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[This corrects the article DOI: 10.1371/journal.ppat.1009330.].
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Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.
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Anticuerpos Antivirales/farmacología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Gripe Humana/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Hemaglutininas/inmunología , Hemaglutininas/farmacología , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/virología , PorcinosRESUMEN
Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.
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Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , COVID-19/diagnóstico , Infecciones por Henipavirus/diagnóstico , Ensayos Analíticos de Alto Rendimiento , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , COVID-19/inmunología , COVID-19/virología , Fusión Celular , Convalecencia , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/virología , Humanos , Sueros Inmunes/química , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Virus Nipah/inmunología , Virus Nipah/patogenicidad , Conformación Proteica , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/inmunología , Virus Sincitial Respiratorio Humano/patogenicidad , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Porcinos , Inhibidores de Proteínas Virales de Fusión/química , Inhibidores de Proteínas Virales de Fusión/metabolismo , Inhibidores de Proteínas Virales de Fusión/farmacología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.
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Nipah virus (NiV) poses a significant threat to human and livestock populations across South and Southeast Asia. Vaccines are required to reduce the risk and impact of spillover infection events. Pigs can act as an intermediate amplifying host for NiV and, separately, provide a preclinical model for evaluating human vaccine candidate immunogenicity. The aim of this study was therefore to evaluate the immunogenicity of an mRNA vectored NiV vaccine candidate in pigs. Pigs were immunized twice with 100 µg nucleoside-modified mRNA vaccine encoding soluble G glycoprotein from the Malaysia strain of NiV, formulated in lipid nanoparticles. Potent antigen-binding and virus neutralizing antibodies were detected in serum following the booster immunization. Antibody responses effectively neutralized both the Malaysia and Bangladesh strains of NiV but showed limited neutralization of the related (about 80% amino acid sequence identity for G) Hendra virus. Antibodies were also capable of neutralizing NiV glycoprotein mediated cell-cell fusion. NiV G-specific T cell cytokine responses were also measurable following the booster immunization with evidence for induction of both CD4 and CD8 T cell responses. These data support the further evaluation of mRNA vectored NiV G as a vaccine for both pigs and humans.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Henipavirus , Virus Nipah , Vacunas Virales , Animales , Virus Nipah/inmunología , Virus Nipah/genética , Porcinos , Infecciones por Henipavirus/prevención & control , Infecciones por Henipavirus/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , ARN Mensajero/genética , ARN Mensajero/inmunología , Inmunogenicidad Vacunal , Inmunización Secundaria , Citocinas/inmunología , Vacunas Sintéticas/inmunología , Liposomas , NanopartículasRESUMEN
Resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (MΦs) or the induction of inflammatory responses that cause lung injury and sepsis. The aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (BVDV) and challenged later with bovine herpesvirus type 1 (BHV-1), evaluating the role of MΦs in the development of pathological lesions in this organ. For this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with BHV-1 through immunohistochemical (MAC387, TNFα, IL-1α, iNOS, COX-2 and Factor-VIII) and ultrastructural studies. Both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. These findings were earlier and more severe in the co-infected group, indicating that the concomitance of BVDV and BHV-1 in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary MΦs. In this regard, the co-infected calves, in spite of presenting a greater number of IMΦs than single-infected group, show a significant decrease in iNOS expression coinciding with the presence of more coagulation lesions. Moreover, animals pre-inoculated with BVDV displayed an alteration in the response of pro-inflammatory cytokines (TNFα and IL-1), which play a key role in activating the immune response, as well as in the local cell-mediated response.
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Diarrea Mucosa Bovina Viral/patología , Coinfección/veterinaria , Virus de la Diarrea Viral Bovina/inmunología , Coagulación Intravascular Diseminada/veterinaria , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/inmunología , Pulmón/patología , Macrófagos Alveolares/metabolismo , Animales , Diarrea Mucosa Bovina Viral/virología , Bovinos , Coinfección/patología , Coinfección/virología , Coagulación Intravascular Diseminada/patología , Coagulación Intravascular Diseminada/virología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Pulmón/virología , Macrófagos Alveolares/citología , Microscopía Electrónica de Transmisión/veterinaria , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
African swine fever (ASF) is a hemorrhagic viral disease of domestic pigs and wild suids (all Sus scrofa) caused by the ASF virus (ASFV). The disease is spreading worldwide without control, threatening pig production due to the absence of licensed vaccine or commercially available treatments. A thorough understanding of the immunopathogenic mechanisms behind ASFV infection is required to better fight the disease. Cytokines are small, non-structural proteins, which play a crucial role in many aspects of the immune responses to viruses, including ASFV. Infection with virulent ASFV isolates often results in exacerbated immune responses, with increased levels of serum pro-inflammatory interleukins (IL-1α, IL-1ß, IL-6), TNF and chemokines (CCL2, CCL5, CXCL10). Increased levels of IL-1, IL-6 and TNF are often detected in several tissues during acute ASFV infections and associated with lymphoid depletion, hemorrhages and oedemas. IL-1Ra is frequently released during ASFV infection to block further IL-1 activity, with its implication in ASFV immunopathology having been suggested. Increased levels of IFN-α and of the anti-inflammatory IL-10 seem to be negatively correlated with animal survival, whereas some correlation between virus-specific IFN-γ-producing cells and protection has been suggested in different studies where different vaccine candidates were tested, although future works should elucidate whether IFN-γ release by specific cell types is related to protection or disease development.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Citocinas/metabolismo , Interleucina-6/metabolismo , Interleucina-1/metabolismo , Sus scrofaRESUMEN
Solid dose vaccine formulation and delivery systems offer potential advantages over traditional liquid vaccine formulations. In addition to enhanced thermostability, needle-free delivery of unit solid dose injectable (USDI) vaccines offers safe, rapid, and error-free administration, with applicability to both human and animal health. Solid dose formulation technologies can be adapted for delivery of different vaccine formats including live attenuated vaccines, which remain the 'gold standard' for many disease targets. Porcine reproductive and respiratory syndrome viruses (PRRSV) cause one of the most economically important diseases affecting the global pig industry. Despite several shortcomings, live attenuated vaccines are widely used to control PRRSV. We optimised a freeze-dried USDI formulation of live attenuated PRRSV-1, which fully retained infectious titre, and evaluated its immunogenicity in comparison to virus delivered in liquid suspension via intramuscular and subcutaneous needle inoculation. Pigs vaccinated with the USDI formulation displayed vaccine viraemia, and PRRSV-specific antibody and T cell responses comparable to animals immunised with the liquid vaccine. The USDI vaccine formulation was stable for at least 6 months when stored refrigerated. These data demonstrate the potential for a solid dose vaccine delivery system as an alternative to conventional needle-syringe delivery of live attenuated PRRSV vaccines.
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There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.
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Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Péptidos/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , Línea Celular , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , PorcinosRESUMEN
Nipah virus (NiV) is an emergent pathogen capable of causing acute respiratory illness and fatal encephalitis in pigs and humans. A high fatality rate and broad host tropism makes NiV a serious public and animal health concern. There is therefore an urgent need for a NiV vaccines to protect animals and humans. In this study we investigated the immunogenicity of bovine herpesvirus (BoHV-4) vectors expressing either NiV attachment (G) or fusion (F) glycoproteins, BoHV-4-A-CMV-NiV-GΔTK or BoHV-4-A-CMV-NiV-FΔTK, respectively in pigs. The vaccines were benchmarked against a canarypox (ALVAC) vector expressing NiV G, previously demonstrated to induce protective immunity in pigs. Both BoHV-4 vectors induced robust antigen-specific antibody responses. BoHV-4-A-CMV-NiV-GΔTK stimulated NiV-neutralizing antibody titers comparable to ALVAC NiV G and greater than those induced by BoHV-4-A-CMV-NiV-FΔTK. In contrast, only BoHV-4-A-CMV-NiV-FΔTK immunized pigs had antibodies capable of significantly neutralizing NiV G and F-mediated cell fusion. All three vectored vaccines evoked antigen-specific CD4 and CD8 T cell responses, which were particularly strong in BoHV-4-A-CMV-NiV-GΔTK immunized pigs and to a lesser extent BoHV-4-A-CMV-NiV-FΔTK. These findings emphasize the potential of BoHV-4 vectors for inducing antibody and cell-mediated immunity in pigs and provide a solid basis for the further evaluation of these vectored NiV vaccine candidates.
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The mechanisms responsible for lymphocyte apoptosis in bovine viral diarrhoea have not yet been clarified. Previous work suggests that bovine viral diarrhea virus (BVDV) is only directly responsible for the destruction of a small number of lymphocytes. The aim of this study was to clarify, in vivo, the role of macrophages in lymphocyte destruction through indirect mechanisms linked to the biosynthetic activation of these immunocompetent cells on ileal Peyer's patches, as well as the distribution and quantification of apoptosis. Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of BVDV genotype 1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (p.i.). The progressive depletion of Peyer's patches was found to be due to massive lymphocyte apoptosis, with an increase in cleaved caspase-3 and TUNEL-positive cells. Lymphoid depletion was accompanied, from 3 days p.i., by a significant rise in macrophage numbers both in lymphoid follicles and in interfollicular areas. Some macrophages showed signs of viral infection, together with subcellular changes indicative of phagocyte activation and, in some cases, of secretory activity. However, the number of macrophages that showed positive immunostaining for tumour necrosis factor-alpha and interleukin-1alpha, cytokines with a proven ability to induce apoptosis, remained low throughout the experiment in lymphoid follicles, where most apoptotic cells were found. These results thus appear to rule out a major involvement of macrophages and macrophage-secreted chemical mediators in the apoptosis of follicular B lymphocytes during BVDV infection.
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Apoptosis , Caspasa 3/inmunología , Virus de la Diarrea Viral Bovina Tipo 1/patogenicidad , Macrófagos/inmunología , Ganglios Linfáticos Agregados/patología , Animales , Bovinos , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Etiquetado Corte-Fin in Situ , Masculino , Factores de TiempoRESUMEN
Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.
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Aves/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Vectores Genéticos/genética , Herpesvirus Meleágrido 1/genética , AnimalesRESUMEN
Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transforming primary chicken spleen and bone marrow cells. Here, by engineering the target sequence of endogenous microRNA miR-142 downstream of the v-rel gene in a Replication-Competent ALV (avian leukosis virus) long terminal repeat (LTR) with a splice acceptor (RCAS) vector and using a v-rel-induced transformation model of chicken embryonic splenocyte cultures, we show that hematopoietic-specific miR-142 can inhibit the v-rel-induced transformation, and that this inhibition effect is due to the silencing of v-rel expression. The data supports the idea that microRNA-targeting can be used to inhibit viral oncogene-induced oncogenesis.
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Carcinogénesis/genética , Transformación Celular Viral , MicroARNs/genética , Proteínas Oncogénicas v-rel/genética , Animales , Virus de la Leucosis Aviar/genética , Células de la Médula Ósea/virología , Células Cultivadas , Pollos , Silenciador del Gen , FN-kappa B/metabolismo , Proteínas Oncogénicas v-rel/antagonistas & inhibidores , Virus de la Reticuloendoteliosis/genética , Bazo/citología , Bazo/virología , Factores de Transcripción/genéticaRESUMEN
Live herpesvirus-vectored vaccines are widely used in veterinary medicine to protect against many infectious diseases. In poultry, three strains of herpesvirus vaccines are used against Marek's disease (MD). However, of these, only the herpesvirus of turkeys (HVT) has been successfully developed and used as a recombinant vaccine vector to induce protection against other avian viral diseases such as infectious bursal disease (IBD), Newcastle disease (ND) or avian influenza (AI). Although effective when administered individually, recombinant HVT vectors have limitations when combined in multivalent vaccines. Thus there is a need for developing additional viral vectors that could be combined with HVT in inducing protection against multiple avian diseases in multivalent vaccines. Gallid herpesvirus 3 (GaHV3) strain SB-1 is widely used by the poultry industry as bivalent vaccine in combination with HVT to exploit synergistic effects against MD. Here, we report the development and application of SB-1 as a vaccine vector to express the VP2 capsid antigen of IBD virus. A VP2 expression cassette was introduced into the SB-1 genome at three intergenic locations (UL3/UL4, UL10/UL11 and UL21/UL22) using recombineering methods on the full-length pSB-1 infectious clone of the virus. We show that the recombinant SB-1 vectors expressing VP2 induced neutralising antibody responses at levels comparable to that of commercial HVT-based VAXXITEKHVT+IBD vaccine. Birds vaccinated with the experimental recombinant SB-1 vaccine were protected against clinical disease after challenge with the very virulent UK661 IBDV isolate, demonstrating its value as an efficient viral vector for developing multivalent vaccines against avian diseases.
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Classical swine fever (CSF) is a highly contagious and often fatal viral disease of domestic pigs and wild boar. Pulmonary oedema and haemorrhages in lung parenchyma are common lesions in the acute forms of CSF that may compromise pig survival and whose pathogenetic mechanisms remain unclear. The appearance of pulmonary lesions in pigs infected with Alfort/187 strain of classical swine fever virus (CSFV) euthanized between 2 and 17 days postinfection (dpi) and the role played by cytokines secreted by different pulmonary macrophage populations in the evolution of lesions was evaluated in this study. Microscopic changes of alveolar septal thickening along with oedema and haemorrhages became more severe at middle-late stages of the experiment. A significant increase in the number of pulmonary macrophages, mainly pulmonary intravascular macrophages (PIMs), was observed coinciding with the onset of alveolar septal thickening from Day 4 pi. PIMs were the main target of CSFV from initial stages of infection while the presence of infected pulmonary alveolar macrophages (PAMs) was scarce and late. Initial infection of PIMs induced phagocytic and biosynthetic activation with subsequent release of chemotactic cytokines. TNFα and, to a lesser extent, IL-1α secreted by PIMs were the major cytokines involved, while IL-6 played only a minor role. On the contrary, results suggested only a secondary role of PAMs as source of cytokines. The presence of vascular changes from Day 9 pi coincided with the highest levels of infected PIMs and the highest number of pro-inflammatory cytokines secreting PIMs. Activation and phagocytosis of platelets were observed in the lungs of infected pigs from early stages, also coinciding with the expression of cytokines with a proven procoagulant activity. The existence of intravascular coagulation phenomena in lung was ruled out.
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Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/patología , Pulmón/irrigación sanguínea , Macrófagos Alveolares/patología , Enfermedades de los Porcinos/patología , Enfermedad Aguda , Animales , Peste Porcina Clásica/etiología , Citocinas/metabolismo , Femenino , Macrófagos Alveolares/metabolismo , Masculino , Porcinos , Enfermedades de los Porcinos/etiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40â¯years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines.
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Sistemas CRISPR-Cas , Vectores Genéticos/genética , Herpesvirus Meleágrido 1/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Animales , Embrión de Pollo , Pollos , Fibroblastos , Edición Génica , Expresión Génica , Técnicas de Sustitución del Gen , Orden Génico , Marcación de Gen , Genes Reporteros , Ingeniería Genética , Ratones , Replicación ViralRESUMEN
OBJECTIVE: To determine serum concentrations of the selected acute-phase proteins (APPs) haptoglobin, serum amyloid A (SAA), and C-reactive protein (CRP) in pigs experimentally inoculated with classical swine fever (CSF) and African swine fever (ASF) viruses. ANIMALS: 8 crossbred (Large White x Landrace) 10-week-old pigs. PROCEDURES: Pigs were allocated to 2 groups (4 pigs/group). One group was inoculated with the CSF virus Alfort 187 strain, whereas the other groupwas inoculated with the ASF virus Spain 70 isolate. Blood samples were collected at various time points. At the end of the study, pigs were euthanized and a complete necropsy was performed, including histologic and immunohistochemical analyses. RESULTS: Serum concentrations of APPs increased in pigs inoculated with CSF and ASF viruses, which suggested an acute-phase response in the course of both diseases. The most noticeable increase in concentration was recorded for SAA in both groups (up to a 300-fold increase for CSF virus and an approx 40-fold increase for ASF virus), followed by CRP and then haptoglobin, which each had only 3- to 4-fold increases. CONCLUSIONS AND CLINICAL RELEVANCE: Serum concentrations of APPs increased significantly in pigs inoculated with CSF and ASF viruses. However, differences were evident in serum concentrations of the proteins evaluated in this study.
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Proteínas de Fase Aguda/metabolismo , Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/inmunología , Fiebre Porcina Africana/sangre , Fiebre Porcina Africana/virología , Animales , Peste Porcina Clásica/sangre , Peste Porcina Clásica/virología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Inmunohistoquímica/veterinaria , Masculino , PorcinosRESUMEN
PRRS control is hampered by the inadequacies of existing vaccines to combat the extreme diversity of circulating viruses. Since immune clearance of PRRSV infection may not be dependent on the development of neutralising antibodies and the identification of broadly-neutralising antibody epitopes have proven elusive, we hypothesised that conserved T cell antigens represent potential candidates for development of a novel PRRS vaccine. Previously we had identified the M and NSP5 proteins as well-conserved targets of polyfunctional CD8 and CD4 T cells. To assess their vaccine potential, peptides representing M and NSP5 were encapsulated in hydrophobically-modified chitosan particles adjuvanted by incorporation of a synthetic multi-TLR2/TLR7 agonist and coated with a model B cell PRRSV antigen. For comparison, empty particles and adjuvanted particles encapsulating inactivated PRRSV-1 were prepared. Vaccination with the particulate formulations induced antigen-specific antibody responses, which were most pronounced following booster immunisation. M and NSP5-specific CD4, but not CD8, T cell IFN-γ reactivity was measurable following the booster immunisation in a proportion of animals vaccinated with peptide-loaded particles. Upon challenge, CD4 and CD8 T cell reactivity was detected in all groups, with the greatest responses being detected in the peptide vaccinated group but with limited evidence of an enhanced control of viraemia. Analysis of the lungs during the resolution of infection showed significant M/NSP5 specific IFN-γ responses from CD8 rather than CD4 T cells. Vaccine primed CD8 T cell responses may therefore be required for protection and future work should focus on enhancing the cross-presentation of M/NSP5 to CD8 T cells.
Asunto(s)
Antígenos Virales/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Linfocitos T/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos/inmunología , Quitosano/química , Péptidos/administración & dosificación , Péptidos/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos , Vacunas Virales/química , Vacunas Virales/normasRESUMEN
Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with lineage mAbs (Lin; CD3, CD8α, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1- cDC (Lin-CD172+ CD1-CD4-) and CD1+ cDC (Lin-CD172a+ CD1+ CD4-), and characterised by phenotypic and functional analyses. CD1+ cDC were distinct from CD1- cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1+ cDC produced IL-8 and IL-10 while CD1- cDC secreted IFN-α, IL-12 and TNF-α. CD1- cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1- and CD1+ populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.
Asunto(s)
Antígenos CD1/análisis , Células Sanguíneas/química , Células Sanguíneas/inmunología , Células Dendríticas/química , Células Dendríticas/inmunología , Animales , Antígenos de Superficie/análisis , Células Sanguíneas/clasificación , Citocinas/metabolismo , Células Dendríticas/clasificación , Citometría de Flujo , Perfilación de la Expresión Génica , Porcinos , Enfermedades de los PorcinosRESUMEN
The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-α and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development.