RESUMEN
There is an urgent need for influenza vaccines providing broader protection that may decrease the need for annual immunization of the human population. We investigated the efficacy of heterologous prime boost immunization with chimpanzee adenovirus (ChAdOx2) and modified vaccinia Ankara (MVA) vectored vaccines, expressing conserved influenza virus nucleoprotein (NP), matrix protein 1 (M1) and neuraminidase (NA) in H1N1pdm09 pre-exposed pigs. We compared the efficacy of intra-nasal, aerosol and intra-muscular vaccine delivery against H3N2 influenza challenge. Aerosol prime boost immunization induced strong local lung T cell and antibody responses and abrogated viral shedding and lung pathology following H3N2 challenge. In contrast, intramuscular immunization induced powerful systemic responses and weak local lung responses but also abolished lung pathology and reduced viral shedding. These results provide valuable insights into the development of a broadly protective influenza vaccine in a highly relevant large animal model and will inform future vaccine and clinical trial design.
RESUMEN
We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies (mAbs). In this study we demonstrated that prophylactic intravenous administration of 15 mg/kg of porcine mAb pb18, against the K160-163 site of the hemagglutinin, significantly reduced lung pathology and nasal virus shedding and eliminated virus from the lung of pigs following H1N1pdm09 challenge. When given at 1 mg/kg, pb18 significantly reduced lung pathology and lung and BAL virus loads, but not nasal shedding. Similarly, when pb18 was given in combination with pb27, which recognized the K130 site, at 1 mg/kg each, lung virus load and pathology were reduced, although without an apparent additive or synergistic effect. No evidence for mAb driven virus evolution was detected. These data indicate that intravenous administration of high doses was required to reduce nasal virus shedding, although this was inconsistent and seldom complete. In contrast, the effect on lung pathology and lung virus load is consistent and is also seen at a one log lower dose, strongly indicating that a lower dose might be sufficient to reduce severity of disease, but for prevention of transmission other measures would be needed.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/tratamiento farmacológico , Administración Intravenosa , Animales , Líquido del Lavado Bronquioalveolar/virología , Modelos Animales de Enfermedad , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/transmisión , Gripe Humana/virología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/patología , Mucosa Nasal/virología , Sus scrofa , Carga Viral/inmunología , Esparcimiento de Virus/efectos de los fármacos , Esparcimiento de Virus/inmunologíaRESUMEN
Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype does not confer interserotype protection. However, some historical data have shown that interserotype protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study, we have investigated the kinetics of the FMDV-specific antibody-secreting cell (ASC) response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar for all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O, A, and Asia1 serotypes) a B cell response to FMDV SAT1 and serotype C was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific ASCs in the absence of circulating FMDV-specific ASCs, indicating the presence of short-lived ASCs, a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid.IMPORTANCE We have demonstrated the development of intraserotype response following a sequential vaccination regime of four different FMDV serotypes. We have found indication of short-lived ASCs in the local lymphoid tissue, further evidence of a TI-2 response to FMDV.
Asunto(s)
Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Protección Cruzada/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Bovinos , Inmunización Secundaria , Serogrupo , Vacunación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Influenza virus infection in pigs is a major farming problem, causing considerable economic loss and posing a zoonotic threat. In addition the pig is an excellent model for understanding immunity to influenza viruses as this is a natural host pathogen system. Experimentally, influenza virus is delivered to pigs intra-nasally, by intra-tracheal instillation or by aerosol, but there is little data comparing the outcome of different methods. We evaluated the shedding pattern, cytokine responses in nasal swabs and immune responses following delivery of low or high dose swine influenza pdmH1N1 virus to the respiratory tract of pigs intra-nasally or by aerosol and compared them to those induced in naturally infected contact pigs. Our data shows that natural infection by contact induces remarkably high innate and adaptive immune response, although the animals were exposed to a very low virus dose. In contacts, the kinetics of virus shedding were slow and prolonged and more similar to the low dose directly infected animals. In contrast the cytokine profile in nasal swabs, antibody and cellular immune responses of contacts more closely resemble immune responses in high dose directly inoculated animals. Consideration of these differences is important for studies of disease pathogenesis and assessment of vaccine protective efficacy.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/veterinaria , Enfermedades de los Porcinos/virología , Administración Intranasal , Aerosoles , Animales , Citocinas/metabolismo , Femenino , Citometría de Flujo/veterinaria , Exposición por Inhalación , Pulmón/patología , Cavidad Nasal/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/patología , Esparcimiento de VirusRESUMEN
Antibodies play a pivotal role against viral infection, and maintenance of protection is dependent on plasma and memory B-cells. Understanding antigen-specific B-cell responses in cattle is essential to inform future vaccine design. We have previously defined T-cell-dependent and -independent B-cell responses in cattle, as a prelude to investigating foot-and-mouth-disease-virus (FMDV)-specific B-cell responses. In this study, we have used an FMDV O-serotype vaccination (O1-Manisa or O SKR) and live-virus challenge (FMDV O SKR) to investigate the homologous and heterologous B-cell response in cattle following both vaccination and live-virus challenge. The FMDV O-serotype vaccines were able to induce a cross-reactive plasma-cell response, specific for both O1-Manisa and O SKR, post-vaccination. Post-FMDV O SKR live-virus challenge, the heterologous O1-Manisa vaccination provided cross-protection against O SKR challenge and cross-reactive O SKR-specific plasma cells were induced. However, vaccination and live-virus challenge were not able to induce a detectable FMDV O-serotype-specific memory B-cell response in any of the cattle. The aim of new FMDV vaccines should be to induce memory responses and increased duration of immunity in cattle.
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Linfocitos B/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Bovinos , Protección Cruzada , Reacciones Cruzadas , Memoria Inmunológica , Vacunas Virales/administración & dosificaciónRESUMEN
Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs.
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Vacunas contra el Adenovirus/administración & dosificación , Anticuerpos Neutralizantes/metabolismo , Fiebre del Valle del Rift/prevención & control , Virus de la Fiebre del Valle del Rift/metabolismo , Proteínas del Envoltorio Viral/genética , Vacunas Virales/administración & dosificación , Vacunas contra el Adenovirus/farmacología , Animales , Camelus , Bovinos , Cabras , Humanos , Pan troglodytes/inmunología , Pan troglodytes/virología , Fiebre del Valle del Rift/inmunología , Virus de la Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/inmunología , Arabia Saudita/epidemiología , Ovinos , Reino Unido/epidemiología , Vacunación , Vacunas Sintéticas/administración & dosificación , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
We have performed a series of studies to investigate the role of CD4(+) T-cells in the immune response to foot-and-mouth disease virus (FMDV) post-vaccination. Virus neutralizing antibody titres (VNT) in cattle vaccinated with killed FMD commercial vaccine were significantly reduced and class switching delayed as a consequence of rigorous in vivo CD4(+) T-cell depletion. Further studies were performed to examine whether the magnitude of T-cell proliferative responses correlated with the antibody responses. FMD vaccination was found to induce T-cell proliferative responses, with CD4(+) T-cells responding specifically to the FMDV antigen. In addition, gamma interferon (IFN-γ) was detected in the supernatant of FMDV antigen-stimulated PBMC and purified CD4(+) T-cells from vaccinated cattle. Similarly, intracellular IFN-γ could be detected specifically in purified CD4(+) T-cells after restimulation. It was not possible to correlate in vitro proliferative responses or IFN-γ production of PBMC with VNT, probably as a consequence of the induction of T-independent and T-dependent antibody responses and antigen non-specific T-cell responses. However, our studies demonstrate the importance of stimulating CD4(+) T-cell responses for the induction of optimum antibody responses to FMD-killed vaccines.
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Linfocitos T CD4-Positivos/inmunología , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/virología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Bovinos , Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Fiebre Aftosa/virología , Interferón gamma/inmunología , Estudios Longitudinales , Activación de Linfocitos/inmunología , Vacunación/veterinaria , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.
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Linfocitos B/inmunología , Bovinos/inmunología , Memoria Inmunológica , Células Plasmáticas/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos/sangre , Antígenos/sangre , Bovinos/sangre , Ensayo de Immunospot Ligado a Enzimas/veterinaria , Ficoll/análogos & derivados , Ficoll/inmunología , Haptenos/inmunología , Inmunoensayo/veterinaria , Leucocitos Mononucleares/fisiología , Masculino , Trinitrobencenos/inmunología , gammaglobulinas/inmunologíaRESUMEN
Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.
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Adyuvantes Inmunológicos/farmacología , Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Subgrupos de Linfocitos T/inmunología , Animales , Línea Celular , Proliferación Celular , Perros , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Fluoresceínas , Inmunoglobulina G/sangre , Leucocitos Mononucleares , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Succinimidas , Sus scrofaRESUMEN
Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in naïve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle.
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Inmunidad Adaptativa , Enfermedades de los Bovinos/inmunología , Citocinas/genética , Virus de la Fiebre Aftosa/fisiología , Fiebre Aftosa/inmunología , Inmunidad Innata , Terapia de Inmunosupresión , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Citocinas/metabolismo , Fiebre Aftosa/microbiología , Recuento de Leucocitos/veterinaria , Masculino , Reacción en Cadena de la Polimerasa/veterinaria , Factores de TiempoRESUMEN
To determine the longevity of protective immunity following a single administration of emergency vaccine, and establish whether the immune response could be enhanced by increasing the antigen payload even further, cattle were vaccinated with an A22 Iraq vaccine containing either 1x antigen payload (field dose) or 5x antigen payload. Six months post-immunisation all cattle received a homologous virus challenge. The magnitude of the virus neutralising antibody response elicited was consistent with the response to similarly formulated A serotype vaccines with a PD(50) greater than 32. All the vaccinated cattle, regardless of antigen payload, were protected from clinical disease following challenge although some cattle in both groups became sub-clinically infected. We conclude that immunisation with a single inoculation of vaccine from the UK emergency reserve can protect cattle from clinical disease for at least 6 months post-vaccination and that a boost may be unnecessary in an outbreak situation. Some animals may become sub-clinically infected but this is likely to be dependent on the severity of challenge. The study confirmed that a booster at 21 days post-vaccination was not necessary to maintain a cell-mediated response in cattle for 6 months. No increased benefits were recognised by increasing the antigen payload of this vaccine 5x.
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Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Bovinos , Inmunización Secundaria , Linfocitos/inmunología , Análisis de Supervivencia , Factores de Tiempo , Reino UnidoRESUMEN
The aim of the study was to build a comprehensive picture of the appearance in the blood stream of Ag-specific plasma cells and memory B cells in the bovine model. For this purpose, we have developed a method allowing the detection and quantification of both cell types within individual calves immunised with ovalbumin. During the primary response, we detected a burst of ovalbumin-specific plasma cells at days 6 and 7 post-immunisation, which was followed by the production of specific Ab, whereas a gradual increase of memory B cells was only detected from day 15. As expected, a boost immunisation performed 7 weeks later induced a quicker and stronger secondary response. Indeed, a burst of plasma cells was detected in the blood at days 3 and 4, which was followed by a strong increase in Ab titres. Furthermore, a burst of memory B cells, and not a gradual increase, was detected at days 5 and 6 post-boost immunisation. Importantly, we showed a strong correlation between the anti-ovalbumin-specific IgG titres detected 5 months after secondary immunisation and the plasma cell numbers detected in the blood at the peak response after secondary immunisation. The detection and quantification of plasma cells following an mmunisation/vaccination strategy could constitute a very effective means for predicting the magnitude and longevity of an Ab response.
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Linfocitos B/inmunología , Bovinos/inmunología , Inmunoensayo/veterinaria , Ovalbúmina/inmunología , Células Plasmáticas/inmunología , Animales , Anticuerpos/sangre , Antígenos/sangre , Bovinos/sangre , Memoria Inmunológica , Leucocitos Mononucleares/fisiologíaRESUMEN
To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the entire polyprotein sequence of the virus, 442 overlapping pentadecapeptides were tested in proliferation assays using lymphocytes from cattle experimentally infected with FMDV. Four months post-infection cells from all investigated animals (n = 4) responded by proliferation and interferon-gamma production to a peptide located on the structural protein 1D (VP1), amino acid residues 66-80. Major histocompatibility complex (MHC) serotyping of the investigated cattle indicated that all animals shared the MHC serotype A31 which comprises the class II allele DRB3 0701. This may explain the common recognition of this newly discovered epitope. Responses to other peptides could only be observed in one animal and rapidly declined during the time course of the study. These observations point to an immunodominant role of this epitope located on the protein 1D in cattle with MHC serotype A31.
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Enfermedades de los Bovinos/inmunología , Epítopos de Linfocito T/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Antígenos de Histocompatibilidad Clase II/inmunología , Epítopos Inmunodominantes/inmunología , Animales , Bovinos , Enfermedades de los Bovinos/virología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/clasificación , Interferón gamma/metabolismo , Leucocitos Mononucleares/metabolismo , MasculinoRESUMEN
Workshop cluster 1(+) gamma delta (WC1(+)gammadelta) T cells have been shown to play important roles in the immune response to infections. WC1 is a transmembrane glycoprotein, uniquely expressed on the surface of gammadelta T cells of ruminants and pigs. A role for WC1 in inducing a reversible growth arrest of gammadelta T cells has been previously demonstrated. WC1-induced growth inhibition has been shown to be overcome following gammadelta T cell activation with Concanavalin A (Con A). However, molecular mechanism(s) by which WC1 signalling might be modulated following activation have not been elucidated. In this paper we show that Con A activation of bovine lymphocytes induces the tyrosine phosphorylation of WC1 in a Src-family kinase-dependent manner. Src family kinases also phosphorylated WC1 in a COS-7 co-transfection system. Furthermore, a glutathione-S-transferase (GST)-WC1 cytoplasmic domain fusion protein was directly phosphorylated by recombinant Lck (rLck) in vitro. The Y(1303) of WC1 was identified by mutational analysis as the only one of the five WC1 tyrosine residues to be critical for Src family phosphorylation. The importance of activation-induced Src family activity for WC1 function was investigated with the Src-family specific inhibitor PP2. These studies show that the surface levels of WC1 are down regulated in a Src-family-dependent manner following activation of bovine lymphocytes. Down regulation of surface WC1 was accompanied by a Src-family-dependent accumulation of intracellular WC1. These data show that WC1 is modulated by activation-induced tyrosine phosphorylation thus providing a new insight into the signalling mechanisms by which WC1 and gammadelta T cell activation are regulated in this important and unique cell population.