Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle

Cattle

Approximately 6-month-old-calves (Friesian and Friesian crosses with Limousin and Angus cattle) were obtained from herds free of bovine tuberculosis and kept in the category 3 biosafety accommodation of the Animal Services Unit, VLA Weybridge. Nine animals from 3 different herds were used in this study.

Experimental infection

Calves were infected with an M. bovis field strain from Great Britain (AF 2122/97) by intratracheal instillation of 2–4 × 10⁴ CFU as described previously. At 18–20 weeks postinfection, single intradermal comparative cervical tuberculin tests were performed as specified in the EEC Directive 80/219EEC, amending directive 64/422/EEC, Annexe B and all animals were tested positive for BTB. Their infectious status was finally confirmed at postmortem examinations performed 20–22 weeks postinfection, where all presented with visible lesions typical of bovine tuberculosis in the lymph nodes of the head and lung regions as well as in the lung itself. In addition, we were able to culture M. bovis from tissue samples collected during the postmortem (data not shown). Heparinized blood samples were obtained at 15–20 weeks after infection but before tuberculin skin tests were performed, when strong and sustained in vitro tuberculin responses were observed.

Antigens and peptides

Bovine (PPD-B) and avian (PPD-A) tuberculins were obtained from the Tuberculin Production Unit at the Veterinary Laboratories Agency-Weybridge and used in culture at 10 μg/ml. Overlapping synthetic peptides were prepared (25 amino acid residues long, overlapping by 10 residues) derived from the sequences of 8 putative RD1 region gene products. Their amino acid sequences were deduced from the nucleic acid sequences of the respective open-reading frames (ORF) present in the RD1 region as described earlier [15]. The peptides, purchased from Genemed Synthesis Inc (San Francisco, USA), were synthesized using Fmoc chemistry; and their sequence fidelity and purity was confirmed by mass spectrometry and analytical HPLC, respectively. These peptides were then formulated into pools of 6–22 peptides (see Table 1). The identification numbers (Rv numbers) of the ORF [16], their positions within the RD1 region of M. tuberculosis, the number of peptides and peptide pools representing them, as well as their potential functions are summarized in Table 1. Peptides were used at 5 μg/ml each peptide when used as components of the peptide cocktail. In preliminary experiments we tested an ESAT-6 derived peptide cocktail (11 overlapping peptides) with doses of up to 200 μg/ml total peptide and did not observe any adverse effects on the immune responses induced (data not shown).

Proliferation assay

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Histopaque-1077 (Sigma) gradient centrifugation and cultured in RPMI 1640 supplemented with 5% Controlled Process Serum Replacement type 1 (CSPR-1, Sigma Poole, UK), nonessential amino acids (Sigma), 5 × 10⁻⁵ M 2- mercaptoethanol, 100 U of penicillin per ml, and 100 μg of streptomycin sulphate per ml. PBMC (2 × 10⁵/well in 0·2-ml aliquots) were cultured in triplicate for 6 days in flat-bottom 96-well microtitre plates in the presence of antigen, radio labelled during the last 16–20 h of culture with 37 kBq of (³H)thymidine (Amersham, Little Chalfont, UK) per well, and harvested onto glass fibre filters. Radioactivity was counted in a scintillation counter (TopCount; Packard, Pangborne, UK). Positive responses are defined by a stimulation index SI (counts per minute with antigen/counts per minute without antigen) of at least 2 (SI ≥ 2).

IFN-γ ELISA assay

Supernatants from PBMC cultures were harvested after 96 h of culture, and gamma interferon (IFN-γ) was quantified using the BOVIGAM enzyme-linked immunosorbent assay (ELISA) kit (CSL, Melbourne, Australia). Results obtained with individual peptides and diagnostic cocktails were deemed positive when the differences in the optical density at 450 nm (OD450) with antigens exceed the OD450 without antigens by at least 0·1 OD unit (ΔOD450 ≥ 0·1). For comparative analysis of PPD-B versus PPD-A responses, a positive result was defined as a value for the OD450 with PPD-B minus the OD450 with PPD-A of at least 0·1 and a value for the OD450 with PPD-B minus the OD450 without stimulation (ΔOD450) ≥0·1.

Immune responses to RD1 region antigens

In a pilot experiment we determined if a pool of peptides would result in responses equivalent to those obtained after stimulation with recombinant proteins using a pool of 11 ESAT-6 derived peptides. All animals responding to the recombinant protein also responded to the peptide cocktail (data not shown). Encouraged by these results, we prepared synthetic peptides corresponding to the eight ORFs of the RD1 region of M. tuberculosis, formulated them into pools of overlapping peptides, and determined their capacity to induce proliferation and IFN-γ production in PBMC from a group of 9 calves experimentally infected with M. bovis. PBMC from all the 9 cattle responded upon stimulation with bovine and avian tuberculin by strong IFN-γ production and strong proliferative responses. The responses to bovine tuberculin PPD-B were substantially stronger than to avian PPD-A and by applying the criteria defined in the instructions accompanying the BOVIGAM^TM IFN-γ EIA kit (OD450 differences between PPD-B and PPD-A responses > 0·1), all 9 animals were classified as positive for BTB (Mean IFN-γ responses ± S.E. PPD-B: 2·773 ± 0·1771; PPD-A: 2·016 ± 0·303. Data were normally distributed on F-test). In addition, all 9 animals tested positive for BTB in comparative tuberculin skin tests carried out 1–2 weeks prior to the postmortem examinations (data not shown). M. bovis infection was confirmed at detailed postmortems and by its culture from lymph node tissue samples collected at postmortem.

All animals were tested for responses to the peptide pools of RD1 region antigens. Representative results of one animal after peptide pool stimulation are shown in Fig. 1. This animal responded to stimulation with peptide pools representing RD1 regions gene products Rv3872, Rv3873, Rv3874, and Rv3875 both by proliferation (Fig. 1a) and IFN-γ production (Fig. 1b). However, the peptide pools representing Rv3872, Rv3874 and Rv3875 were recognized most strongly (Fig. 1).

The analysis of results from all 9 animals showed responses to peptide pools representing 5 of 8 ORFs tested (Rv3871, Rv3872, Rv3873, Rv3874 CFP-10], Rv3875 ESAT-6]) in both proliferation and IFN-γ assays (Fig. 2). Animals generally responded by both proliferation and IFN-γ production, although some animals responded either by proliferation or IFN-γ production alone. This is particularly notable in the case of Rv3871, which induced proliferative responses in 5 of 9 animals but IFN-γ in only 2. The responder frequencies underneath in Fig. 2 combine the total numbers of animals recognizing peptides by either test.

Rv3874 (CFP10) and Rv3875 (ESAT-6) were recognized most frequently by 8 of 9 and 6 of 9 calves respectively in proliferation or IFN-γ assays (Fig. 2). These data confirm and reinforce the previously reported immunodominant status of these two antigens in M. bovis infected cattle. In addition, each of the peptide pools representing Rv3872 and Rv3873 were recognized by T cells from 5 of 9 and 6 of 9 infected animals, respectively (Fig. 2). Although 5 of 9 responded to the peptide pool representing Rv3871 in proliferation or IFN-γ assays, the responses were generally weaker (Fig. 2). A degree of individual variability in the ability of T cells from calves tested to recognize different ORFs was apparent, since not all calves recognized all 5 antigens simultaneously (Fig. 1 and data not shown).

To determine whether the individual antigens improved the sensitivity of tests diagnosing BTB, we compared the responses induced by them in relation to the responses observed with tuberculin and in relation to ESAT-6 (Rv3875), the antigen previously described as giving the highest test sensitivity when used individually [17,18]. As shown in Table 2, all 9 animals were diagnosed as positive for BTB when bovine and avian tuberculin PPD were applied either in comparative skin tests or in in vitro tests determining IFN-γ and proliferative responses (Table 2). As described above, ESAT-6 alone induced IFN-γ responses in 6 animals and proliferative responses in 5. CFP-10 (Rv3874) was recognized by all the animals that recognized ESAT-6. In addition, CFP-10 induced proliferative responses in 3 animals, and IFN-γ responses in 2 animals that did not recognize ESAT-6 (Table 2). In total, 8 of 9 animals were correctly identified as having BTB when the results from CFP-10 and ESAT-6 were combined. Rv3871 and Rv3873 were recognized by one animal not responding to ESAT-6 suggesting that both could complement ESAT-6 or CFP-10 as part of a more specific diagnostic test (Table 2). However, the single animal not responding to ESAT-6 and CFP-10 did also not recognize Rv3871 or Rv3873 (data not shown), and only large-scale field evaluation will determine the relative merits of the antigens identified in this study in future specific diagnostic tests for BTB.