scholarly journals Optimization in the expression of ASFV proteins for the development of subunit vaccines using poxviruses as delivery vectors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jaime Lopera-Madrid ◽  
Lex G. Medina-Magües ◽  
Douglas P. Gladue ◽  
Manuel V. Borca ◽  
Jorge E. Osorio
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Expression Vectors ◽  
Complex Nature ◽  
Hemorrhagic Disease ◽  
Mrna Levels ◽  
Subunit Vaccines ◽  
Protective Antigens ◽  
Delivery Vector ◽  
Efficacious Treatment

AbstractAfrican swine fever virus (ASFV) causes a highly contagious hemorrhagic disease that affects domestic pig and Eurasian wild boar populations. To date, no safe and efficacious treatment or vaccine against ASF is available. Nevertheless, there are several reports of protection elicited by experimental vaccines based on live attenuated ASFV and some levels of protection and reduced viremia in other approaches such as DNA, adenovirus, baculovirus, and vaccinia-based vaccines. Current ASF subunit vaccine research focuses mainly on delivering protective antigens and antigen discovery within the ASFV genome. However, due to the complex nature of ASFV, expression vectors need to be optimized to improve their immunogenicity. Therefore, in the present study, we constructed several recombinant MVA vectors to evaluate the efficiency of different promoters and secretory signal sequences in the expression and immunogenicity of the p30 protein from ASFV. Overall, the natural poxvirus PrMVA13.5L promoter induced high levels of both p30 mRNA and specific anti-p30 antibodies in mice. In contrast, the synthetic PrS5E promoter and the S E/L promoter linked to a secretory signal showed lower mRNA levels and antibodies. These findings indicate that promoter selection may be as crucial as the antigen used to develop ASFV subunit vaccines using MVA as the delivery vector.

Optimization in the Expression of ASFV Proteins for the Development of Subunit Vaccines Using Poxviruses As Delivery Vectors.

2021 ◽  
Author(s):  
Jaime Lopera-Madrid ◽  
Lex Medina-Magües ◽  
Douglas P. Gladue ◽  
Manuel V. Borca ◽  
Jorge E. Osorio
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Expression Vectors ◽  
Complex Nature ◽  
Hemorrhagic Disease ◽  
Mrna Levels ◽  
Subunit Vaccines ◽  
Protective Antigens ◽  
Delivery Vector ◽  
Efficacious Treatment

Abstract African swine fever virus (ASFV) causes a highly contagious hemorrhagic disease that affects domestic pig and Eurasian wild boar populations. To date, no safe and efficacious treatment or vaccine against ASF is available. Nevertheless, there are several reports of protection elicited by experimental vaccines based on live attenuated ASFV and some levels of protection and reduced viremia in other approaches such as DNA, adenovirus, baculovirus, and vaccinia-based vaccines. Current ASF subunit vaccine research focuses mainly on delivering protective antigens and antigen discovery within the ASFV genome. However, due to the complex nature of ASFV, expression vectors need to be optimized to improve their immunogenicity. Therefore, in the present study, we constructed several recombinant MVA vectors to evaluate the efficiency of different promoters and secretory signal sequences in the expression and immunogenicity of the p30 protein from ASFV. Overall, the natural poxvirus PrMVA13.5L promoter induced high levels of both p30 mRNA and specific anti-p30 antibodies in mice. In contrast, the synthetic PrS5E promoter and the S E/L promoter linked to a secretory signal showed lower mRNA levels and antibodies. These findings indicate that promoter selection may be as crucial as the antigen used to develop ASFV subunit vaccines using MVA as the delivery vector.

scholarly journals Computational Analysis of African Swine Fever Virus Protein Space for the Design of an Epitope-Based Vaccine Ensemble

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1078 ◽  
Author(s):  
Albert Ros-Lucas ◽  
Florencia Correa-Fiz ◽  
Laia Bosch-Camós ◽  
Fernando Rodriguez ◽  
Julio Alonso-Padilla
Keyword(s):  
T Cell ◽  
Vaccine Development ◽  
De Novo ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Virus Protein ◽  
Hemorrhagic Disease ◽  
Starting Point

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

scholarly journals African Swine Fever Laboratory Diagnosis—Lessons Learned from Recent Animal Trials

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 177
Author(s):  
Jutta Pikalo ◽  
Paul Deutschmann ◽  
Melina Fischer ◽  
Hanna Roszyk ◽  
Martin Beer ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Laboratory Diagnosis ◽  
Lessons Learned ◽  
Hemorrhagic Disease ◽  
Passive Surveillance ◽  
Animal Trials ◽  
Fit For Purpose ◽  
The Impact

African swine fever virus (ASFV) causes a hemorrhagic disease in pigs with high socio-economic consequences. To lower the impact of disease incursions, early detection is crucial. In the context of experimental animal trials, we evaluated diagnostic workflows for a high sample throughput in active surveillance, alternative sample matrices for passive surveillance, and lateral flow devices (LFD) for rapid testing. We could demonstrate that EDTA blood is significantly better suited for early ASFV detection than serum. Tissues recommended by the respective diagnostic manuals were in general comparable in their performance, with spleen samples giving best results. Superficial lymph nodes, ear punches, and different blood swabs were also evaluated as potential alternatives. In summary, all matrices yielded positive results at the peak of clinical signs and could be fit for purpose in passive surveillance. However, weaknesses were discovered for some matrices when it comes to the early phase of infection or recovery. The antigen LFD showed variable results with best performance in the clinical phase. The antibody LFD was quite comparable with ELISA systems. Concluding, alternative approaches are feasible but have to be embedded in control strategies selecting test methods and sample materials following a “fit-for-purpose” approach.

scholarly journals Two African Swine Fever Virus Proteins Derived from a Common Precursor Exhibit Different Nucleocytoplasmic Transport Activities

2004 ◽  
Vol 78 (18) ◽  
pp. 9731-9739 ◽  
Author(s):  
A. Eulálio ◽  
I. Nunes-Correia ◽  
A. L. Carvalho ◽  
C. Faro ◽  
V. Citovsky ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Mammalian Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Nucleocytoplasmic Transport ◽  
Proteolytic Processing ◽  
Fever Virus ◽  
Hemorrhagic Disease ◽  
Common Precursor ◽  
Green Fluorescent

ABSTRACT African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

Bacterial lipoprotein based expression vectors as tools for the characterisation of African swine fever virus (ASFV) antigens

2000 ◽  
Vol 145 (8) ◽  
pp. 1639-1657 ◽  
Author(s):  
A. Leitão ◽  
A. Malur ◽  
C. Cartaxeiro ◽  
G. Vasco ◽  
B. Cruz ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Expression Vectors ◽  
Bacterial Lipoprotein

scholarly journals Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

2022 ◽  
Author(s):  
Pengfei Wang ◽  
Chunguo Liu ◽  
Shida Wang ◽  
Lili Wen ◽  
Zhibin Shi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immunohistochemical Staining ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Diagnostic Tools ◽  
Hemorrhagic Disease ◽  
Structural Protein ◽  
Linear Epitopes ◽  
Group 2 ◽  
Basic And Applied Research

AbstractAfrican swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

scholarly journals African Swine Fever Virus Multigene Family 360 and 530 Genes Affect Host Interferon Response

2004 ◽  
Vol 78 (4) ◽  
pp. 1858-1864 ◽  
Author(s):  
C. L. Afonso ◽  
M. E. Piccone ◽  
K. M. Zaffuto ◽  
J. Neilan ◽  
G. F. Kutish ◽  
...  
Keyword(s):  
Viral Infection ◽  
Multigene Family ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Cdna Clones ◽  
Type I ◽  
Mrna Levels ◽  
Type I Ifn ◽  
Transcriptional Responses

ABSTRACT African swine fever virus (ASFV) multigene family 360 and 530 (MGF360/530) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol. 75:3066-3076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF360/530 deletion mutant (Pr4Δ35). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4Δ35 at 3 and 6 h postinfection (hpi). While at 3 hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, 38 genes had significantly increased (>2.0-fold, P < 0.05) mRNA levels in Pr4Δ35-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr4Δ35 up-regulated genes were part of a type I interferon (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein 3, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG43, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4Δ35 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-α mRNA and secreted IFN-α levels at 3, 8, and 24 hpi revealed undetectable IFN-α in mock- and Pr4-infected macrophages but significant IFN-α levels at 24 hpi in Pr4Δ35-infected macrophages. The absence of IFN-α in Pr4-infected macrophages suggests that MGF360/530 genes either directly or indirectly suppress a type I IFN response. An inability to suppress host type I IFN responses may account for the growth defect of Pr4Δ35 in macrophages and its attenuation in swine.

Pathogenesis of African swine fever virus inOrnithodorosticks

2001 ◽  
Vol 2 (2) ◽  
pp. 121-128 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Glen A. Scoles
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Sub Saharan Africa ◽  
Hemorrhagic Disease ◽  
Sylvatic Cycle ◽  
Infectious Dose ◽  
Domestic Pigs ◽  
Ornithodoros Porcinus Porcinus ◽  
Sub Saharan

AbstractAfrican swine fever virus (ASFV) is the only known DNA arbovirus and the sole member of the family Asfarviridae. It causes a lethal, hemorrhagic disease in domestic pigs. ASFV is enzootic in sub-Saharan Africa and is maintained in a sylvatic cycle by infecting both wild members of the Suidae (e.g. warthogs) and the argasid tickOrnithodoros porcinus porcinus. The pathogenesis of ASFV inO. porcinus porcinusticks is characterized by a low infectious dose, lifelong infection, efficient transmission to both pigs and ticks, and low mortality until after the first oviposition. ASFV pathogenesis in warthogs is characterized by an inapparent infection with transient, low viremic titers. ThusO. porcinus porcinusticks probably constitute the most important natural vector of ASFV, although both the mammalian and tick hosts are probably required for the maintenance of ASFV in the sylvatic cycle. The mechanism of ASFV transmission from the sylvatic cycle to domestic pigs is probably through infected ticks feeding on pigs. In addition toO. porcinus porcinus, a number of North American, Central American and Caribbean species ofOrnithodoroshave been shown to be potential vectors of ASFV.

scholarly journals Persistent domestic circulation of African swine fever virus in Tanzania, 2015–2017

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Clara M. Yona ◽  
Merijn Vanhee ◽  
Edgar Simulundu ◽  
Mariam Makange ◽  
Hans J. Nauwynck ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Composite Likelihood ◽  
Control Measures ◽  
Disease Spread ◽  
Hemorrhagic Disease ◽  
Sylvatic Cycle ◽  
East African Community ◽  
Domestic Pigs

Abstract Background African swine fever (ASF) is a highly fatal viral hemorrhagic disease of domestic pigs that threatens livelihoods and food security. In Africa, ASF virus (ASFV) circulates in sylvatic (transmission between warthogs and soft argasid ticks) and domestic (transmission between domestic pigs) cycles, with outbreaks resulting from ASFV spill-over from sylvatic cycle. A number of outbreaks were reported in different parts of Tanzania between 2015 and 2017. The present study investigated ASFV transmission patterns through viral DNA sequencing and phylogenetic analysis. A total of 3120 tissue samples were collected from 2396 domestic pigs during outbreaks at different locations in Tanzania between 2015 and 2017. Partial sequencing of the B646L (p72) gene was conducted for diagnostic confirmation and molecular characterization of ASFV. Phylogenetic analysis to study the relatedness of current ASFV with those that caused previous outbreaks in Tanzania and representatives of all known 24 ASFV was performed using the Maximum Composite Likelihood model with 1000 bootstrap replications in MEGA 6.0. Results ASFV was confirmed to cause disease in sampled domestic pigs. ASFV genotypes II, IX, and X were detected from reported outbreaks in 2015–2017. The current ASFV isolates were similar to those recently documented in the previous studies in Tanzania. The similarities of these isolates suggests for continuous circulation of ASFV with virus maintenance within the domestic pigs. Conclusions Genetic analysis confirmed the circulation of ASFV genotypes II, IX, and X by partial B646L (p72) gene sequencing. The similarities of current isolates to previously isolated Tanzanian isolates and pattern of disease spread suggest for continuous circulation of ASF with virus’ maintenance in the domestic pigs. Although certain viral genotypes seem to be geographically restricted into certain zones within Tanzania, genotype II seems to expand its geographical range northwards with the likelihood of spreading to other states of the East African Community. The spread of ASFV is due to breach of quarantine and transportation of infected pigs via major highways. Appropriate control measures including zoosanitary measures and quarantine enforcement are recommended to prevent ASF domestic circulation in Tanzania.

scholarly journals Fecal microbiota transplantation from warthog to pig confirms the influence of the gut microbiota on African swine fever susceptibility

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jinya Zhang ◽  
Fernando Rodríguez ◽  
Maria Jesus Navas ◽  
Mar Costa-Hurtado ◽  
Vanessa Almagro ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Fecal Microbiota Transplantation ◽  
African Swine Fever Virus ◽  
Differential Susceptibility ◽  
African Swine Fever ◽  
Preliminary Evidence ◽  
Fecal Microbiota ◽  
Host Susceptibility ◽  
Hemorrhagic Disease ◽  
Domestic Pigs

Abstract African swine fever virus (ASFV) is the causative agent of a devastating hemorrhagic disease (ASF) that affects both domestic pigs and wild boars. Conversely, ASFV circulates in a subclinical manner in African wild pigs, including warthogs, the natural reservoir for ASFV. Together with genetic differences, other factors might be involved in the differential susceptibility to ASF observed among Eurasian suids (Sus scrofa) and African warthogs (Phacochoerus africanus). Preliminary evidence obtained in our laboratory and others, seems to confirm the effect that environmental factors might have on ASF infection. Thus, domestic pigs raised in specific pathogen-free (SPF) facilities were extremely susceptible to highly attenuated ASFV strains that were innocuous to genetically identical domestic pigs grown on conventional farms. Since gut microbiota plays important roles in maintaining intestinal homeostasis, regulating immune system maturation and the functionality of the innate/adaptive immune responses, we decided to examine whether warthog fecal microbiota transplantation (FMT) to domestic pigs affects host susceptibility to ASFV. The present work demonstrates that warthog FMT is not harmful for domestic weaned piglets, while it modifies their gut microbiota; and that FMT from warthogs to pigs confers partial protection against attenuated ASFV strains. Future work is needed to elucidate the protective mechanisms exerted by warthog FMT.

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