Low molecular weight sulfated chitosan efficiently reduces infection capacity of porcine circovirus type 2 (PCV2) in PK15 cells.

BACKGROUND: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to vaccines, the availability of antiviral polymers provides an efficient and safe option for reducing the impact of these diseases. By virtue of their molecular weight and repetitious structure, polymers possess properties not found in small-molecule drugs. In this perspective, we focus on chitosan, a ubiquitous biopolymer, that adjusts the molecular weight and sulfated-mediated functionality can act as an efficient antiviral polymer by mimicking PCV2-cell receptor interactions. METHODS: Sulfated chitosan (Chi-S) polymers of two molecular weights were synthesized and characterized by FTIR, SEM-EDS and elemental analysis. The Chi-S solutions were tested against PCV2 infection in PK15 cells in vitro and antiviral activity was evaluated by measuring the PCV2 DNA copy number, TCID50 and capsid protein expression, upon application of different molecular weights, sulfate functionalization, and concentrations of polymer. In addition, to explore the mode of action of the Chi-S against PCV2 infection, experiments were designed to elucidate whether the antiviral activity of the Chi-S would be influenced by when it was added to the cells, relative to the time and stage of viral infection. RESULTS: Chi-S significantly reduced genomic copies, TCID50 titers and capsid protein of PCV2, showing specific antiviral effects depending on its molecular weight, concentration, and chemical functionalization. Assays designed to explore the mode of action of the low molecular weight Chi-S revealed that it exerted antiviral activity through impeding viral attachment and penetration into cells. CONCLUSIONS: These findings help better understanding the interactions of PCV2 and porcine cells and reinforce the idea that sulfated polymers, such as Chi-S, represent a promising candidates for use in antiviral therapies against PCV2-associated diseases. Further studies in swine are warranted.

Rate of Transfer of Infectious Anaemia Maternal Antibodies from Broiler Breeders To the Progeny: a Field Evaluation

Clinical manifestation of the disease caused by the chicken anaemia virus (CAV) occurs when chicken chicks are vertically contaminated or before the second week of life. CAV control is based on the vaccination of broiler breeders in order to promote progeny protection through maternal antibodies. This work aims to evaluate, under field conditions, the antibody title in commercial broiler breeders at 28, 48, and 68 weeks of age, the rate of transference to the progeny, as well as the duration of antibodies in the progeny up to 21 days of age. Thus, a total of 92 sera samples from 93,000 broiler breeders vaccinated with a live vaccine for CAV at 14 weeks of age and 366 sera samples from their respective progeny were analyzed using ELISA. Breeders' antibody title for CAV ranged between 5051 and 8660, and these titles may provide sufficient protection for their progeny. On average, 63% of the maternal antibodies were transferred to the progeny and lasted up to the second week of chick's life. It is possible to conclude that the vaccine and the vaccination procedure used by this company for breeders against CAV seems to be effective in inducing high antibody levels in the breeders and transfering protective maternal antibodies to the progeny.(AU)

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Short communication: a modified Vaccinia virus Ankara-based Porcine circovirus 2 vaccine elicits strong antibody response upon prime-boost homologous immunization in a preclinical model.

Porcine circovirus 2 (PCV2) infections are related to a number of syndromes and clinical manifestations, generally known as Porcine circovirus-associated diseases, which are related to losses in the swine industry. There are commercially available vaccines and new vaccines being tested, however, persistency of the PCV2 as an important pig pathogen, and the growing number of affected farms in different countries have suggested that there is room for vaccine improvement. In this study, we describe the construction and testing of a recombinant live vaccine based on a modified Vaccinia virus Ankara (MVA) vector expressing the PCV2b capsid protein (CAP). Using a two-dose homologous vaccination regimen, in mice, we demonstrated that the vaccine induced high titers of anti-PCV2 antibodies. The vaccine is stable upon lyophilization, and, together with the good immunogenicity potential observed, the results support further evaluation of the MVA-CAP vaccine in the target species.

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs.

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51-CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.

Chitosan microparticles loaded with yeast-derived PCV2 virus-like particles elicit antigen-specific cellular immune response in mice after oral administration.

BACKGROUND: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to improved management and husbandry practices, the availability of several anti-PCV2 vaccines provides an efficient immunological option for reducing the impact of these diseases. Most anti-PCV2 vaccines are marketed as injectable formulations. Although these are effective, there are problems associated with the use of injectable products, including laborious and time-consuming procedures, the induction of inflammatory responses at the injection site, and treatment-associated stress to the animals. Oral vaccines represent an improvement in antigen delivery technology; they overcome the problems associated with injection management and facilitate antigen boosting when an animals' immunity falls outside the protective window. METHODS: Chitosan microparticles were used as both a vehicle and mucosal adjuvant to deliver yeast-derived PCV2 virus-like particles (VLPs) in an attempt to develop an oral vaccine. The physical characteristics of the microparticles, including size, Zeta potential, and polydispersity, were examined along with the potential to induce PCV2-specific cellular immune responses in mice after oral delivery. RESULTS: Feeding mice with PCV2 VLP-loaded, positively-charged chitosan microparticles with an average size of 2.5 µm induced the proliferation of PCV2-specific splenic CD4+/CD8+ lymphocytes and the subsequent production of IFN-γ to levels comparable with those induced by an injectable commercial formulation. CONCLUSION: Chitosan microparticles appear to be a safe, simple system on which to base PCV2 oral vaccines. Oral chitosan-mediated antigen delivery is a novel strategy that efficiently induces anti-PCV2 cellular responses in a mouse model. Further studies in swine are warranted.

Estratégias de controle da circovirose suína / Strategies to control swine circovirosis

A circovirose suína é uma virose causada pelo Circovirus que acomete suínos mundialmente, levando a manifestação da Sindrome Multisistemica definhante dos suíno, mioclonia congênita, síndrome dermatite nefropatia além de estar relacionada com Alteraçoes reprodutivas e respiratórias. O vírus é resistente a diferentes desinfetantes e ao calor o que dificulta a sua eliminação do ambiente, aliado a isto, ele esta presente em altas prevalências nos rebanhos mundiais, causando prejuízos por perdas de animais, condenações de carcaças bem como gastos com tratamentos principalmente de infecções secundarias, tendo em vista sua ação imunosupressora. As medidas de controle são a implantação dos 20 pontos de Madec nas granjas, uso de desinfetantes, ingestão de colostro, ingestão de plasma suíno e recentemente adoção de vacinas, as quais tem se mostrado eficazes na redução da viremia, eliminação do vírus e melhora dos índices produtivos. Portanto o controle desta enfermidade deve ser focado nos esquemas de vacinação e na biosegurança.

The circovirosis swine is caused by a virus that affects pigs worldwide Circovirus, leading to multisystem syndrome manifestation of the withering of swine, congenital myoclonus, syndrome dermatitis nephropathy besides being related changes reproductive and respiratory. The virus is resistant to various disinfectants and heat which complicates their disposal environment, allied to this, it is present in high prevalence herds in the world, causing damages for loss of animals, carcass condemnations and expenses treatments mainly secondary infections, given its immunosuppressive action. Control measures are the implementation of the 20 points of Madec on farms, use of disinfectants, colostrum intake, intake of porcine plasma and recently adopting vaccines, which have proven effective in reducing viremia, virus elimination and improvement of production indices. Therefore the control of this disease should be focused on vaccination schedules and biosafety.

Estratégias de controle da circovirose suína / Strategies to control swine circovirosis

A circovirose suína é uma virose causada pelo Circovirus que acomete suínos mundialmente, levando a manifestação da Sindrome Multisistemica definhante dos suíno, mioclonia congênita, síndrome dermatite nefropatia além de estar relacionada com Alteraçoes reprodutivas e respiratórias. O vírus é resistente a diferentes desinfetantes e ao calor o que dificulta a sua eliminação do ambiente, aliado a isto, ele esta presente em altas prevalências nos rebanhos mundiais, causando prejuízos por perdas de animais, condenações de carcaças bem como gastos com tratamentos principalmente de infecções secundarias, tendo em vista sua ação imunosupressora. As medidas de controle são a implantação dos 20 pontos de Madec nas granjas, uso de desinfetantes, ingestão de colostro, ingestão de plasma suíno e recentemente adoção de vacinas, as quais tem se mostrado eficazes na redução da viremia, eliminação do vírus e melhora dos índices produtivos. Portanto o controle desta enfermidade deve ser focado nos esquemas de vacinação e na biosegurança. (AU)

The circovirosis swine is caused by a virus that affects pigs worldwide Circovirus, leading to multisystem syndrome manifestation of the withering of swine, congenital myoclonus, syndrome dermatitis nephropathy besides being related changes reproductive and respiratory. The virus is resistant to various disinfectants and heat which complicates their disposal environment, allied to this, it is present in high prevalence herds in the world, causing damages for loss of animals, carcass condemnations and expenses treatments mainly secondary infections, given its immunosuppressive action. Control measures are the implementation of the 20 points of Madec on farms, use of disinfectants, colostrum intake, intake of porcine plasma and recently adopting vaccines, which have proven effective in reducing viremia, virus elimination and improvement of production indices. Therefore the control of this disease should be focused on vaccination schedules and biosafety. (AU)

The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts.

Porcine circovirus type 2 (PCV2)-associated diseases are considered to be the biggest problem for the worldwide swine industry. The PCV2 capsid protein (Cap) is an important antigen for development of vaccines. At present, most anti-PCV2 vaccines are produced as injectable formulations. Although effective, these vaccines have certain drawbacks, including stress with concomitant immunosuppresion, and involve laborious and time-consuming procedures. In this study, Saccharomyces cerevisiae was used as a vehicle to deliver PCV2 antigen in a preliminary attempt to develop an oral vaccine, and its immunogenic potential in mice was tested after oral gavage-mediated delivery. The cap gene with a yeast-optimized codon usage sequence (opt-cap) was chemically synthesized and cloned into Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2, under the control of the Gal1 promoter. Intracellular expression of the Cap protein was confirmed by Western blot analysis and its antigenic properties were compared with those of baculovirus/insect cell-produced Cap protein derived from the native PCV2 cap gene. It was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to insect cell-derived VLPs. Feeding raw yeast extract containing Cap protein to mice elicited both serum- and fecal-specific antibodies against the antigen. These results show that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles, and that oral yeast-mediated antigen delivery is an alternative strategy to efficiently induce anti-PCV2 antibodies in a mouse model, which is worthy of further investigation in swine.