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A Modified Bacillus Calmette-Guérin (BCG) Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence.

Shoen CM, DeStefano MS, Hager CC, Tham KT, Braunstein M, Allen AD, Gates HO, Cynamon MH, Kernodle DS - Vaccines (Basel) (2013)

Bottom Line: The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase.In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis.The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

View Article: PubMed Central - PubMed

Affiliation: Veterans Affairs Medical Center, Syracuse, NY 13212, USA. shoenc@cnyrc.org.

ABSTRACT
Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

No MeSH data available.


Related in: MedlinePlus

Construction of 4dBCG. (A) Hexameric ring representing half of the enzymatically-active dodecameric form of GlnA1 (left) and enlargement (right) of the area within the rectangle to show the deleted amino acids in relationship to the active site manganese atom, represented by the black dot, with amino acids numbered according to the glutamine synthetase of Salmonella [30]. As the active site comprises residues from adjacent monomers, insertion of one ∆D50∆E327 dnGlnA1 monomer into the ring is predicted to inactivate two active sites. (B) SDS-PAGE (upper) and immunoblot (lower) for GlnA1 of lysates of Bacillus Calmette-Guérin (BCG), 3dBCG and 4dBCG. Lanes are labeled on the figures as L (lysate) and AS (ammonium sulfate-treated lysate) for each strain, kDaM = Kilodalton markers. (C) Graph of a representative experiment comparing the glutamine synthetase activity of undiluted and diluted AS preparations from 3dBCG and 4dBCG as determined by monitoring A540 over time.
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vaccines-01-00034-f001: Construction of 4dBCG. (A) Hexameric ring representing half of the enzymatically-active dodecameric form of GlnA1 (left) and enlargement (right) of the area within the rectangle to show the deleted amino acids in relationship to the active site manganese atom, represented by the black dot, with amino acids numbered according to the glutamine synthetase of Salmonella [30]. As the active site comprises residues from adjacent monomers, insertion of one ∆D50∆E327 dnGlnA1 monomer into the ring is predicted to inactivate two active sites. (B) SDS-PAGE (upper) and immunoblot (lower) for GlnA1 of lysates of Bacillus Calmette-Guérin (BCG), 3dBCG and 4dBCG. Lanes are labeled on the figures as L (lysate) and AS (ammonium sulfate-treated lysate) for each strain, kDaM = Kilodalton markers. (C) Graph of a representative experiment comparing the glutamine synthetase activity of undiluted and diluted AS preparations from 3dBCG and 4dBCG as determined by monitoring A540 over time.

Mentions: Details of the construction of 3dBCG, with three genetic modifications of the parent BCG Tice vaccine strain, have been previously reported [12]. Two of the modifications involved allelic inactivation of sigH, the oxidative stress sigma factor [20,26,27] and secA2, the secretion channel for iron co-factored superoxide dismutase (SodA) [17,18]. A third modification involved recombinant expression of a dominant-negative ∆H28∆H76 mutant of sodA that reduced SodA activity by more than 90% compared to the parent vaccine. GlnA1 is another microbial factor implicated in immune suppression that has been shown to confer resistance to killing of M. tuberculosis by human macrophages [24,25]. As GlnA1 is essential for the growth of M. tuberculosis [28] we partially lowered GlnA1 activity by using dominant-negative interference techniques while preserving potentially important epitopes for immune recognition [29]. First we deleted two amino acids that are important for enzymatic activity, an aspartic acid at position 50 and glutamic acid at position 327 (Figure 1A). Then we inserted the allele encoding the ∆D50∆E327 GlnA1 monomer on a plasmid vector into 3dBCG to yield 4dBCG. Immunoblotting demonstrated that enzyme activity was reduced 8-fold in 4dBCG, verifying a dominant-negative effect (Figure 1B,C).


A Modified Bacillus Calmette-Guérin (BCG) Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence.

Shoen CM, DeStefano MS, Hager CC, Tham KT, Braunstein M, Allen AD, Gates HO, Cynamon MH, Kernodle DS - Vaccines (Basel) (2013)

Construction of 4dBCG. (A) Hexameric ring representing half of the enzymatically-active dodecameric form of GlnA1 (left) and enlargement (right) of the area within the rectangle to show the deleted amino acids in relationship to the active site manganese atom, represented by the black dot, with amino acids numbered according to the glutamine synthetase of Salmonella [30]. As the active site comprises residues from adjacent monomers, insertion of one ∆D50∆E327 dnGlnA1 monomer into the ring is predicted to inactivate two active sites. (B) SDS-PAGE (upper) and immunoblot (lower) for GlnA1 of lysates of Bacillus Calmette-Guérin (BCG), 3dBCG and 4dBCG. Lanes are labeled on the figures as L (lysate) and AS (ammonium sulfate-treated lysate) for each strain, kDaM = Kilodalton markers. (C) Graph of a representative experiment comparing the glutamine synthetase activity of undiluted and diluted AS preparations from 3dBCG and 4dBCG as determined by monitoring A540 over time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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vaccines-01-00034-f001: Construction of 4dBCG. (A) Hexameric ring representing half of the enzymatically-active dodecameric form of GlnA1 (left) and enlargement (right) of the area within the rectangle to show the deleted amino acids in relationship to the active site manganese atom, represented by the black dot, with amino acids numbered according to the glutamine synthetase of Salmonella [30]. As the active site comprises residues from adjacent monomers, insertion of one ∆D50∆E327 dnGlnA1 monomer into the ring is predicted to inactivate two active sites. (B) SDS-PAGE (upper) and immunoblot (lower) for GlnA1 of lysates of Bacillus Calmette-Guérin (BCG), 3dBCG and 4dBCG. Lanes are labeled on the figures as L (lysate) and AS (ammonium sulfate-treated lysate) for each strain, kDaM = Kilodalton markers. (C) Graph of a representative experiment comparing the glutamine synthetase activity of undiluted and diluted AS preparations from 3dBCG and 4dBCG as determined by monitoring A540 over time.
Mentions: Details of the construction of 3dBCG, with three genetic modifications of the parent BCG Tice vaccine strain, have been previously reported [12]. Two of the modifications involved allelic inactivation of sigH, the oxidative stress sigma factor [20,26,27] and secA2, the secretion channel for iron co-factored superoxide dismutase (SodA) [17,18]. A third modification involved recombinant expression of a dominant-negative ∆H28∆H76 mutant of sodA that reduced SodA activity by more than 90% compared to the parent vaccine. GlnA1 is another microbial factor implicated in immune suppression that has been shown to confer resistance to killing of M. tuberculosis by human macrophages [24,25]. As GlnA1 is essential for the growth of M. tuberculosis [28] we partially lowered GlnA1 activity by using dominant-negative interference techniques while preserving potentially important epitopes for immune recognition [29]. First we deleted two amino acids that are important for enzymatic activity, an aspartic acid at position 50 and glutamic acid at position 327 (Figure 1A). Then we inserted the allele encoding the ∆D50∆E327 GlnA1 monomer on a plasmid vector into 3dBCG to yield 4dBCG. Immunoblotting demonstrated that enzyme activity was reduced 8-fold in 4dBCG, verifying a dominant-negative effect (Figure 1B,C).

Bottom Line: The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase.In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis.The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

View Article: PubMed Central - PubMed

Affiliation: Veterans Affairs Medical Center, Syracuse, NY 13212, USA. shoenc@cnyrc.org.

ABSTRACT
Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

No MeSH data available.


Related in: MedlinePlus