RESUMEN
The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1ß. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1ß reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Pulmón , Infecciones por Orthomyxoviridae , Análisis de la Célula Individual , Animales , Porcinos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Pulmón/inmunología , Pulmón/virología , Vacunas contra la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Inmunización , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/virologíaRESUMEN
Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.
Asunto(s)
Antígenos de Histocompatibilidad Clase I , Antígenos de Histocompatibilidad Menor , Células T Invariantes Asociadas a Mucosa , Especificidad de la Especie , Animales , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Invariantes Asociadas a Mucosa/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ratones , Bovinos , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/química , Porcinos , Macaca , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009330.].
RESUMEN
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.
Asunto(s)
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
A vaccine providing both powerful Ab and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. In this study, we evaluated i.m., aerosol (Aer), and simultaneous systemic and respiratory immunization (SIM) by both routes in Babraham pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization, pigs were challenged with H1N1pdm09 virus. i.m.-immunized pigs generated a high titer of neutralizing Abs but poor T cell responses, whereas Aer induced powerful respiratory tract T cell responses but a low titer of Abs. SIM pigs combined high Ab titers and strong local T cell responses. SIM showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T/inmunología , Aerosoles , Animales , Formación de Anticuerpos , Modelos Animales de Enfermedad , Resistencia a la Enfermedad , Humanos , Inmunidad Celular , Inmunización , Inyecciones Intramusculares , PorcinosRESUMEN
Swine influenza A virus (swIAV) infection causes substantial economic loss and disease burden in humans and animals. The 2009 pandemic H1N1 (pH1N1) influenza A virus is now endemic in both populations. In this study, we evaluated the efficacy of different vaccines in reducing nasal shedding in pigs following pH1N1 virus challenge. We also assessed transmission from immunized and challenged pigs to naive, directly in-contact pigs. Pigs were immunized with either adjuvanted, whole inactivated virus (WIV) vaccines or virus-vectored (ChAdOx1 and MVA) vaccines expressing either the homologous or heterologous influenza A virus hemagglutinin (HA) glycoprotein, as well as an influenza virus pseudotype (S-FLU) vaccine expressing heterologous HA. Only two vaccines containing homologous HA, which also induced high hemagglutination inhibitory antibody titers, significantly reduced virus shedding in challenged animals. Nevertheless, virus transmission from challenged to naive, in-contact animals occurred in all groups, although it was delayed in groups of vaccinated animals with reduced virus shedding.IMPORTANCE This study was designed to determine whether vaccination of pigs with conventional WIV or virus-vectored vaccines reduces pH1N1 swine influenza A virus shedding following challenge and can prevent transmission to naive in-contact animals. Even when viral shedding was significantly reduced following challenge, infection was transmissible to susceptible cohoused recipients. This knowledge is important to inform disease surveillance and control strategies and to determine the vaccine coverage required in a population, thereby defining disease moderation or herd protection. WIV or virus-vectored vaccines homologous to the challenge strain significantly reduced virus shedding from directly infected pigs, but vaccination did not completely prevent transmission to cohoused naive pigs.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza/administración & dosificación , Infecciones por Orthomyxoviridae/transmisión , Enfermedades de los Porcinos/transmisión , Esparcimiento de Virus , Adyuvantes Inmunológicos/administración & dosificación , Animales , Femenino , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Infecciones por Orthomyxoviridae/prevención & control , Porcinos , Enfermedades de los Porcinos/prevención & control , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas de Productos Inactivados/administración & dosificaciónRESUMEN
Influenza is a major cause of mortality and morbidity worldwide. Despite numerous studies of the pathogenesis of influenza in humans and animal models the dynamics of infection and transmission in individual hosts remain poorly characterized. In this study, we experimentally modelled transmission using the H1N1pdm09 influenza A virus in pigs, which are considered a good model for influenza infection in humans. Using an experimental design that allowed us to observe individual transmission events occurring within an 18-hr period, we quantified the relationships between infectiousness, shed virus titre and antibody titre. Transmission event was observed on 60% of occasions when virus was detected in donor pig nasal swabs and transmission was more likely when donor pigs shed more virus. This led to the true infectious period (mean 3.9 days) being slightly shorter than that predicted by detection of virus (mean 4.5 days). The generation time of infection (which determines the rate of epidemic spread) was estimated for the first time in pigs at a mean of 4.6 days. We also found that the latent period of the contact pig was longer when they had been exposed to smaller amount of shed virus. Our study provides quantitative information on the time lines of infection and the dynamics of transmission that are key parts of the evidence base needed to understand the spread of influenza viruses though animal populations and, potentially, in humans.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/transmisión , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Infecciones por Orthomyxoviridae/virología , Porcinos , Factores de Tiempo , Esparcimiento de VirusRESUMEN
mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/tratamiento farmacológico , PorcinosRESUMEN
There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Sistema Respiratorio/inmunología , Aerosoles , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Epítopos/química , Epítopos/genética , Femenino , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Endogamia , Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/transmisión , Masculino , Modelos Animales , Modelos Moleculares , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/veterinaria , Sus scrofa/genética , Sus scrofa/inmunología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Vacunación/métodos , Vacunación/veterinariaRESUMEN
Influenza is a major health threat, and a broadly protective influenza vaccine would be a significant advance. Signal Minus FLU (S-FLU) is a candidate broadly protective influenza vaccine that is limited to a single cycle of replication, which induces a strong cross-reactive T cell response but a minimal Ab response to hemagglutinin after intranasal or aerosol administration. We tested whether an H3N2 S-FLU can protect pigs and ferrets from heterosubtypic H1N1 influenza challenge. Aerosol administration of S-FLU to pigs induced lung tissue-resident memory T cells and reduced lung pathology but not the viral load. In contrast, in ferrets, S-FLU reduced viral replication and aerosol transmission. Our data show that S-FLU has different protective efficacy in pigs and ferrets, and that in the absence of Ab, lung T cell immunity can reduce disease severity without reducing challenge viral replication.
Asunto(s)
Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Hurones , Hemaglutininas/inmunología , Humanos , Inmunidad/inmunología , Memoria Inmunológica/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Porcinos , Linfocitos T/inmunología , Vacunación/métodos , Replicación Viral/inmunologíaRESUMEN
Designing improved vaccines against mutable viruses such as dengue and influenza would be helped by a better understanding of how the B-cell memory compartment responds to variant antigens. Towards this we have recently shown, after secondary immunization of mice with a widely variant dengue virus envelope protein with only 63% amino acid identity, that IgM+ memory B cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, further investigation of memory responses to variant proteins, using more closely related influenza virus haemagglutinins (HA) that were 82% identical, produced a variant-induced boost response in the GC dominated by highly mutated B cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over a certain level of antigenic differences, cross-reactive memory B-cell populations have reduced competency for affinity maturation. Combined with our previous observations, these findings also provide new parameters of success and failure in antibody memory responses.
Asunto(s)
Linfocitos B/fisiología , Centro Germinal/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Receptores de Antígenos de Linfocitos B/genética , Animales , Afinidad de Anticuerpos , Reacciones Cruzadas , Femenino , Hemaglutininas Virales/genética , Hemaglutininas Virales/metabolismo , Humanos , Inmunidad Humoral , Inmunización Secundaria , Memoria Inmunológica , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Polimorfismo GenéticoRESUMEN
The pestivirus noncytopathic bovine viral diarrhea virus (BVDV) can suppress IFN production in the majority of cell types in vitro. However, IFN is detectable in serum during acute infection in vivo for â¼5-7 d, which correlates with a period of leucopoenia and immunosuppression. In this study, we demonstrate that a highly enriched population of bovine plasmacytoid dendritic cells (DCs) produced IFN in response to BVDV in vitro. We further show that the majority of the IFN produced in response to infection both in vitro and in vivo is type III IFN and acid labile. Further, we show IL-28B (IFN-λ3) mRNA is induced in this cell population in vitro. Supernatant from plasmacytoid DCs harvested postinfection with BVDV or recombinant bovine IFN-α or human IL-28B significantly reduced CD4(+) T cell proliferation induced by tubercle bacillus Ag 85-stimulated monocyte-derived DCs. Furthermore, these IFNs induced IFN-stimulated gene expression predominantly in monocyte-derived DCs. IFN-treated immature DCs derived from murine bone marrow also had a reduced capacity to stimulate T cell proliferative responses to tubercle bacillus Ag 85. Immature DCs derived from either source had a reduced capacity for Ag uptake following IFN treatment that is dose dependent. Immunosuppression is a feature of a number of pestivirus infections; our studies suggest type III IFN production plays a key role in the pathogenesis of this family of viruses. Overall, in a natural host, we have demonstrated a link between the induction of type I and III IFN after acute viral infection and transient immunosuppression.
Asunto(s)
Diarrea Mucosa Bovina Viral/inmunología , Células Dendríticas/inmunología , Virus de la Diarrea Viral Bovina/inmunología , Inmunidad Celular , Interferón-alfa/inmunología , Interleucinas/inmunología , Aciltransferasas/inmunología , Animales , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Bovinos , Línea Celular , Proliferación Celular , Humanos , Tolerancia Inmunológica , Interferón-alfa/sangre , Interferones , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , Proteínas Recombinantes/inmunología , Sus scrofaRESUMEN
Influenza A viruses are a major health threat to livestock and humans, causing considerable mortality, morbidity, and economic loss. Current inactivated influenza vaccines are strain specific and new vaccines need to be produced at frequent intervals to combat newly arising influenza virus strains, so that a universal vaccine is highly desirable. We show that pandemic H1N1 influenza virus in which the hemagglutinin signal sequence has been suppressed (S-FLU), when administered to pigs by aerosol can induce CD4 and CD8 T cell immune responses in blood, bronchoalveolar lavage (BAL), and tracheobronchial lymph nodes. Neutralizing Ab was not produced. Detection of a BAL response correlated with a reduction in viral titer in nasal swabs and lungs, following challenge with H1N1 pandemic virus. Intratracheal immunization with a higher dose of a heterologous H5N1 S-FLU vaccine induced weaker BAL and stronger tracheobronchial lymph node responses and a lesser reduction in viral titer. We conclude that local cellular immune responses are important for protection against influenza A virus infection, that these can be most efficiently induced by aerosol immunization targeting the lower respiratory tract, and that S-FLU is a promising universal influenza vaccine candidate.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Infecciones por Orthomyxoviridae/prevención & control , Carga Viral , Aerosoles , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunidad Celular , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Humana/sangre , Gripe Humana/inmunología , Gripe Humana/virología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Nariz/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Pandemias/prevención & control , Sus scrofa , Vacunación , Vacunas de Productos Inactivados/administración & dosificación , 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.
Asunto(s)
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
Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.
Asunto(s)
Epítopos/inmunología , Vectores Genéticos , Muromegalovirus , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Epítopos/genética , Femenino , Antígeno de Histocompatibilidad H-2D/genética , Antígeno de Histocompatibilidad H-2D/inmunología , Interleucinas/genética , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/genética , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patologíaRESUMEN
CD8(+) T cell memory inflation, first described in murine CMV (MCMV) infection, is characterized by the accumulation of high-frequency, functional Ag-specific CD8(+) T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of Ag is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus's low-level persistence and stochastic reactivation. We developed a new model of memory inflation based on a ß-galactosidase (ßgal)-recombinant adenovirus vector. After i.v. administration in C57BL/6 mice, we observed marked memory inflation in the ßgal96 epitope, whereas a second epitope, ßgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype, and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC class II. As in MCMV, only the inflating epitope showed immunoproteasome independence. These data define a new model for memory inflation, which is fully replication independent, internally controlled, and reproduces the key immunologic features of the CD8(+) T cell response. This model provides insight into the mechanisms responsible for memory inflation and, because it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Memoria Inmunológica , Trastornos de la Memoria/inmunología , Trastornos de la Memoria/virología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Linfocitos T CD8-positivos/patología , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Memoria Inmunológica/genética , Masculino , Trastornos de la Memoria/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Muromegalovirus/genética , Muromegalovirus/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Monoclonal antibodies (mAbs) can be used to complement immunization for the therapy of influenza virus infection. We have established the pig, a natural large animal host for influenza A, with many physiological, immunological, and anatomical similarities to humans, as an appropriate model for testing mAbs. We have evaluated the protective efficacy of the strongly neutralizing human anti-hemagglutinin mAb, 2-12C in the pig influenza model. Intravenous administration of recombinant 2-12C reduced virus load and lung pathology, however, it did not prevent virus nasal shedding and, consequently, transmission. This may be because the pigs were directly infected intranasally with a high dose of the H1N1pdm09 virus. To address this, we developed a contact challenge model in which the animals were given 2-12C and one day later co-housed with donor pigs previously infected intra-nasally with H1N1pdm09. 2-12C pre-treatment completely prevented infection. We also administered a lower dose of 2-12C by aerosol to the respiratory tract, but this did not prevent shedding in the direct challenge model, although it abolished lung infection. We propose that the direct contact challenge model of pig influenza may be useful for evaluating candidate mAbs and emerging delivery platforms prior to clinical trials.
Asunto(s)
Gripe Humana , Infecciones por Orthomyxoviridae , Porcinos , Humanos , Animales , Anticuerpos Monoclonales , Aerosoles y Gotitas Respiratorias , HemaglutininasRESUMEN
Cattle possess three IgG subclasses. However, the key immune functions, including complement and NK cell activation, and enhancement of phagocytosis, are not fully described for bovine IgG1, 2 and 3. We produced chimeric monoclonal antibodies (mAbs) consisting of a defined variable region linked to the constant regions of bovine IgG1, 2 and 3, and expressed His-tagged soluble recombinant bovine Fc gamma receptors (FcγRs) IA (CD64), IIA (CD32A), III (CD16) and Fcγ2R. Functional assays using bovinized mAbs were developed. IgG1 and IgG3, but not IgG2, activated complement-dependent cytotoxicity. Only IgG1 could activate cattle NK cells to mobilize CD107a after antigen crosslinking, a surrogate assay for antibody-dependent cell cytotoxicity. Both IgG1 and IgG2 could trigger monocyte-derived macrophages to phagocytose fluorescently labelled antigen-expressing target cells. IgG3 induced only weak antibody-dependent cellular phagocytosis (ADCP). By contrast, monocytes only exhibited strong ADCP when triggered by IgG2. IgG1 bound most strongly to recombinant FcγRs IA, IIA and III, with weaker binding by IgG3 and none by IgG2, which bound exclusively to Fcγ2R. Immune complexes containing IgG1, 2 and 3 bound differentially to leukocyte subsets, with IgG2 binding strongly to neutrophils and monocytes and all subclasses binding platelets. Differential expression of the FcγRs on leukocyte subsets was demonstrated by surface staining and/or RT-qPCR of sorted cells, e.g., Fcγ2R mRNA was expressed in monocytes/macrophages, neutrophils, and platelets, potentially explaining their strong interactions with IgG2, and FcγRIII was expressed on NK cells, presumably mediating IgG1-dependent NK cell activation. These data reveal differences in bovine IgG subclass functionality, which do not correspond to those described in humans, mice or pigs, which is relevant to the study of these IgG subclasses in vaccine and therapeutic antibody development.
Asunto(s)
Inmunoglobulina G , Receptores de IgG , Humanos , Bovinos , Animales , Ratones , Porcinos , Factores Inmunológicos , Macrófagos , Fagocitosis , Anticuerpos Monoclonales , AntígenosRESUMEN
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.