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Particle tracking analysis for the intracellular trafficking of nanoparticles modified with African swine fever virus protein p54-derived peptide.

ABSTRACT: Previous studies showed that the cytoplasmic transport of nanoparticles to the nucleus is driven by a vesicular sorting system. Artificial approaches for targeting a microtubule-associating motor complex is also a challenge. We describe herein the development of a liposomal nanoparticle, the surface of which is modified with stearylated octa-arginine (STR-R8), and a dynein light chain (LC8)-associated peptide derived from an African swine fever virus protein p54 (p54(149-161)) with polyethyleneglycol (PEG) as a spacer (p54(149-161)-PEG/R8-liposomal nanoparticles (LNPs)). The p54(149-161)-PEG/R8-LNPs preferentially gain access to the nucleus, resulting in a one- to two-order of magnitude higher transfection activity in comparison with p54(149-161)-free nanoparticles (PEG/R8-LNPs). Further studies of particle tracking in HeLa cells stably expressing green fluorescent protein (GFP)-tagged tubulin (GFP/Tub-HeLa) indicate that p54(149-161) stimulated the transport of nanoparticles along fibrous tubulin structures. Moreover, a part of the p54(149-161)-PEG/R8-LNPs appeared to undergo quasi-straight transport without sharing the tracks corresponding to PKH67, the plasma membrane of which had been prestained with a marker just before transfection, while corresponding movement was never observed in the case of PEG/R8-LNPs. These findings suggest that a portion of the p54(149-161)-modified nanoparticles can use microtubule-dependent transport without the need for an assist by a vesicular sorting system.

SUBMITTER: Akita H

PROVIDER: S-EPMC3594009 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:African swine fever (ASF) has received great attention from the swine industry due to the pandemic and the lack of vaccines or effective treatments. In the present study, 13 African swine fever virus (ASFV) p54-specific nanobodies (Nbs) were successfully screened based on Bactrian camel immunization of p54 protein and phage display technology, and their reactivity with the p54 C-terminal domain (p54-CTD) was determined; however, only Nb8-horseradish peroxidase (Nb8-HRP) exhibited the best reactivity. Immunoperoxidase monolayer assay (IPMA) and immunofluorescence assay (IFA) results indicated that Nb8-HRP specifically reacted with ASFV-infected cells. Then, the possible epitopes of p54 were identified using Nb8-HRP. The results showed that Nb8-HRP could recognize p54-CTD truncated mutant p54-T1. Then, 6 overlapping peptides covering p54-T1 were synthesized to determine the possible epitopes. Dot blot and peptide-based enzyme-linked immunosorbent assay (ELISA) results suggested that one novel minimal linear B cell epitope, 76QQWVEV81, which had never been reported before, was identified. Alanine-scanning mutagenesis revealed that 76QQWV79 was the core binding site for Nb8. Epitope 76QQWVEV81 was highly conserved among genotype II ASFV strains and could react with inactivated ASFV antibody-positive serum from naturally infected pigs, indicating that it was a natural linear B cell epitope. These findings provide valuable insights for vaccine design and p54 as an effective diagnostic tool. IMPORTANCE The ASFV p54 protein plays an important role in inducing neutralization antibodies in vivo after viral infection and is often used as a candidate protein for subunit vaccine development. The full understanding of the p54 protein epitope provides a sufficient theoretical basis for p54 as a vaccine candidate protein. The present study uses a p54-specific nanobody as a probe to identify a highly conserved antigenic epitope, 76QQWVEV81, among different ASFV strains, and it can induce humoral immune responses in pigs. This is the first report using virus-specific nanobodies as a tool to identify some special epitopes that cannot be recognized by conventional monoclonal antibodies. This study opens up nanobodies as a new tool for identifying epitopes and also provides a theoretical basis for understanding p54-induced neutralizing antibodies.

Project description:Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

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Particle tracking analysis for the intracellular trafficking of nanoparticles modified with African swine fever virus protein p54-derived peptide.

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