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Hydrophobic Mycobacterial Antigens Elicit Polyfunctional T Cells in Mycobacterium bovis Immunized Cattle: Association With Protection Against Challenge?

ABSTRACT: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic disease of cattle with a detrimental impact on food quality and production. Research on bTB vaccines has predominantly been focused on proteinaceous antigens. However, mycobacteria have a thick and intricate lipid outer layer and lipids as well as lipopeptides are important for immune-evasion and virulence. In humans, lipid extracts of M. tuberculosis have been shown to elicit immune responses effective against M. tuberculosis in vitro. Chloroform-methanol extraction (CME) was applied to M. bovis BCG to obtain a hydrophobic antigen extract (CMEbcg) containing lipids and lipopeptides. CMEbcg stimulated IFN-?⁺IL-2⁺ and IL-17A⁺IL-22⁺ polyfunctional T cells and elicited T cell responses with a Th1 and Th17 cytokine release profile in both M. bovis BCG vaccinated and M. bovis challenged calves. Lipopeptides were shown to be the immunodominant antigens in CMEbcg, stimulating CD4 T cells via MHC class II. CMEbcg expanded T cells killed CMEbcg loaded monocytes and the CMEbcg-specific CD3 T cell proliferative response following M. bovis BCG vaccination was the best predictor for reduced pathology following challenge with M. bovis. Although the high predictive value of CMEbcg-specific immune responses does not confirm a causal relationship with protection against M. bovis challenge, when taking into account the in vitro antimycobacterial phenotype of CMEbcg-specific T cells (e.g. Th1/Th17 cytokine profile), it is indicative that CMEbcg-specific immune responses could play a functional role in immunity against M. bovis. Based on these findings we conclude that lipopeptides of M. bovis are potential novel subunit vaccine candidates and that further studies into the functional characterization of lipopeptide-specific immune responses together with their role in protection against bovine tuberculosis are warranted.

SUBMITTER: Benedictus L

PROVIDER: S-EPMC7688591 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Hydrophobic Mycobacterial Antigens Elicit Polyfunctional T Cells in Mycobacterium bovis Immunized Cattle: Association With Protection Against Challenge?

Benedictus Lindert L Steinbach Sabine S Holder Thomas T Bakker Douwe D Vrettou Christina C Morrison W Ivan WI Vordermeier Martin M Connelley Timothy T

Frontiers in immunology 20201112

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic disease of cattle with a detrimental impact on food quality and production. Research on bTB vaccines has predominantly been focused on proteinaceous antigens. However, mycobacteria have a thick and intricate lipid outer layer and lipids as well as lipopeptides are important for immune-evasion and virulence. In humans, lipid extracts of M. tuberculosis have been shown to elicit immune responses effective against ...[more]

PMID: 33281817

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Project description:Mycobacterium bovis (M. bovis) causes bovine tuberculosis (bTB). The challenges in controlling and eradicating this zoonotic disease are compounded by our incomplete understanding of the host immune response. In this study, we used high-throughput bulk RNA sequencing (RNA-seq) to characterise the response profiles of γδ T cells to antigenic stimulation using purified protein derivate from M. bovis (PPDb). γδ T cells are a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. These cells are usually classified based on the expression of a pathogen-recognition receptor, Workshop Cluster 1 (WC1), into two main subsets: WC1.1+ and WC1.2+. Previous studies have identified a preferential transcriptomic response in WC1.1+ cells during natural bTB infection, suggesting a subset-specific response to mycobacterial antigens. This follow on study tested the hypothesis that a subset specific response would also be apparent from γδ T cells from infected cattle after repeat stimulation. Peripheral blood was collected from Holstein-Friesian cattle naturally infected with M. bovis, confirmed by a single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA and stimulated with 10 μg/ml PPDb for 6 hours. After whole blood stimulation, WC1.1+ and WC1.2+ γδ T cell subsets were isolated using magnetic cell sorting (n = 5 per group). High-quality RNA was extracted from each purified lymphocyte subset (WC1.1+ and WC1.2+) to generate transcriptomes using bulk RNA sequencing, resulting in 20 RNA-seq libraries. Transcriptomic analysis revealed 111 differentially expressed genes (DEGs) common to both WC1.1+ and WC1.2+ γδ T cell compartments, including upregulation of IL1A, IL1B, IL6, IL17A, IL17F, and IFNG genes (FDR-Padj. < 0.1). Interestingly, the WC1.2+ cells showed upregulation of IL10, CCL22, and GZMA (log2FC ≥ 1.5, and FDR-Padj. < 0.1). In conclusion, while WC1.1+ and WC1.2+ γδ T cells exhibit a conserved inflammatory response to PPDb, differences in anti-inflammatory and antimicrobial gene expression between these cell subsets provide new insights into their effector functions in response to mycobacterial antigens.

Project description:BackgroundBabesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis.ResultsThe B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas.ConclusionsThe data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Project description:Coccidiosis is a parasitic disease in the intestine caused by the genus Eimeria that poses a substantial economic threat to the broiler breeding industry. The misuse of chemoprophylaxis and live oocyst vaccines has a negative impact on chicken reproductivity. Therefore, there is a pressing need to develop safe, convenient, and effective vaccines. Lactic acid bacteria can be used as a means to deliver mucosal vaccines against intestinal pathogens, which is a promising strategy. In this study, a recombinant Lactobacillus plantarum (L. plantarum) with surface-expressed antigens constructed from the fusion of Eimeria tenella (E. tenella) antigen profilin and the Salmonella enterica serovar Typhimurium flagellin protein FliC was created. After oral immunization with the recombinant L. plantarum, T-cell differentiation was analyzed by flow cytometry, and specific antibody levels were determined via indirect ELISA. Oocyst shedding, body weight, and cecum lesions were assessed as measures of protective immunity after challenge with E. tenella. The results of this study demonstrate the effectiveness of recombinant L. plantarum as an immunization agent for chickens. Specific IgA titers in the intestine and specific IgG antibody titers in the serum were significantly higher in chickens immunized with recombinant L. plantarum (P < 0.001). Additionally, the levels of IL-2 (P < 0.05) and IFN-γ (P < 0.01) in the serum were markedly increased. Recombinant L. plantarum induced T-cell differentiation, resulting in a higher proportion of CD4+ and CD8+ T cells in splenocytes (P < 0.001). Fecal oocyst shedding in the immunized group was significantly reduced (P < 0.001). Additionally, recombinant L. plantarum significantly relieved pathological damage in the cecum, as evidenced by lesion scores (P < 0.01) and histopathological cecum sections. In conclusion, the present study provides evidence to support the possibility of using L. plantarum as a promising carrier for the delivery of protective antigens to effectively protect chickens against coccidiosis.

Dataset Information

Hydrophobic Mycobacterial Antigens Elicit Polyfunctional T Cells in Mycobacterium bovis Immunized Cattle: Association With Protection Against Challenge?

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Hydrophobic Mycobacterial Antigens Elicit Polyfunctional T Cells in <i>Mycobacterium bovis</i> Immunized Cattle: Association With Protection Against Challenge?

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