Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

International Atomic Energy Agency

The establishment of a panel of immune markers is of paramount importance to understand the different transcription patterns of infectious diseases in livestock. The array of commercially available immunological assays for cattle and sheep is currently limited, due to the lack of antibodies for these species. Even though SYBR Green based real time quantitative PCR (qPCR) is the most commonly used method to study cytokine transcription in ruminants, a lack of standardization impairs its implementation in the study of different ruminant diseases. In order to obtain reliable qPCR results, several variables need to be considered: choice of reference genes for optimal normalization, variation of annealing temperature among primer sets, and assay specificity and sensitivity. In this study, we developed and validated a panel of immune markers in bovine and ovine samples using SYBR Green based qPCR in a cost-effective way with multiple primer sets optimised to amplify at a common thermal cycling temperature. Twenty primer sets were designed to quantify immune markers (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, TNF-α, IFN-γ, IFN-α, Ki-67, NFkB-65, TLR-3, TLR-4, TLR-8 and Rig-1) in ovine and bovine templates. For optimal normalization and selection of suitable reference genes, primer sets that measure the transcription of five reference genes were also included in the panel. The amplification efficiency, linearity and specificity was validated for all target genes. Optimal amplification conditions were achieved in both ovine and bovine samples for all gene targets, with the exception of Ki67. Relative quantification studies were performed on ovine and bovine mRNA obtained from sheep peripheral blood mononuclear cells (PBMCs) stimulated with three different treatments (PMA/Ionomycin, Concanavalin A (Con A) and pokeweed mitogen (PWM)). Pokeweed and ConA efficiently induced gene transcription of most of the targeted genes, while PMA/Ionomycin showed a weaker induction. Finally, we further assessed usability of our panel by running it on bovine monocyte derived dendritic cells (MoDCs) stimulated with different vaccines. Results confirmed the induction of a specific pro-inflammatory gene transcription pattern by rabies vaccine, which resembles the one occurring during viral infection. Altogether, we validated the efficiency and usability of an extended real-time PCR panel that gives the possibility to rapidly measure a broad spectrum of ovine and bovine immune markers by using a single set of reagents and protocol thus representing a valid and cost-effective tool for research purposes.

Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep.

Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC .

Avian pathogenic Escherichia coli (APEC) causes colibacillosis with different clinical manifestations. The disease is associated with compromised animal welfare and results in substantial economic losses in poultry production worldwide. So far, immunological mechanisms of protection against colibacillosis are not comprehensively resolved. Therefore, the present study aimed to use an ex vivo model applying chicken mononuclear cells stimulated by live and inactivated APEC. For this purpose, an 8-color flow cytometry panel was set up to target viable chicken immune cells including CD45, CD8α, CD4, TCR-γδ, Bu-1 cells and monocytes/macrophages along with the cytokines interferon gamma (IFN-γ) or interleukin 17A (IL-17A). The 8-color flow cytometry panel was applied to investigate the effect of live and two different types of inactivated APEC (formalin-killed APEC and irradiated APEC) on the cellular immune response. For that, mononuclear cells from spleen, lung and blood of 10-week-old specific pathogen-free layer birds were isolated and stimulated with live, irradiated or killed APEC. Intracellular cytokine staining and RT-qPCR assays were applied for the detection of IFN-γ and IL-17A protein level, as well as at mRNA level for spleenocytes. Ex vivo stimulation of isolated splenocytes, lung and peripheral blood mononuclear cells (PBMCs) from chickens with live, irradiated or killed APEC showed an increasing number of IFN-γ and IL-17A producing cells at protein and mRNA level. Phenotyping of the cells from blood and organs revealed that IFN-γ and IL-17A were mainly produced by CD8α, TCR-γδ T cells as well as CD4 T cells following stimulation with APEC. Expression level of cytokines were very similar following stimulation with live and irradiated APEC but lower when killed APEC were applied. Consequently, in the present study, an ex vivo model using mononuclear cells of chickens was applied to investigate the cellular immune response against APEC. The results suggest the relevance of IFN-γ and IL-17A production in different immune cells following APEC infection in chickens which needs to be further investigated in APEC primed birds.

Production of interferon gamma and interleukin 17A in chicken T-cell subpopulations hallmarks the stimulation with live, irradiated and killed avian pathogenic Escherichia coli.

African swine fever (ASF) is among the most devastating viral diseases of pigs and wild boar worldwide. In recent years, the disease has spread alarmingly. Despite intensive research activities, a commercialized vaccine is still not available, and efficacious live attenuated vaccine candidates raise safety concerns. From a safety perspective, inactivated preparations would be most favourable. However, both historical and more recent trials with chemical inactivation did not show an appreciable protective effect. Under the assumption that the integrity of viral particles could enhance presentation of antigens, we used gamma irradiation for inactivation. To this means, gamma irradiated ASFV "Estonia 2014" was adjuvanted with either Polygen™ or Montanide™ ISA 201 VG, respectively. Subsequently, five weaner pigs per preparation were immunized twice with a three-week interval. Six weeks after the first immunization, all animals were challenged with the highly virulent ASFV strain "Armenia 2008". Although ASFV p72-specific IgG antibodies were detectable in all vaccinated animals prior challenge, no protection could be observed. All animals developed an acute lethal course of ASF and had to be euthanized at a moderate humane endpoint within six days. Indeed, the vaccinated pigs showed even higher clinical scores and a higher inner body temperature than the control group. However, significantly lower viral loads were detectable in spleen and liver of immunized animals at the time point of euthanasia. This phenomenon suggests an immune mediated disease enhancement that needs further investigation.

Vaccination With a Gamma Irradiation-Inactivated African Swine Fever Virus Is Safe But Does Not Protect Against a Challenge.

H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection.

Protective Efficacy of H9N2 Avian Influenza Vaccines Inactivated by Ionizing Radiation Methods Administered by the Parenteral or Mucosal Routes.

Escherichia coli causes colibacillosis in chickens, which has severe economic and public health consequences. For the first time, we investigated the efficacy of gamma-irradiated E. coli to prevent colibacillosis in chickens considering different strains and application routes. Electron microscopy, alamarBlue assay and matrix assisted laser desorption/ionization time-of- flight mass spectrometry showed that the cellular structure, metabolic activity and protein profiles of irradiated and non-treated E. coli PA14/17480/5-ovary (serotype O1:K1) were similar. Subsequently, three animal trials were performed using the irradiated E. coli and clinical signs, pathological lesions and bacterial colonization in systemic organs were assessed. In the first animal trial, the irradiated E. coli PA14/17480/5-ovary administered at 7 and 21 days of age via aerosol and oculonasal routes, respectively, prevented the occurrence of lesions and systemic bacterial spread after homologous challenge, as efficient as live infection or formalin-killed cells. In the second trial, a single aerosol application of the same irradiated strain in one-day old chickens was efficacious against challenges with a homologous or a heterologous strain (undefined serotype). The aerosol application elicited better protection as compared to oculonasal route. Finally, in the third trial, efficacy against E. coli PA15/19103-3 (serotype O78:K80) was shown. Additionally, previous results of homologous protection were reconfirmed. The irradiated PA15/19103-3 strain, which also showed lower metabolic activity, was less preferred even for the homologous protection, underlining the importance of the vaccine strain. In all the trials, the irradiated E. coli did not provoke antibody response indicating the importance of innate or cell mediated immunity for protection. In conclusion, this proof-of-concept study showed that the non-adjuvanted single aerosol application of irradiated "killed but metabolically active" E. coli provided promising results to prevent colibacillosis in chickens at an early stage of life. The findings open new avenues for vaccine production with E. coli in chickens using irradiation technology.

Richard Thiga Kangethe

International Atomic Energy Agency

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells