Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells

Abstract

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

Figure 1

Responses of Mtb11-specific T cell clone (4E4) to either purified rMtb11 or varying numbers of E. coli expressing either vector alone or rMtb11. 10⁴ 4E4 cells were cultured for 3 d with either (A) 10⁴ autologous macrophages or (B) 10⁴ autologous monocyte-derived DCs that had been incubated with either E. coli or purified rMtb11. Supernatants were assessed for IFN-γ levels by ELISA. The highest concentrations of E. coli and rMtb11 were ∼10⁶ bacteria per well and 10 μg/ml, respectively.

Figure 2

Reactivities of donor 160 DC-4 and DC-6 T cell lines generated to autologous DCs infected with Mtb to (A) purified rMtb antigens (10 μg/ml) and (B) E. coli expressing rMtb11 either neat or diluted 1:25 in control E. coli.

Figure 3

Screen of 48 pools of ∼50–80 recombinants per pool from Mtb Erdman library with donor 160 DC-6 T cell line. Data are the mean of duplicate wells. (A) Proliferation. (B) IFN-γ. (C) Confirmation of single positive clone derived from pool 31.

Figure 4

Responses of donor 160 DC-6 T cell line to either (A) synthetic peptides generated from Mtb9.9A (10 μg/ml) or (B) rMtb9.9A.

Figure 5

Southern blot analysis of Mtb9.9 genes. Genomic DNA from various mycobacterial strains was digested with PstI, separated by agarose gel electrophoresis, and blotted on Nytran^®. The mtb9.9a gene was labeled with ³²P and used as a probe. Size markers (M) are in kb.

Figure 6

Predicted amino acid sequence of Mtb9.9 proteins. (A) Comparison of the predicted amino acid sequences of the five Mtb9.9 family members, and (B) overlapping synthetic peptides generated comprising portions of these sequences (peptides derived from Mtb9.9A denoted by number only). The Mtb H37Rv nucleotide sequences of mtb9.9a, mtb9.9c, mtb9.9d, and mtb9.9e are available from GenBank under accession nos. CAA17714, CAB07821, CAB06842, and CAB06161, respectively. The Mtb Erdman nucleotide sequence of mtb9.9b is available from GenBank under accession no. AF226277.

Figure 8

Response of PBMCs from (A and C) 12 healthy PPD⁺ and (B and D) 12 normal PPD⁻ donors to rMtb9.9A. A and B show proliferation data; C and D show IFN-γ data. CFPs, r85B, and rMtb9.9A were used at 10 μg/ml, and Tetanus Toxoid (Tet Tox) was used at 0.1 LP/ml.

Figure 7

Western blot analysis of Mtb9.9 protein. The blots were probed with (A) preimmune rabbit serum, (B) antiserum to rMtb9.9A, and (C) antiserum to antigen r85B. Mtb lysate and CFPs were run at 5 μg/lane and rMtb9.9A at 50 ng/lane.

Figure 9

Response of PBMCs from four PPD⁺ donors to rMtb9.9A and overlapping synthetic peptides derived from Mtb9.9A, Mtb9.9B, and Mtb9.9C sequences (peptides derived from Mtb9.9A denoted by number only). Peptides and rMtb9.9A were used at 10 μg/ml.

Figure 10

Response of donor 201 T cell line to rMtb9.9A and Mtb9.9A, Mtb9.9B and Mtb9.9C peptides (10 μg/ml).