Limits...
Vaccination against Streptococcus pneumoniae using truncated derivatives of polyhistidine triad protein D.

Plumptre CD, Ogunniyi AD, Paton JC - PLoS ONE (2013)

Bottom Line: In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein.This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised.These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity.

View Article: PubMed Central - PubMed

Affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.

ABSTRACT
Polyhistidine triad protein D (PhtD) has been described as a promising vaccine candidate for use against Streptococcus pneumoniae, but there has been a lack of examination of its structure and of which region(s) of the protein are targeted by protective immune responses. In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein. This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised. The truncated derivatives were tested as vaccine antigens in mouse models of pneumococcal sepsis and colonisation, using alum and E. coli heat labile toxin B subunit respectively as adjuvants. These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity. These results are significant for the potential for PhtD to be used in novel vaccines, which are currently being tested in clinical trials.

Show MeSH

Related in: MedlinePlus

Presence and exposure of Pht proteins in strain P9 compared to D39.A: Western blotting using pneumococcal cell lysates with anti-PhtD (left) or anti-PhtABDE (right) polyclonal sera. Mobilities of molecular size markers are indicated. B and C: detection of PhtD on the surface of D39 (B) and P9 (C) by flow cytometry using antisera generated against PhtD truncated derivatives. NMS, normal mouse serum (negative control). Median fluorescence intensities for 10,000 events are displayed.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814962&req=5

pone-0078916-g007: Presence and exposure of Pht proteins in strain P9 compared to D39.A: Western blotting using pneumococcal cell lysates with anti-PhtD (left) or anti-PhtABDE (right) polyclonal sera. Mobilities of molecular size markers are indicated. B and C: detection of PhtD on the surface of D39 (B) and P9 (C) by flow cytometry using antisera generated against PhtD truncated derivatives. NMS, normal mouse serum (negative control). Median fluorescence intensities for 10,000 events are displayed.

Mentions: To test the effectiveness of immunisation with truncated derivatives of PhtD against disease caused by a different strain from D39, we next examined the clinically relevant serotype 6A strain P9 (recently isolated as part of the Program for Appropriate Technology in Health [PATH] pneumococcal vaccines project). The presence and immunological cross-reactivity of Pht proteins in this strain with Pht proteins of D39 was checked by Western blotting using whole-cell lysates of wild-type P9 and D39, as well as of a D39 ΔphtABDE mutant, with anti-PhtD or anti-PhtABDE (from animals immunised with a combination of all four Pht proteins) polyclonal murine sera. As shown in Figure 7A, there were only small differences in the presence or reactivity of bands corresponding to Pht proteins between D39 and P9, implying that murine antibodies generated by immunisation with Pht proteins (which were cloned from D39) were likely to be able to bind P9 in the sepsis model. A band at around 80 kDa was detected in both Western blots; it is not clear whether this represents PhtA (which has a predicted molecular weight of approximately 92 kDa) or a partly degraded form of PhtD.


Vaccination against Streptococcus pneumoniae using truncated derivatives of polyhistidine triad protein D.

Plumptre CD, Ogunniyi AD, Paton JC - PLoS ONE (2013)

Presence and exposure of Pht proteins in strain P9 compared to D39.A: Western blotting using pneumococcal cell lysates with anti-PhtD (left) or anti-PhtABDE (right) polyclonal sera. Mobilities of molecular size markers are indicated. B and C: detection of PhtD on the surface of D39 (B) and P9 (C) by flow cytometry using antisera generated against PhtD truncated derivatives. NMS, normal mouse serum (negative control). Median fluorescence intensities for 10,000 events are displayed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814962&req=5

pone-0078916-g007: Presence and exposure of Pht proteins in strain P9 compared to D39.A: Western blotting using pneumococcal cell lysates with anti-PhtD (left) or anti-PhtABDE (right) polyclonal sera. Mobilities of molecular size markers are indicated. B and C: detection of PhtD on the surface of D39 (B) and P9 (C) by flow cytometry using antisera generated against PhtD truncated derivatives. NMS, normal mouse serum (negative control). Median fluorescence intensities for 10,000 events are displayed.
Mentions: To test the effectiveness of immunisation with truncated derivatives of PhtD against disease caused by a different strain from D39, we next examined the clinically relevant serotype 6A strain P9 (recently isolated as part of the Program for Appropriate Technology in Health [PATH] pneumococcal vaccines project). The presence and immunological cross-reactivity of Pht proteins in this strain with Pht proteins of D39 was checked by Western blotting using whole-cell lysates of wild-type P9 and D39, as well as of a D39 ΔphtABDE mutant, with anti-PhtD or anti-PhtABDE (from animals immunised with a combination of all four Pht proteins) polyclonal murine sera. As shown in Figure 7A, there were only small differences in the presence or reactivity of bands corresponding to Pht proteins between D39 and P9, implying that murine antibodies generated by immunisation with Pht proteins (which were cloned from D39) were likely to be able to bind P9 in the sepsis model. A band at around 80 kDa was detected in both Western blots; it is not clear whether this represents PhtA (which has a predicted molecular weight of approximately 92 kDa) or a partly degraded form of PhtD.

Bottom Line: In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein.This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised.These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity.

View Article: PubMed Central - PubMed

Affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.

ABSTRACT
Polyhistidine triad protein D (PhtD) has been described as a promising vaccine candidate for use against Streptococcus pneumoniae, but there has been a lack of examination of its structure and of which region(s) of the protein are targeted by protective immune responses. In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein. This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised. The truncated derivatives were tested as vaccine antigens in mouse models of pneumococcal sepsis and colonisation, using alum and E. coli heat labile toxin B subunit respectively as adjuvants. These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity. These results are significant for the potential for PhtD to be used in novel vaccines, which are currently being tested in clinical trials.

Show MeSH
Related in: MedlinePlus