Elsevier

Vaccine

Volume 24, Issue 6, 6 February 2006, Pages 835-845
Vaccine

Factors influencing the protective efficacy of a BCG homologous prime-boost vaccination regime against tuberculosis

https://doi.org/10.1016/j.vaccine.2005.07.033Get rights and content

Abstract

Tuberculosis due to Mycobacterium spp. continues to represent a major threat to human and animal health, prompting the search for effective vaccines. We have previously reported a sequential prime and boost homologous vaccination regime, using live avirulent M. bovis bacille Calmette-Guerin (BCG) strain 1173P2, that can provide significant protection to red deer (Cervus elaphus) against virulent M. bovis infection. Here, we have investigated the influence of varying the time-periods during and following the vaccination regime on the subsequent outcome of disease following post-vaccination pathogen challenge. Deer vaccinated using the standard regime of BCG-prime (week 0) and BCG-boost (week 8) followed by M. bovis challenge (week 14–16) were highly protected, showing significant reductions in the incidence of M. bovis infection and tuberculous lesions, as well as reduced pathogen burdens in sentinel lymphatic tissues. Decreasing the time-period between primary and secondary immunisations from 8 to 4 weeks had no significant impact on the protective efficacy afforded by BCG vaccination, while increasing this period to 43 weeks largely ablated protection. Increasing the time-period between secondary immunisation and M. bovis challenge from 6 to 26 or 52 weeks also had no significant impact on protection, suggesting that an appropriately timed BCG prime-boost vaccination regime can establish long-lasting protective immunological memory in deer. Finally, increasing the time-period between virulent M. bovis challenge and the subsequent post-mortem examination of disease outcome indicated that – once vaccinated by the standard prime-boost regime – deer remain refractory to disease if challenged and maintained for up to 52 weeks, suggesting that vaccinated animals harbouring low numbers of virulent M. bovis organisms do not succumb to disease activation over time. These findings are discussed in relation to concurrent measurements of in vivo and ex vivo immunological markers of disease and protection, as well as the wider implications of a standardised vaccine regimen for practical use in animal health.

Introduction

Tuberculosis (TB), due to virulent species of Mycobacterium, continues to represent a major threat to human and animal health on a global scale. Tuberculosis in humans, predominantly due to M. tuberculosis, represents an on-going health problem in developed and developing countries alike, with an estimated annual mortality rate of over two million people [1]. Tuberculosis in farmed animals, caused by M. bovis infection, represents a problem in developing countries where disease may cycle via horizontal transmission between and among several animal species, and also in developed countries where on-going transmission to livestock is assured by the persistence of TB among wildlife reservoirs of the disease [2]. Research in the last 20 years has focussed on the development and testing of protective vaccines against TB, based on sub-unit, live conventional or recombinant micro-organism vaccines [3], [4]; delivered via mucosal or parenteral routes [5], [6], [7]; and administered in single-dose or prime-boost regimes [8], [9]. In all of these cases, one of the most effective vaccination agents remains parenterally administered inocula of the avirulent M. bovis strain bacille Calmette-Guerin (BCG), particularly when this is given in a homologous or heterologous prime-boost regime [10], [11], [12], [13].

Several ruminant species are highly susceptible to TB as a result of infection with virulent strains of M. bovis. Parenterally administered M. bovis BCG vaccine has been shown to confer significant protection to cattle [14], [15] and deer [16], [17] against experimental challenge infection, in terms of reduced tissue burdens of pathogenic bacteria, a lower incidence of infection and disease, and/or reduced disease severity. In deer, vaccination using live (but not killed) BCG micro-organisms confers protection against virulent M. bovis challenge [17], and it has been shown that a homologous prime-boost regime – administering two sequential sub-cutaneous doses between 5 × 104 and 5 × 107 BCG at 8 week intervals apart – significantly reduces tissue pathogen burdens and the incidence of infection and TB disease [17], [18], [19]. However, despite the potential of the BCG vaccine as a prophylactic measure in animal health, several issues and concerns remain. First, the duration of anti-tuberculous immunological memory is uncertain, which is an important issue for livestock production where animals may need to be protected from birth over several years. Moreover, recent evidence from murine models of TB suggests that the longevity of BCG vaccine-mediated protection may be limited, particularly in the absence of boosting [20] or in the absence of continued BCG bacterial survival in vivo [21]. Secondly, when considering a prime-boost regime, the optimum interval between primary and secondary vaccination is unknown. This is important, since evidence from human studies suggests that a long period between BCG primary vaccination and subsequent boosting may compromise vaccine efficacy. Thirdly, while BCG vaccination can invoke sterilising anti-TB immunity in deer [18], [19], it is known that some vaccinates may still harbour low numbers of virulent M. bovis bacilli up to 21 weeks post-challenge [18]; the fate of these animals in the long-term is uncertain. Do they maintain a capacity to limit disease, eliminate infection entirely or progress to disseminated infection and TB? Finally, the interference of BCG vaccination with current TB immunodiagnostic tests is uncertain, but highly likely given that vaccine and virulent strains of M. bovis are antigenically similar [22] and that BCG vaccination has been reported to confound the interpretation of immunodiagnostic tests in other ruminant species [23].

The present study has aimed to address these issues by conducting a targeted series of homologous BCG prime-boost vaccination/M. bovis challenge studies in red deer (Cervus elaphus). In particular, the study focussed on the ability of homologous prime-boost vaccination to extend the period of immunological memory beyond the currently recognised period of 6–8 weeks post-vaccination, and sought to determine whether shortening or lengthening the time interval between priming and boosting doses could influence protective efficacy. Further, the phenomenon of low-level M. bovis infection in BCG-vaccinated deer was investigated to determine whether disease activation, resolution or status quo would occur after an extended period post-challenge. Finally, the predictive potential of current in vivo and ex vivo immunodiagnostic tests was examined to identify M. bovis infection and/or disease following experimental pathogen challenge, with a key focus on determining whether any or all such tests are likely to be compromised by vaccination. For these studies, the low-dose intra-tonsilar challenge model of M. bovis infection in deer, which has been researched thoroughly over the past decade [17], [18], [24], [25], was used. This produces high and reproducible rates of TB in deer, along with pathology consistent with that observed in field cases of naturally acquired TB in wild and farmed deer.

Section snippets

Animals

Healthy outbred farmed red deer (Cervus elaphus) were obtained from herds that had been certified TB-free for at least 10 years prior to this study. Female weaners were used for all experimental purposes (average age 4 months, average weight 50 kg, range 41–60 kg at commencement of study). Prior to experimentation, animals were screened for reactivity to mycobacterial antigens using the standard BTB diagnostic test [26]; animals showing a high degree of pre-existing lymphocyte and/or antibody

Results

In the present study, three major experiments were conducted, based on the prime-boost-challenge model that we have previously described for determining the protective efficacy of BCG vaccination in deer [17], [18], [19], [25]. In essence, these experiments were designed to address issues of periodicity during the vaccination regime, memory following the vaccination regime, and persistence of stable protection following challenge.

Discussion

In the present study, we have investigated the effects of varying the times during and following a BCG homologous prime-boost vaccination regime on the consequent protection against virulent M. bovis challenge. As reported in previous studies [17], [18], [19], [24], [25], the low-dose intra-tonsilar challenge route here provided a highly reproducible disease model, with 41/45 (91%) of non-vaccinated control animals (across all three main experiments) returning post-challenge M. bovis

Acknowledgements

We would like to thank Dr. Marina Gheorghiu and the Institut Pasteur, Paris, France, for providing the seedstock of the BCG 1173P2. We also acknowledge the staff of the Disease Research Laboratory for technical assistance. Technical support from Dr. Geoffrey de Lisle, AgResearch, Wallaceville, for primary culture of M. bovis and typing of the mycobacterial isolates, is gratefully acknowledged. Histopathological analysis of TB lesions was carried out by Dr. Hugh Montgomery. This project was

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