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O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Nandakumar S, Kannanganat S, Dobos KM, Lucas M, Spencer JS, Fang S, McDonald MA, Pohl J, Birkness K, Chamcha V, Ramirez MV, Plikaytis BB, Posey JE, Amara RR, Sable SB - PLoS Pathog. (2013)

Bottom Line: Glycosylation is the most abundant post-translational polypeptide chain modification in nature.Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood.These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

ABSTRACT
Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

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Characterization of nApa-specific T cell hybridoma clone (4C3).(A–B) The T cell hybridoma 4C3 was generated using draining LN cells of nApa-FIA vaccinated mice. The Ag and epitope specificity of 4C3 T cell clone was determined using IL-2 assay. (A) The 4C3 T cells (5×104 cells/well) were cultured with APCs pulsed with indicated amounts of nApa, rApa or whole trypsin digests using T cell to APC ratio of 1∶1 for 24 h and culture supernatants were evaluated using IL-2 ELISA. (B) Identification of peptide/epitope specific for 4C3 T cell clone. The nApa was digested with trypsin and the peptide fragments were resolved by RP-HPLC, total 25 fractions were collected. Fractions 7–25 containing nApa peptides were used to screen the biological activity of 4C3 clone in IL-2 assay. The whole digest of nApa and fractions 19, 21, and 22 containing single N-terminal glycopeptide (residues p40-145) produced a positive IL-2 response when used for Ag pulsing of APCs. The fraction 19 is characterized by LC-MS (Figure S5B). The data (A and B) are means (O.D. 405 nm) of culture supernatant evaluations from two separate cultures in IL-2 ELISA. The experiment was repeated with similar results.
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ppat-1003705-g005: Characterization of nApa-specific T cell hybridoma clone (4C3).(A–B) The T cell hybridoma 4C3 was generated using draining LN cells of nApa-FIA vaccinated mice. The Ag and epitope specificity of 4C3 T cell clone was determined using IL-2 assay. (A) The 4C3 T cells (5×104 cells/well) were cultured with APCs pulsed with indicated amounts of nApa, rApa or whole trypsin digests using T cell to APC ratio of 1∶1 for 24 h and culture supernatants were evaluated using IL-2 ELISA. (B) Identification of peptide/epitope specific for 4C3 T cell clone. The nApa was digested with trypsin and the peptide fragments were resolved by RP-HPLC, total 25 fractions were collected. Fractions 7–25 containing nApa peptides were used to screen the biological activity of 4C3 clone in IL-2 assay. The whole digest of nApa and fractions 19, 21, and 22 containing single N-terminal glycopeptide (residues p40-145) produced a positive IL-2 response when used for Ag pulsing of APCs. The fraction 19 is characterized by LC-MS (Figure S5B). The data (A and B) are means (O.D. 405 nm) of culture supernatant evaluations from two separate cultures in IL-2 ELISA. The experiment was repeated with similar results.

Mentions: Since nApa is also glycosylated with complex mannose modifications at its N-terminus (Figure S3B), the presence of N-terminal glycopeptide(s)-specific T cells in our nApa vaccinated mice samples remained a possibility. Our preliminary attempt to synthesize and evaluate N-terminal glycopeptide (39-mer) was unsuccessful, and whether nApa glycopeptide-specific T cells are generated following subunit vaccination was not clear. Therefore, to identify nApa-specific T cell clones we employed a specialized protocol of T-cell hybridoma generation, using a single dose nApa vaccination of BALB/c mice in Freund's incomplete adjuvant (FIA). Individual T cell clones responding to nApa were purified by serial dilutions and tested for reactivity to nApa or rApa using an IL-2 capture ELISA after co-culture with APCs (i.e., syngeneic bone marrow derived dendritic cells) pulsed with Ag. Of the total 17 Apa-reactive hybridoma clones developed, 7 clones recognized both nApa and rApa while 10 clones recognized only nApa and not rApa (data not shown). Three of the 7 clones that recognized both nApa and rApa responded to a specific synthetic, nonmodified 15-mer peptide (two reactive to peptides spanning p271-288, and one to peptide p70-84, data not shown), while none of the 10 nApa reactive clones recognized any of the synthetic, overlapping, nonmodified peptides. Of the 10 clones only reacting to nApa, one clone, 4C3, reacted with a glycopeptide fraction of trypsin digested nApa fractionated by a reversed phase-HPLC column chromatography. Clone 4C3 produced significant amount of IL-2 when cultured with APCs pulsed with the whole digest of nApa but not with the digest of rApa (Figure 5A), indicating that the Ag presentation of nApa to 4C3 T cell clone was not inhibited by trypsin digestion. The RP-HPLC fractions consisting of the N-terminal 106 amino acid glycopeptide (residues p40-145) of nApa only demonstrated reactivity in IL-2 assay (Figure 5B, Figure S5A and S5B). Glycosylation of the N-terminus of nApa from Mtb likely explains specific recognition of the 4C3 clone to the nApa peptide. The lack of biological activity of T cell clone 4C3 when presented with rApa or synthetic nonglycosylated peptides collectively suggest that N-terminal glycopeptide-specific T cells are generated after nApa subunit vaccination. Further confirmation of these results will require identification of the precise epitope and synthesis of the N-terminal glycopeptide.


O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Nandakumar S, Kannanganat S, Dobos KM, Lucas M, Spencer JS, Fang S, McDonald MA, Pohl J, Birkness K, Chamcha V, Ramirez MV, Plikaytis BB, Posey JE, Amara RR, Sable SB - PLoS Pathog. (2013)

Characterization of nApa-specific T cell hybridoma clone (4C3).(A–B) The T cell hybridoma 4C3 was generated using draining LN cells of nApa-FIA vaccinated mice. The Ag and epitope specificity of 4C3 T cell clone was determined using IL-2 assay. (A) The 4C3 T cells (5×104 cells/well) were cultured with APCs pulsed with indicated amounts of nApa, rApa or whole trypsin digests using T cell to APC ratio of 1∶1 for 24 h and culture supernatants were evaluated using IL-2 ELISA. (B) Identification of peptide/epitope specific for 4C3 T cell clone. The nApa was digested with trypsin and the peptide fragments were resolved by RP-HPLC, total 25 fractions were collected. Fractions 7–25 containing nApa peptides were used to screen the biological activity of 4C3 clone in IL-2 assay. The whole digest of nApa and fractions 19, 21, and 22 containing single N-terminal glycopeptide (residues p40-145) produced a positive IL-2 response when used for Ag pulsing of APCs. The fraction 19 is characterized by LC-MS (Figure S5B). The data (A and B) are means (O.D. 405 nm) of culture supernatant evaluations from two separate cultures in IL-2 ELISA. The experiment was repeated with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795050&req=5

ppat-1003705-g005: Characterization of nApa-specific T cell hybridoma clone (4C3).(A–B) The T cell hybridoma 4C3 was generated using draining LN cells of nApa-FIA vaccinated mice. The Ag and epitope specificity of 4C3 T cell clone was determined using IL-2 assay. (A) The 4C3 T cells (5×104 cells/well) were cultured with APCs pulsed with indicated amounts of nApa, rApa or whole trypsin digests using T cell to APC ratio of 1∶1 for 24 h and culture supernatants were evaluated using IL-2 ELISA. (B) Identification of peptide/epitope specific for 4C3 T cell clone. The nApa was digested with trypsin and the peptide fragments were resolved by RP-HPLC, total 25 fractions were collected. Fractions 7–25 containing nApa peptides were used to screen the biological activity of 4C3 clone in IL-2 assay. The whole digest of nApa and fractions 19, 21, and 22 containing single N-terminal glycopeptide (residues p40-145) produced a positive IL-2 response when used for Ag pulsing of APCs. The fraction 19 is characterized by LC-MS (Figure S5B). The data (A and B) are means (O.D. 405 nm) of culture supernatant evaluations from two separate cultures in IL-2 ELISA. The experiment was repeated with similar results.
Mentions: Since nApa is also glycosylated with complex mannose modifications at its N-terminus (Figure S3B), the presence of N-terminal glycopeptide(s)-specific T cells in our nApa vaccinated mice samples remained a possibility. Our preliminary attempt to synthesize and evaluate N-terminal glycopeptide (39-mer) was unsuccessful, and whether nApa glycopeptide-specific T cells are generated following subunit vaccination was not clear. Therefore, to identify nApa-specific T cell clones we employed a specialized protocol of T-cell hybridoma generation, using a single dose nApa vaccination of BALB/c mice in Freund's incomplete adjuvant (FIA). Individual T cell clones responding to nApa were purified by serial dilutions and tested for reactivity to nApa or rApa using an IL-2 capture ELISA after co-culture with APCs (i.e., syngeneic bone marrow derived dendritic cells) pulsed with Ag. Of the total 17 Apa-reactive hybridoma clones developed, 7 clones recognized both nApa and rApa while 10 clones recognized only nApa and not rApa (data not shown). Three of the 7 clones that recognized both nApa and rApa responded to a specific synthetic, nonmodified 15-mer peptide (two reactive to peptides spanning p271-288, and one to peptide p70-84, data not shown), while none of the 10 nApa reactive clones recognized any of the synthetic, overlapping, nonmodified peptides. Of the 10 clones only reacting to nApa, one clone, 4C3, reacted with a glycopeptide fraction of trypsin digested nApa fractionated by a reversed phase-HPLC column chromatography. Clone 4C3 produced significant amount of IL-2 when cultured with APCs pulsed with the whole digest of nApa but not with the digest of rApa (Figure 5A), indicating that the Ag presentation of nApa to 4C3 T cell clone was not inhibited by trypsin digestion. The RP-HPLC fractions consisting of the N-terminal 106 amino acid glycopeptide (residues p40-145) of nApa only demonstrated reactivity in IL-2 assay (Figure 5B, Figure S5A and S5B). Glycosylation of the N-terminus of nApa from Mtb likely explains specific recognition of the 4C3 clone to the nApa peptide. The lack of biological activity of T cell clone 4C3 when presented with rApa or synthetic nonglycosylated peptides collectively suggest that N-terminal glycopeptide-specific T cells are generated after nApa subunit vaccination. Further confirmation of these results will require identification of the precise epitope and synthesis of the N-terminal glycopeptide.

Bottom Line: Glycosylation is the most abundant post-translational polypeptide chain modification in nature.Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood.These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

ABSTRACT
Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Show MeSH
Related in: MedlinePlus