Shelley Rhodes

Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis.

ABSTRACT Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.

Cellular immune responses induced in cattle by heterologous prime–boost vaccination using recombinant viruses and bacille Calmette–Guérin

The development of novel vaccine strategies to replace or supplement bacille Calmette–Guérin (BCG) is urgently required. Here we study, in cattle, the use of heterologous prime–boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A). Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5). Vaccine‐induced levels of cellular immunity were assessed by determining interferon‐γ (IFN‐γ) responses in vitro. Prime–boost protocols, using recombinant MVA and BCG in combination (groups 1–3), resulted in significantly higher frequencies of Ag85‐specific IFN‐γ‐secreting cells than the two viral vectors used in combination (P=0·0055), or BCG used alone (groups 2 and 3, P=0·04). The T‐cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization. In a second experiment, the effects of BCG\MVA85A heterologous prime–boost vaccination were compared with BCG\BCG homologous revaccination. The results suggested a higher Ag85A‐specific response with a wider T‐cell repertoire in the MVA85A‐boosted calves than in the BCG\BCG‐vaccinated calves. In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime–boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis.

Minimum Infective Dose of Mycobacterium bovis in Cattle

ABSTRACT The aim of this work was to determine the minimum infective dose of Mycobacterium bovis necessary to stimulate specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by the intratracheal route with 1,000, 100, 10, or 1 CFU of M. bovis. Specific immune responses (gamma interferon [IFN-γ] and interleukin-4 [IL-4] responses) to mycobacterial antigens were monitored throughout the study, and the responses to the tuberculin skin test were assessed at two times. Rigorous post mortem examinations were performed to determine the presence of pathology, and samples were taken for microbiological and histopathological confirmation of M. bovis infection. One-half of the animals infected with 1 CFU of M. bovis developed pulmonary pathology typical of bovine tuberculosis. No differences in the severity of pathology were observed for the different M. bovis doses. All animals that developed pathology were skin test positive and produced specific IFN-γ and IL-4 responses. No differences in the sizes of the skin test reactions, the times taken to achieve a positive IFN-γ result, or the levels of the IFN-γ and IL-4 responses were observed for the different M. bovis doses, suggesting that diagnostic assays (tuberculin skin test and IFN-γ test) can detect cattle soon after M. bovis infection regardless of the dose. This information should be useful in modeling the dynamics of bovine tuberculosis in cattle and in assessing the risk of transmission.

Antigen Recognition and Immunomodulation by γδ T Cells in Bovine Tuberculosis

This report describes the in vitro proliferative responses of peripheral blood γδ T cells to defined mycobacterial protein Ags and the immunomodulatory effect of γδ T cells in cattle infected with Mycobacterium bovis. γδ T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating γδ T cell cultures produced enhanced levels of IFN-γ and TGF-β, but not IL-2 in response to the more immunodominant mycobacterial Ags. Depletion of γδ T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of γδ T cells upon other (αβ) T cell responses. Because γδ T cells constitute a major T cell population in the peripheral blood of cattle, the activities of γδ T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.

Repeat tuberculin skin testing leads to desensitisation in naturally infected tuberculous cattle which is associated with elevated interleukin-10 and decreased interleukin-1 beta responses

The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-γ (IFN-γ) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1β (IL-1β) whilst confirming stable IFN-γ and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1β and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-γ responses.

Distinct Response Kinetics of Gamma Interferon and Interleukin-4 in Bovine Tuberculosis

ABSTRACT This study shows that gamma interferon (IFN-γ) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected with Mycobacterium bovis. The different kinetics of the IFN-γ and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN-γ was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose of M. bovis which did not result in the development of lesions, positive DTH skin test, or substantial IFN-γ responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors showed increased IFN-γ and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of theM. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematode Onchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.

Antigen Specificity in Experimental Bovine Tuberculosis

ABSTRACT This report describes the kinetics of T-cell responses to a panel of mycobacterial antigens (PPD-M, PPD-A, ESAT-6, Ag85, 38kD, MPB64, MPB70, MPB83, hsp16.1, hsp65, and hsp70) following experimental infection of cattle with Mycobacterium bovis. Increased antigen-specific lymphocyte proliferation, gamma interferon, and interleukin-2 responses were observed in all calves following infection. Positive lymphocyte proliferation and cytokine responses to PPD-M and ESAT-6 were observed throughout the infection period studied. In contrast, responses to all other antigens were more variable and were not constantly present, suggesting that antigen cocktails rather than individual antigens should be used for immunodiagnosis. The detection of cytokine responses in the absence of lymphocyte proliferation, particularly during the early stages of infection, suggests a role for antigen-specific cytokine readout systems in the early identification of M. bovis infection in cattle.

Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4(+) T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.

Bovine tuberculosis: immune responses in the peripheral blood and at the site of active disease.

This report describes a comparison of immune responses in the peripheral blood and at the site of active disease in cattle 20 weeks after experimental infection with Mycobacterium bovis. Lymphocyte proliferation, and the production of interferon‐γ (IFN‐γ) and interleukin (IL)‐2 were measured in response to tuberculin and a number of mycobacterial antigens, including ESAT‐6, MPB64, MPB70, MPB83, hsp 16.1, hsp 65, hsp 70 and the 38 000 MW lipoprotein antigen. The level of transforming growth factor‐β (TGF‐β) was measured following stimulation of cells with tuberculin. Our results suggest little difference in the responses of peripheral blood and lymph node cells to most of the antigens used. However, tuberculin purified protein derivative (PPD) and ESAT‐6 elicited stronger responses in the peripheral blood compared with lymph node cells. Investigation of the responding T‐cell subpopulations in the peripheral blood showed that both CD4+ and, to a lesser extent, γδ T‐cell receptor‐positive (TCR+) T cells contributed to these responses. This is the first report to compare peripheral and local immune responses in bovine tuberculosis. Unlike cases of human tuberculosis where immune activity at the site of disease and anergy in the peripheral blood have been reported, our results suggest that for bovine tuberculosis immune responses occurring in the peripheral blood reflect those at the site of disease.

Is ‘timing’ important for cytokine polarization?

Abstract A comparison of the longitudinal cytokine responses of cattle to infection with intracellular Mycobacterium bovis and extracellular Onchocerca ochengi illustrates the development of reciprocal interferon-γ and interleukin-4 responses, which result ultimately in an infection-induced type-1 or type-2 polarization, respectively. These kinetic studies of natural host–pathogen relationships show that the cytokine responses to infection fluctuate over time, resulting in periods of polarization and nonpolarization before the establishment of a chronic infection. Here, we discuss our data from cattle in the light of the current understanding of cytokine polarization towards infection in mouse models and humans.