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Bovine tuberculosis (BTB), caused by Mycobacterium bovis, is a zoonotic disease and was the cause of approximately 6% of total human deaths due to BTB in the 1930s and 1940s (5, 6). The introduction of pasteurization of milk in developed countries in the 1930s dramatically reduced the transmission from cattle to humans, although BTB is still a major human health problem in developing countries. Compulsory BTB eradication programs were introduced in many countries based on the slaughter of infected cattle detected by the single intradermal comparative tuberculin skin test. The implementation of this control strategy resulted in the dramatic reduction of BTB in Great Britain. However, possibly due to a wildlife reservoir, the incidence of TB in cattle caused by M. bovis has exponentially increased over the last two decades in the British national herd. This increase constitutes a significant economic and potential public health problem (17). To control this zoonotic disease, better and more specific diagnostic reagents, as well as effective vaccines for cattle, are urgently needed. The diagnosis of BTB in cattle is at present almost exclusively based on the use of tuberculin purified protein derivative (PPD) in skin tests. In addition, a blood-based test measuring tuberculin-induced production of gamma interferon (IFN-γ) is now also in limited field use (34). The specificity of these tuberculin-based tests is limited due to the undefined and cross-reactive nature of PPD. Furthermore, the specificity of tuberculin-based reagents is also compromised following vaccination with the human TB vaccine M. bovis BCG (16). Diagnostic reagents allowing the differential diagnosis of M. bovis-infected and -vaccinated animals are therefore a precondition for the development of novel TB vaccines in cattle (17). The specificity of diagnostic reagents can be improved by using antigens that are highly expressed by M. bovis yet whose genes have been deleted from the genome of BCG. The antigens ESAT-6 and CFP10, which are encoded in the RD1 region of M. bovis-Mycobacterium tuberculosis—which is deleted in all strains of BCG (2, 19)—have shown particular promise as such improved diagnostic reagents when used as recombinant proteins or synthetic peptides in the IFN-γ test (3, 4, 28, 31). Such antigens not only allowed the differential diagnosis of infected and BCG-vaccinated cattle but also improved the specificity of the test per se compared to tuberculin PPD in the absence of vaccination (22, 23, 31). An attractive recent approach to effectively delivering proteins to the immune system is through nonreplicating protein vectors such as bacterial toxins (20). The Bordetella pertussis adenylate cyclase (CyaA) is such a vector system that has shown promise in mouse models (21, 25, 26). Indeed, we have recently demonstrated that CD11b/CD18, a member of the β2-integrin family, is a specific cell receptor for this toxin (14). CD11b/CD18, also known as complement type 3 receptor or MAC-1, is expressed on macrophages, neutrophils, dendritic cells, and NK cells. Thus, the cellular specificity of CyaA allows its selective targeting to CD11c+ CD11bhigh dendritic cells in vivo (13). Peptide and small proteins can be inserted into this protein and expressed as fusion proteins (11, 12, 21, 25, 27). CyaA facilitates direct translocation of these inserted antigens across the plasma membrane of target cells (15). Importantly, it has been shown that CyaA vaccination can not only induce major histocompatibility complex (MHC) class I-restricted CD8+-T-cell responses (8, 10, 12, 21, 25, 27) but also result in the presentation of CD4+-T-cell epitopes restricted by MHC class II (8, 18). Thus, CyaA fusion proteins that contain mycobacterial antigens could constitute not only candidates for subunit vaccines but also diagnostic antigens, particularly if they will be recognized in cattle more effectively than conventional recombinant proteins, thereby enhancing sensitivity, or are recognized at lower protein concentrations. The latter consideration could have major cost benefits because this could significantly reduce the amount of antigen that would have to be produced to implement testing. Consequently, the experiments conducted in this study have been performed to determine whether CyaA-based recombinant proteins fused with either ESAT-6 or CFP10 are recognized by bovine T cells more efficiently than the corresponding nonfusion proteins and to establish the mechanisms of this improved recognition.

Cattle were given Mycobacterium bovis bacillus Calmette-Guerin (BCG) in a lipid-based formulation via the oral route and tested for immune responses and protection against a challenge with virulent M. bovis . Calves were vaccinated by orally administering a pellet containing 10 8 colony forming units (CFU) of BCG, or 10 pellets containing a total of 10 9 CFU of BCG, whereas positive controls were injected subcutaneously with 10 6 CFU of BCG. All of the subcutaneously vaccinated calves produced positive responses in the caudal fold tuberculin skin test at 8 weeks after vaccination, whereas only 3/9 of the low dose and 6/10 of the high dose orally-vaccinated animals produced positive reactions. None of the animals produced positive reactions to the mycobacterial antigens, ESAT-6 and CFP10 in the interferon-γ (IFN-γ) test and only a total of four of the BCG-vaccinated animals produced positive responses in either the standard IFN-γ or comparative cervical skin test. Oral administration of 10 pellets of lipid-formulated BCG to cattle induced a significant level of protection against bovine tuberculosis compared to that observed in non-vaccinated animals and this level was similar to that seen in the BCG subcutaneously vaccinated animals. Oral vaccination of BCG in a lipid-formulation to calves was shown to induce some positive tuberculin skin test reactions, but could also induce protection against bovine tuberculosis.

The identification of tuberculosis vaccines and vaccination strategies, which in small animal models appear to be more effective than BCG, offer some exciting possibilities for control of human tuberculosis in the future. However, some major problems remain including selecting which vaccines should go into human trials and the length of time it will take for testing these vaccines in humans. The cattle model is well suited for the secondary screening of tuberculosis vaccines as there is a strong similarity between the disease in cattle and humans and outbred animals are used. Moreover, there are many similarities in the results from field trials of BCG in both cattle and humans, with BCG often failing to protect when the trials are extended over a number of years. In addition, calves, like human infants, are immunocompetent at birth. Recent studies in calves have shown that BCG vaccination of calves within hours of birth is highly effective in protecting animals against bovine tuberculosis, but BCG revaccination at 6 weeks of age is contraindicated. Prime boost vaccination strategies using BCG and DNA vaccines have provided evidence that these combinations may give better protection in calves than either vaccine alone. Based on antigens whose genes are absent from the BCG genome, advances have also been made to develop diagnostic reagents distinguishing infected and vaccinated animals (differential diagnosis). The cattle model has been particularly useful in prioritizing such antigens for testing in humans. Finally, there is an urgent need to identify an immunological correlate of protection against tuberculosis. The cattle model can be particularly helpful in this area as it is relatively easy to collect large volumes of blood from calves at intervals following vaccination and challenge, and a large number of immunological reagents are now available for cattle.

Vaccine development and our understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by definition of the immunological correlates of protection and/or pathology. In this study we analyzed humoral immune responses in Mycobacterium bovis BCG-vaccinated and control cattle (in particular, the relationship between the intradermal comparative tuberculin skin test and serum immunoglobulin G [IgG] responses) against a range of mycobacterial antigens (MPB59, MPB64, MPB70, MPB83, ESAT-6, CFP-10, Acr1, and PstS-1) by multiantigen print immunoassay and conventional enzyme-linked immunosorbent assay. Following M. bovis infection, the comparative tuberculin skin test strongly boosted IgG, IgG1, and IgG2 antibody responses, particularly against MPB83 and MPB70, in unvaccinated cattle but failed to boost these responses, or did so only weakly, in BCG-vaccinated calves. In addition, the skin test-induced increases in MPB83-specific IgG responses correlated positively with bacterial loads and ESAT-6-induced in vitro gamma interferon responses. In conclusion, both the negative correlation of skin test-enhanced MPB83-specific antibody responses with BCG-induced protection and their positive correlation with bacterial loads can serve as useful markers for vaccine efficacy after challenge.

The variable efficacy of bacillus Calmette-Guérin ( Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-γ) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-γ responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-γ responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.

A scientific review for the government of the United Kingdom has recommended that the development of a cattle vaccine against bovine tuberculosis holds the best prospects to control this disease in the national herd. As BCG vaccination of cattle results in variable degrees of protection, novel vaccine strategies that could replace or supplement BCG are required. In this study, the mycobacterial antigen HSP65 was used to determine whether priming cattle with a plasmid DNA vaccine and subsequently boosting with the recombinant protein in adjuvant (heterologous prime-boost approach) would result in improved and more homogenous immune responses over immunising with plasmid DNA or protein in adjuvant alone. The results demonstrated that strong, and compared to protein or DNA vaccination protocols alone, more homogenous, cellular immune responses were induced in cattle vaccinated with the prime-boost regimen. In addition, DNA prime-protein boost vaccination as well as protein vaccination resulted in stronger humoral immune responses with a balanced IgG profile compared to DNA vaccination alone. Importantly, none of the vaccination protocols sensitised cattle to the intradermal tuberculin test suggesting that TB subunit vaccines can be designed to allow the continued use of the tuberculin test to discriminate between vaccinated cattle and those infected with Mycobacterium bovis.

Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) and play relevant roles in several chronic infections, including human TB and HIV. However, the assessment of this response in bovine infections was not feasible due to the lack of monoclonal antibodies that recognize bovine IL-2. Recently, an antibody specific for bovine IL-2 enabled the evaluation of polyfunctional T cells in cattle. The objective of the present study was to access antigen-specific polyfunctional ex vivo responses after aerosol Mycobacterium bovis infection of cattle. Peripheral blood mononuclear cells (PBMCs) were collected from infected cattle and stimulated with rESAT-6:CFP-10 (E:C), media or pokeweed (PWM) for 16 hours. After stimulation, the expression of CD4, CD45RO, CCR7, IL-2, IFN-γ, TNF-α and cell viability were evaluated. The ex vivo response to E:C consisted of 63% effectors (CD4+ CD45RO- CCR7-), 32% effector memory (CD4+ CD45RO+ CCR7-), and 9% central memory (CD4+ CD45RO+ CCR7+) phenotypes. In regard to the cytokine profile, 70% of cells producing cytokines expressed both IFN-γ and TNF-α, 31% expressed all three cytokines, 6% expressed both TNF-α and IL-2, and 3% expressed IL-2 and IFN-γ. Cells producing only IFN-γ, IL-2, or TNF-α represented, 5%, 8% and 1%, respectively. These findings demonstrate that the polyfunctional response consists mainly of effector cells co-producing IFN-γ and TNF-α.

Long-term (i.e., 14 days) cultured IFN-γ ELISPOT assays measure central memory T cell (Tcm) responses in both humans and cattle. With bovine tuberculosis, vaccine-elicited long-term IFN-γ ELISPOT responses correlate with protection. In other species, Tcm’s posses low activation threshold and are highly proliferative. It has been shown that Tcm’s accounts for 75% of long-term cultured cells. The objective of this study was to access the proliferative capability of Tcm cells compared to 6 day cultured cells (n = 6) in response to aerosol Mycobacterium bovis infection. Long-term cultured PBMCs from infected cattle were stimulated with M. bovis purified protein derivative (PPDb), rESAT-6:CFP10 (E:C), rTB10.4 and rAg85A for 13 days. Next, cells were stained with CellTrace dye and re-stimulated with E:C in the presence of fresh autologous adherent cells for an additional six days (20 days). In response to E:C, long-term cultured were mainly CD4+ cells and were more proliferative (~ 48%) than 6d cultured cells (27%; p=0.001). Long-term cultured cells were: ~30% Tcm (CCR7+, CD45RO+), ~48% Tem (CCR7-, CD45RO+), ~8% effector (CCR7-, CD45RO-) and

Leprosy research has predominantly been concerned with the dichotomy between immunological events in tuberculoid leprosy and lepromatous leprosy. Thus, distinct cytokine-secreting profiles have been reported for cloned CD4 and CD8 T cells (30), and lepromatous leprosy patients were found to respond to some purified antigens while remaining anergic to whole mycobacterial extracts (21, 26, 34). Analysis of the specificity of “split anergy” at the level of individual antigenic determinants showed skewing of T-cell responses toward Mycobacterium tuberculosis-specific epitopes, accompanied by relative anergy to the cross-reactive common mycobacterial peptides (13). However, consistent differences in either the specificities or phenotypes of T cells between the sensitized healthy contacts and tuberculoid leprosy patients have so far not been revealed. Previous studies of HLA associations reported the association of tuberculoid leprosy with DR2 in India (18, 37), Japan (11, 23), Thailand (31), and Korea (16); with DR3 in Venezuela and Surinam (36); or with DQ1 (11, 23, 31). On the basis of molecular modelling, the presence of arginine (R) and absence of negatively charged amino acids at positions 13 or 70–71 in pocket 4 of the DRB1 alleles (e.g., DR15) has been associated with susceptibility to tuberculoid leprosy (40), but the molecular source of the corresponding peptide specificity has not been identified. Proteins with a molecular mass of 10 to 12 kDa from Mycobacterium leprae (10) and M. tuberculosis (22) were originally found to carry separate species-specific epitopes identified with monoclonal antibodies. Their sequence analysis showed 90% identity between M. leprae and M. tuberculosis and 44% homology with the GroES heat shock protein of Escherichia coli (1, 20, 33), and the protein was localized to the cell wall and cytosol of M. leprae (29). The protein induced strong DTH and Th1 cytokine production, and limiting cell dilution analysis showed a high frequency of responding T cells from blood or from lepromin-induced Mitsuda reactions (17, 19, 35), but it also reacted with human “suppressor” T-cell clones (27) and was reported either to suppress (32) or to induce (9) DTH responses in lepromatous leprosy patients. This study was performed at the level of individual peptide determinants, with additional emphasis on the analysis of HLA class II genetic associations. Using a comprehensive set of GroES peptides of the M. leprae and M. tuberculosis sequences, the aim of this study has been to identify any differences in the proliferation of blood mononuclear cells between paucibacillary or multibacillary leprosy patients and healthy leprosy contacts or medical staff in Thailand. Possible HLA associations were ascertained on the basis of typing for HLA-DR and -DQ alleles and by peptide binding to purified DR molecules.

orrigendum to ‘‘Bovine tuberculosis in Europe from the perspective f an officially tuberculosis free country: Trade, surveillance and iagnostics’’ [Vet. Microbiol. 151 (2011) 152–159] ene Schiller *, W. Ray Waters , H. Martin Vordermeier , Thomas Jemmi , ichael Welsh , Nicolas Keck , Adam Whelan , Eamonn Gormley , aria Laura Boschiroli , Jean Louis Moyen , Carmen Vela , Monica Cagiola , yce M. Buddle , Mitchell Palmer , Tyler Thacker , Bruno Oesch l

Background Most countries carrying out campaigns of bovine tuberculosis (TB) eradication impose a ban on the use of mycobacterial vaccines in cattle. However, vaccination against paratuberculosis (PTB) in goats is often allowed even when its effect on TB diagnosis has not been fully evaluated. To address this issue, goat kids previously vaccinated against PTB were experimentally infected with TB. Results Evaluation of interferon-γ (IFN-γ) secretion induced by avian and bovine tuberculins (PPD) showed a predominant avian PPD-biased response in the vaccinated group from week 4 post-vaccination onward. Although 60% of the animals were bovine reactors at week 14, avian PPD-biased responses returned at week 16. After challenge with M. caprae, the IFN-γ responses radically changed to show predominant bovine PPD-biased responses from week 18 onward. In addition, cross-reactions with bovine PPD that had been observed in the vaccinated group at week 14 were reduced when using the M. tuberculosis complex-specific antigens ESAT-6/CFP-10 and Rv3615c as new DIVA (differentiation of infected and vaccinated animals) reagents, which further maintained sensitivity post-challenge. Ninety percent of the animals reacted positively to the tuberculin cervical comparative intradermal test performed at 12 weeks post-infection. Furthermore, post-mortem analysis showed reductions in tuberculous lesions and bacterial burden in some vaccinated animals, particularly expressed in terms of the degree of extrapulmonary dissemination of TB infection. Conclusions Our results suggest a degree of interference of PTB vaccination with current TB diagnostics that can be fully mitigated when using new DIVA reagents. A partial protective effect associated with vaccination was also observed in some vaccinated animals.

A peptide-based approach towards improving the immunodiagnosis of, and vaccination against, tuberculosis faces the problems of MHC restriction of T cell recognition and the poor immunogenicity of peptides in the absence of adjuvant. We sought to compensate this by the use of synthetic branched polypeptides of the poly[Lys-(Xi-DL-Alam)] type, containing a glutamic acid residue (EAK), and further modified either by succinylation (SucEAK) or acetylation (AcEAK). These carriers were conjugated to two permissively recognized peptides of Mycobacterium tuberculosis. The 38p350‐369-SucEAK conjugate enhanced IFN- production more than 13-fold (from 22.6 to 294 pg/ml, p = 0.001) in peripheral blood mononuclear cells from healthy subjects, and 8.7-fold (p = 0.012) in cells from tuberculosis patients. The effect was dependent on the carrier used and on covalent linkage of SucEAK to 38p350‐369. An increased response occurred best in cells from subjects bearing at least one HLA-DR allele for which 38p350‐369 had high binding affinity and required cellular processing of the conjugate as inhibitors (chloroquine and wortmannin) blocked the IFNresponse. SucEAK conjugation of peptide 16p91‐110 did not significantly increase IFNproduction, indicating that the ability of conjugation to enhance the response was peptide structure dependent. These data indicate that the use of SucEAK polymer coupled with permissively recognized peptides could contribute to the development of an improved immunodiagnostic or vaccine reagent for tuberculosis.