RESUMO
Foot-and-mouth disease (FMD) is a highly contagious, economically devastating disease of cloven-hooved animals. The development of long-lasting effective FMD vaccines would greatly benefit the global FMD control programme. Deep analysis of adaptive immunity in cattle vaccinated against FMD is technically challenging due to the lack of species-specific tools. In this study, we aimed to identify CD4+ T-cell epitopes in the FMD virus (FMDV) capsid and to phenotype the CD4+ T cells that recognize them using bovine major histocompatibility complex (BoLA) class II tetramer. A BoLA class II tetramer based on the DRA/DRB3*020:02 allele and FMDV antigen-stimulated PBMCs from bovine vaccinates were used to successfully identify four epitopes in the FMDV capsid, three of which have not been previously reported; two epitopes were identified in the structural protein VP1, one in VP3 and one in VP4. Specificity of the three novel epitopes was confirmed by proliferation assay. All epitope-expanded T-cell populations produced IFN-γ in vitro, indicating a long-lasting Th1 cell phenotype after FMD vaccination. VP3-specific CD4+ T cells exhibited the highest frequency amongst the identified epitopes, comprising >0·004% of the CD4+ T-cell population. CD45RO+ CCR7+ defined central memory CD4+ T-cell subpopulations were present in higher frequency in FMDV-specific CD4+ T-cell populations from FMD-vaccinated cattle ex vivo. This indicates an important role in maintaining cell adaptive immunity after FMD vaccination. Notably, FMDV epitope-loaded tetramers detected the presence of FMDV-specific CD4+ T cells in bovine PBMC more than four years after vaccination. This work contributes to our understanding of vaccine efficacy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/virologia , Proteínas do Capsídeo/imunologia , Bovinos , Células Cultivadas , Epitopos de Linfócito T/imunologia , Febre Aftosa/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Sorogrupo , Vacinação/métodosRESUMO
African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centers of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centers, probang samples (oropharyngeal scrapings), and tonsil swabs to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1 coding region of the SAT1 virus inoculum clustered around 2 subpopulations differing by 22 single-nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions, depending on the day postinfection (dpi). There was a significant difference between the populations of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral populations in samples from individual animals, germinal centers, and epithelial tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to postmortem showed less within-sample variability of VP1 than that of tissue sample sequences analyzed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralize the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study, there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo.IMPORTANCE Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven-hoofed animals and is recognized as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV, and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low-frequency sequence variants over time, there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.
Assuntos
Búfalos , Portador Sadio/veterinária , Evolução Molecular , Vírus da Febre Aftosa/crescimento & desenvolvimento , Febre Aftosa/imunologia , Febre Aftosa/virologia , Evasão da Resposta Imune , Animais , Proteínas do Capsídeo/genética , Portador Sadio/imunologia , Portador Sadio/virologia , Epitélio/virologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Instabilidade Genômica , Centro Germinativo/virologia , Mutação , Orofaringe/virologia , Análise de Sequência de DNARESUMO
Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.
Assuntos
Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/fisiologia , Vacinas Virais/genética , Substituição de Aminoácidos , Animais , Vírus da Febre Aftosa/imunologia , Instabilidade Genômica , Concentração de Íons de Hidrogênio , Imunogenicidade da Vacina , Íons , Cinética , Mutação , Sorogrupo , Cloreto de Sódio/farmacologia , Temperatura , Potência de Vacina , Vacinas Virais/químicaRESUMO
UNLABELLED: Foot-and-mouth disease (FMD) virus (FMDV) circulates as multiple serotypes and strains in many regions of endemicity. In particular, the three Southern African Territories (SAT) serotypes are maintained effectively in their wildlife reservoir, the African buffalo, and individuals may harbor multiple SAT serotypes for extended periods in the pharyngeal region. However, the exact site and mechanism for persistence remain unclear. FMD in buffaloes offers a unique opportunity to study FMDV persistence, as transmission from carrier ruminants has convincingly been demonstrated for only this species. Following coinfection of naive African buffaloes with isolates of three SAT serotypes from field buffaloes, palatine tonsil swabs were the sample of choice for recovering infectious FMDV up to 400 days postinfection (dpi). Postmortem examination identified infectious virus for up to 185 dpi and viral genomes for up to 400 dpi in lymphoid tissues of the head and neck, focused mainly in germinal centers. Interestingly, viral persistence in vivo was not homogenous, and the SAT-1 isolate persisted longer than the SAT-2 and SAT-3 isolates. Coinfection and passage of these SAT isolates in goat and buffalo cell lines demonstrated a direct correlation between persistence and cell-killing capacity. These data suggest that FMDV persistence occurs in the germinal centers of lymphoid tissue but that the duration of persistence is related to virus replication and cell-killing capacity. IMPORTANCE: Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in domestic livestock and wildlife species. African buffaloes (Syncerus caffer) are the primary carrier hosts of FMDV in African savannah ecosystems, where the disease is endemic. We have shown that the virus persists for up to 400 days in buffaloes and that there is competition between viruses during mixed infections. There was similar competition in cell culture: viruses that killed cells quickly persisted more efficiently in passaged cell cultures. These results may provide a mechanism for the dominance of particular viruses in an ecosystem.
Assuntos
Búfalos/virologia , Portador Sadio/veterinária , Vírus da Febre Aftosa/fisiologia , Vírus da Febre Aftosa/patogenicidade , Febre Aftosa/virologia , África/epidemiologia , Animais , Animais Selvagens/virologia , Anticorpos Antivirais/sangue , Portador Sadio/virologia , Coinfecção/epidemiologia , Coinfecção/veterinária , Coinfecção/virologia , Ensaio de Imunoadsorção Enzimática , Febre Aftosa/epidemiologia , Febre Aftosa/imunologia , Febre Aftosa/transmissão , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Genoma Viral , Tonsila Palatina/virologia , Sorogrupo , Virulência , Replicação ViralRESUMO
Foot-and-mouth disease (FMD) has a major economic impact throughout the world and is a considerable threat to food security. Current FMD virus (FMDV) vaccines are made from chemically inactivated virus and need to contain intact viral capsids to maximize efficacy. FMDV exists as seven serotypes, each made up by a number of constantly evolving subtypes. A lack of immunological cross-reactivity between serotypes and between some strains within a serotype greatly complicates efforts to control FMD by vaccination. Thus, vaccines for one serotype do not afford protection against the others, and multiple-serotype-specific vaccines are required for effective control. The FMDV serotypes exhibit variation in their thermostability, and the capsids of inactivated preparations of the O, C and SAT serotypes are particularly susceptible to dissociation at elevated temperature. Methods to quantify capsid stability are currently limited, lack sensitivity and cannot accurately reflect differences in thermostability. Thus, new, more sensitive approaches to quantify capsid stability would be of great value for the production of more stable vaccines and to assess the effect of production conditions on vaccine preparations. Here we have investigated the application of a novel methodology (termed PaSTRy) that utilizes an RNA-binding fluorescent dye and a quantitative (q)PCR machine to monitor viral genome release and hence dissociation of the FMDV capsid during a slow incremental increase in temperature. PaSTRy was used to characterize capsid stability of all FMDV serotypes. Furthermore, we have used this approach to identify stabilizing factors for the most labile FMDV serotypes.
Assuntos
Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/imunologia , Animais , Capsídeo/imunologia , Linhagem Celular , Cricetinae , Febre Aftosa/imunologia , Febre Aftosa/virologia , Genoma Viral/genética , Cabras/virologia , Temperatura Alta , Reação em Cadeia da Polimerase , Sorogrupo , VacinaçãoRESUMO
Laboratory animal models have provided valuable insight into foot-and-mouth disease virus (FMDV) pathogenesis in epidemiologically important target species. While not perfect, these models have delivered an accelerated time frame to characterize the immune responses in natural hosts and a platform to evaluate therapeutics and vaccine candidates at a reduced cost. Further expansion of these models in mice has allowed access to genetic mutations not available for target species, providing a powerful and versatile experimental system to interrogate the immune response to FMDV and to target more expensive studies in natural hosts. The purpose of this review is to describe commonly used FMDV infection models in laboratory animals and to cite examples of when these models have failed or successfully provided insight relevant for target species, with an emphasis on natural and vaccine-induced immunity.
Assuntos
Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinas Virais/uso terapêutico , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Bovinos , Febre Aftosa/virologia , Imunidade Celular , Imunidade Humoral , Imunidade Inata , Camundongos , Modelos Animais , Sus scrofa , Linfócitos T/imunologiaRESUMO
Field isolates of foot-and-mouth disease virus (FMDV) have a restricted cell tropism which is limited by the need for certain RGD-dependent integrin receptors. In contrast, cell culture-adapted viruses use heparan sulfate (HS) or other unidentified molecules as receptors to initiate infection. Here, we report several novel findings resulting from cell culture adaptation of FMDV. In cell culture, a virus with the capsid of the A/Turkey/2/2006 field isolate gained the ability to infect CHO and HS-deficient CHO cells as a result of a single glutamine (Q)-to-lysine (K) substitution at VP1-110 (VP1-(Q)110(K)). Using site-directed mutagenesis, the introduction of lysine at this same site also resulted in an acquired ability to infect CHO cells by type O and Asia-1 FMDV. However, this ability appeared to require a second positively charged residue at VP1-109. CHO cells express two RGD-binding integrins (α5ß1 and αvß5) that, although not used by FMDV, have the potential to be used as receptors; however, viruses with the VP1-(Q)110(K) substitution did not use these integrins. In contrast, the VP1-(Q)110(K) substitution appeared to result in enhanced interactions with αvß6, which allowed a virus with KGE in place of the normal RGD integrin-binding motif to use αvß6 as a receptor. Thus, our results confirmed the existence of nonintegrin, non-HS receptors for FMDV on CHO cells and revealed a novel, non-RGD-dependent use of αvß6 as a receptor. The introduction of lysine at VP1-110 may allow for cell culture adaptation of FMDV by design, which may prove useful for vaccine manufacture when cell culture adaptation proves intractable.
Assuntos
Adaptação Biológica , Vírus da Febre Aftosa/fisiologia , Receptores Virais/metabolismo , Inoculações Seriadas , Tropismo Viral , Animais , Células CHO , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cricetinae , Análise Mutacional de DNA , Vírus da Febre Aftosa/genética , Mutagênese Sítio-DirigidaRESUMO
Foot-and-mouth disease virus (FMDV) is one of the most extensively studied animal pathogens because it remains a major threat to livestock economies worldwide. However, the dynamics of FMDV infection are still poorly understood. The application of reverse genetics provides the opportunity to generate molecular tools to further dissect the FMDV life cycle. Here, we have used reverse genetics to determine the capsid packaging limitations for a selected insertion site in the FMDV genome. We show that exogenous RNA up to a defined length can be stably introduced into the FMDV genome, whereas larger insertions are excised by recombination events. This led us to construct a recombinant FMDV expressing the fluorescent marker protein, termed iLOV. Characterization of infectious iLOV-FMDV showed the virus has a plaque morphology and rate of growth similar to the parental virus. In addition, we show that cells infected with iLOV-FMDV are easily differentiated by flow cytometry using the inherent fluorescence of iLOV and that cells infected with iLOV-FMDV can be monitored in real-time with fluorescence microscopy. iLOV-FMDV therefore offers a unique tool to characterize FMDV infection in vitro, and its applications for in vivo studies are discussed.
Assuntos
Epitélio/virologia , Vírus da Febre Aftosa/patogenicidade , Proteínas de Fluorescência Verde/metabolismo , Recombinação Genética , Animais , Células Cultivadas , Efeito Citopatogênico Viral , Citometria de Fluxo , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/fisiologia , Cabras , Proteínas de Fluorescência Verde/genéticaRESUMO
Foot-and-mouth disease (FMD) is a highly contagious viral disease of livestock which is prevalent across Africa, the Middle East, Asia, and South America where it has a severe economic impact on the agriculture industry. Vaccination with inactivated viral vaccines is used as the main control measure in these endemic regions of the world, however the presence of multiple serotypes, subtypes, and the continual emergence of new, antigenically divergent strains limits its effectiveness. East Africa (EA) has been identified as a region that would particularly benefit from updated FMD vaccines, since those currently in use contain older strains which do not provide good protection against contemporary strains. Four serotypes are currently circulating in EA, necessitating the development of a quadrivalent vaccine containing representative strains of each serotype. A key consideration in the selection of vaccine strains is the stability of the virus particle, since the capsids readily dissociate on exposure to elevated temperatures, but only intact capsids induce protective immunity to FMD. Therefore, with a view to producing a more stable, updated quadrivalent vaccine for EA, we recently screened a panel of foot-and-mouth disease virus (FMDV) isolates from the region to select strains with naturally higher thermostabilities and confirmed their immunogenicity in cattle. Herein we describe the formulation and serological assessment of wild-type and recombinant quadrivalent vaccine candidates comprising these stable strains, and demonstrate that both vaccines generate high neutralising antibody titres against the homologous strains and also to heterologous strains from EA. Importantly, the vaccine passed the criteria set by the AgResults vaccine challenge project and offers good cross-protection against a panel of regional FMDV strains.
RESUMO
Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.
Assuntos
Proteínas do Capsídeo/metabolismo , Epitopos , Vírus da Febre Aftosa/classificação , Febre Aftosa/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Febre Aftosa/virologia , Modelos Moleculares , Conformação Proteica , RNA Viral , Coloração e Rotulagem , Vacinas de DNA , Vacinas de Produtos InativadosRESUMO
Type I interferons (alpha/beta interferons [IFN-α/ß]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.
Assuntos
Doenças dos Bovinos/imunologia , Células Dendríticas/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Interferon Tipo I/metabolismo , Animais , Antígenos CD/análise , Bovinos , Centrifugação com Gradiente de Concentração , Células Dendríticas/química , Citometria de FluxoRESUMO
Foot-and-mouth disease is an economically devastating disease of livestock caused by foot-and-mouth disease virus (FMDV). Vaccination is the most effective control measure in place to limit the spread of the disease; however, the success of vaccination campaigns is hampered by the antigenic diversity of FMDV and the rapid rate at which new strains emerge that escape pre-existing immunity. FMDV has seven distinct serotypes, and within each serotype are multiple strains that often induce little cross-protective immunity. The diversity of FMDV is a consequence of the high error rate of the RNA-dependent RNA polymerase, accompanied by extensive recombination between genomes during co-infection. Since multiple serotypes and strains co-circulate in regions where FMDV is endemic, co-infection is common, providing the conditions for recombination, and also for other events such as trans-encapsidation in which the genome of one virus is packaged into the capsid of the co-infecting virus. Here, we demonstrate that the co-infection of cells with two FMDVs of different serotypes results in trans-encapsidation of both viral genomes. Crucially, this facilitates the infection of new cells in the presence of neutralizing antibodies that recognize the capsid that is encoded by the packaged genome.
Assuntos
Coinfecção , Vírus da Febre Aftosa , Febre Aftosa , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais , Proteínas do Capsídeo/genética , Vírus da Febre Aftosa/genética , SorogrupoRESUMO
Foot-and-mouth disease (FMD) is endemic in large parts of sub-Saharan Africa, Asia and South America, where outbreaks in cloven-hooved livestock threaten food security and have severe economic impacts. Vaccination in endemic regions remains the most effective control strategy. Current FMD vaccines are produced from chemically inactivated foot-and-mouth disease virus (FMDV) grown in suspension cultures of baby hamster kidney 21 cells (BHK-21). Strain diversity means vaccines produced from one subtype may not fully protect against circulating disparate subtypes, necessitating the development of new vaccine strains that "antigenically match". However, some viruses have proven difficult to adapt to cell culture, slowing the manufacturing process, reducing vaccine yield and limiting the availability of effective vaccines, as well as potentiating the selection of undesired antigenic changes. To circumvent the need to cell culture adapt FMDV, we have used a systematic approach to develop recombinant suspension BHK-21 that stably express the key FMDV receptor integrin αvß6. We show that αvß6 expression is retained at consistently high levels as a mixed cell population and as a clonal cell line. Following exposure to field strains of FMDV, these recombinant BHK-21 facilitated higher virus yields compared to both parental and control BHK-21, whilst demonstrating comparable growth kinetics. The presented data supports the application of these recombinant αvß6-expressing BHK-21 in future FMD vaccine production.
Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Animais , Linhagem Celular , Vírus da Febre Aftosa/genética , Vacinação , Vacinas Virais/genéticaRESUMO
Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is an economically devastating disease affecting several important livestock species. FMDV is antigenically diverse and exists as seven serotypes comprised of many strains which are poorly cross-neutralised by antibodies induced by infection or vaccination. Co-infection and recombination are important drivers of antigenic diversity, especially in regions where several serotypes co-circulate at high prevalence, and therefore experimental systems to study these events in vitro would be beneficial. Here we have utilised recombinant FMDVs containing an HA or a FLAG epitope tag within the VP1 capsid protein to investigate the products of co-infection in vitro. Co-infection with viruses from the same and from different serotypes was demonstrated by immunofluorescence microscopy and flow cytometry using anti-tag antibodies. FLAG-tagged VP1 and HA-tagged VP1 could be co-immunoprecipitated from co-infected cells, suggesting that newly synthesised capsids may contain VP1 proteins from both co-infecting viruses. Furthermore, we provide the first demonstration of trans-encapsidation of an FMDV genome into capsids comprised of proteins encoded by a co-infecting heterologous virus. This system provides a useful tool for investigating co-infection dynamics in vitro, particularly between closely related strains, and has the advantage that it does not depend upon the availability of strain-specific FMDV antibodies.
Assuntos
Capsídeo/metabolismo , Vírus da Febre Aftosa/fisiologia , Febre Aftosa/virologia , RNA Viral/metabolismo , Empacotamento do Genoma Viral , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Coinfecção , Epitopos , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Genoma Viral , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , RNA Viral/genética , SorogrupoRESUMO
Foot-and-mouth disease (FMD) is a global burden on the livestock industry. The causative agent, FMD virus (FMDV), is highly infectious and exists in seven distinct serotypes. Vaccination remains the most effective control strategy in endemic regions and current FMD vaccines are made from inactivated preparations of whole virus. The inherent instability of FMDV and the emergence of new strains presents challenges to efficacious vaccine development. Currently, vaccines available in East Africa are comprised of relatively historic strains with unreported stabilities. As an initial step to produce an improved multivalent FMD vaccine we have identified naturally stable East African FMDV strains for each of the A, O, SAT1 and SAT2 serotypes and investigated their potential for protecting ruminants against strains that have recently circulated in East Africa. Interestingly, high diversity in stability between and within serotypes was observed, and in comparison to non-African A serotype viruses reported to date, the East African strains tested in this study are less stable. Candidate vaccine strains were adapted to propagation in BHK-21 cells with minimal capsid changes and used to generate vaccinate sera that effectively neutralised a panel of FMDV strains selected to improve FMD vaccines used in East Africa. This work highlights the importance of combining tools to predict and assess FMDV vaccine stability, with cell culture adaptation and serological tests in the development of FMD vaccines.
Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Animais , Proteínas do Capsídeo/genética , Febre Aftosa/prevenção & controle , SorogrupoRESUMO
Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.
Assuntos
Proteínas do Capsídeo/imunologia , Quimera/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Linhagem Celular , Quimera/genética , Cricetinae , Febre Aftosa/imunologia , Vírus da Febre Aftosa/genética , Cabras , SuínosRESUMO
Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus. There are applications for the use of Nano-FMDV such as real-time monitoring of FMDV replication in vitro and in vivo.
Assuntos
Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/fisiologia , Genes Reporter , Luciferases/biossíntese , Coloração e Rotulagem/métodos , Replicação Viral , Animais , Linhagem Celular , Instabilidade Genômica , Medições Luminescentes , Ensaio de Placa Viral , Cultura de Vírus , VirosesRESUMO
Intact (146S) foot-and-mouth disease virus (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH-M332F-that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA) employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3-4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.
RESUMO
Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvß6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvß6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas do Capsídeo/metabolismo , Vírus da Febre Aftosa/metabolismo , Integrinas/metabolismo , Oligopeptídeos/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Capsídeo/metabolismo , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos/química , Ligação Proteica , Receptores Virais/metabolismo , Replicação ViralRESUMO
Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.