophilized ASFV-G-ΔI177L. Understanding the stability of different formulations of vaccines is information necessary for deployment of vaccines to ASF outbreak areas, particularly those that do not have a reliable well established cold chain to ensure conservation of vaccine quality. In this report, we determined that ASFV-G-ΔI177L, when lyophilized under specific conditions, is stable for up to one year at 4 °C, with similar vaccine titers after storage. Next-generation sequencing analysis also determined that lyophilization and long-term storage under these conditions had no effect on the genome of ASFV as the genome remained genetically identical to the original non-lyophilized form.

Science & Technology - Other Topics↗

DOE OSTI2024

African Swine Fever Virus Protein–Protein Interaction Prediction

The African swine fever virus (ASFV) is an often deadly disease in swine and poses a threat to swine livestock and swine producers. With its complex genome containing more than 150 coding regions, developing effective vaccines for this virus remains a challenge due to a lack of basic knowledge about viral protein function and protein–protein interactions between viral proteins and between viral and host proteins. In this work, we identified ASFV-ASFV protein–protein interactions (PPIs) using artificial intelligence-powered protein structure prediction tools. We benchmarked our PPI identification workflow on the Vaccinia virus, a widely studied nucleocytoplasmic large DNA virus, and found that it could identify gold-standard PPIs that have been validated in vitro in a genome-wide computational screening. We applied this workflow to more than 18,000 pairwise combinations of ASFV proteins and were able to identify seventeen novel PPIs, many of which have corroborating experimental or bioinformatic evidence for their protein–protein interactions, further validating their relevance. Two protein–protein interactions, I267L and I8L, I267L__I8L, and B175L and DP79L, B175L__DP79L, are novel PPIs involving viral proteins known to modulate host immune response.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 10 4 HAD 50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 10 4 or 10 6 HAD 50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 10 4 HAD 50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 10 6 HAD 50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-ΔI177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals

The African swine fever virus (ASFV) mutant ASFV-G-ΔI177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-ΔI177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-ΔI177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-ΔI177L/ΔEP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-ΔI177L. Domestic pigs intramuscularly (IM) inoculated with either 10 2 or 10 6 HAD 50 of ASFV-G-ΔI177L/ΔEP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 10 2 HAD 50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-ΔI177L/ΔEP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-ΔI177L/ΔEP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-ΔI177L, ASFV-G-ΔMGF, or ASFV-G-Δ9GL/ΔUK. Therefore, ASFV-G-ΔI177L/ΔEP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Reclassification of ASFV into 7 Biotypes Using Unsupervised Machine Learning

In 2007, an outbreak of African swine fever (ASF), a deadly disease of domestic swine and wild boar caused by the African swine fever virus (ASFV), occurred in Georgia and has since spread globally. Historically, ASFV was classified into 25 different genotypes. However, a newly proposed system recategorized all ASFV isolates into 6 genotypes exclusively using the predicted protein sequences of p72. However, ASFV has a large genome that encodes between 150–200 genes, and classifications using a single gene are insufficient and misleading, as strains encoding an identical p72 often have significant mutations in other areas of the genome. We present here a new classification of ASFV based on comparisons performed considering the entire encoded proteome. A curated database consisting of the protein sequences predicted to be encoded by 220 reannotated ASFV genomes was analyzed for similarity between homologous protein sequences. Weights were applied to the protein identity matrices and averaged to generate a genome-genome identity matrix that was then analyzed by an unsupervised machine learning algorithm, DBSCAN, to separate the genomes into distinct clusters. We conclude that all available ASFV genomes can be classified into 7 distinct biotypes.

59 BASIC BIOLOGICAL SCIENCES↗